Biochemistry Of Normal And Leukemic Leucocytes, Thrombocytes, And Bone Marrow Cells

BIOCHEMISTRY OF NORMAL AND LEUKEMIC LEUCOCYTES. THROMBOCYTES. AND BONE MARROW CELLS I. F. Seitz Scientific Research Institute for Blood Transfusion. Leningrad. U.S.S.R.

I . Introduction . . . . . . . . . . . . . I1. Leucocytes . . . . . . . . . . . . . . A . General Indexes of Metabolic L\rtivity of Leucocytes . . . . B. Content and Resynthesis of A T P in Normal and Leukemic Human . . . . . . . . . . . . . Leucocytes . C. Effect of Metabolic Poisons on Leurocyte Metabolism . . . . D . Characteristics of t.he bletabolisrn of Inorganic Phosphate in Leucocytes . . . . . . . . of Normal and Pathological Blood E. Nucleic Acids, Phospholipids, and Phosphoproteins in Human Leucocytes . . . . . . . . . . . . . . . F. Relationship between Energy Metabolism and Anabolism in Leucocytes G . Substrate of Respiration in Leucocytes . . . . . . . H . Glycogen Metabolism in Leucocytes of Normal Persons and Patients . . . . . . . . with Leukemia and Polycythemia . I. Other Enzymatic Activhies in Normal and Leukemic Human Leucocytes I11. ’I hrombocytes . . . . . . . . . . . . . . A . Respiration, Glycolysis, and Resynthesis of Adenosine Triphosphate in Human Thrombocytes . . . . . . . . . . . 13. Nurleic Acids, Phospholipids, and I’hosphoproteins in Thrombocytes . C . Experiments with Glacose-Cl4: Assimilation of Labeled Fragments from Gli1cose-C1~ by Thrombocytes . . . . . . . . . . . . D . Glycogen and Its Metabolism in Human Thrombocytes . E . Other Enzymatic Activities in Human Thrombocytes . . . . IV Bone Marrow . . . . . . . . . . . . . . A . Energy Metabolimi of Bone Marrow in Normal Subjects and Leukemic Patients . . . . . . . . . . . . . . B . Enzyme Systems Participating in Glycogen Synthesis in Normal and Leukemic Bone Marrow . . . . . . . . . . . C . Some Other Data on the Biochemistry of the Bone Marrow Which Are Characteristic of the Leukemic Process . . . . . . . . V . Discussion and Conclusions . . . . . . . . . . . A . Aerobic Glycolysis . . . . . . . . . . . . . . . . . . . . . B. Aerobic Glycolysis and Cancer . . . . . . C. Aerobic Glycolysis and Functional “Stress” . V I . Summary . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

.

303

304 305 306 323 326 331 331 335 338 340 366 369 370 375 376 377 383 386 386 393 394 395 395 397 401 402 404

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I. F. SEITZ

1. Introduction

Cancer and leukemia present perhaps the most serious challenge offered by nature to science. The disappointing experience in the practical control of these horrible diseases suggests that success can be achieved only on the basis of a deep and multifaceted understanding of the nature of neoplastic cells. If biochemical research in this field is regarded, not from the standpoint of the rather modest achievements gained to date, but in the light of the possibilities which have been created for the immediate future by some astounding recent discoveries, then the perspective appears encouraging. There are important reasons to believe that the part played by biochemistry will be decisive in the final success. Essentially, the problem of malignant tumors is a cellular problem. The primary impulses, leading t o the generalization of the process and finally to the tragic end, apparently originate a t the level of the cell or its elements. In leukemia, the first changes occur in the blood. Therefore, the study of the blood cells and of their source, the bone marrow, may yield the most valuable data for an understanding of the nature of the leukemic transformation. Although the first studies on the chemistry and metabolism of the leucocytes were carried out in the last century and a t the beginning of this century, systematic biochemical studies of these cells were begun only in the 1930’s and 1940’s, and particularly in recent years. A considerable number of the earlier studies were based on a methodology which was on an insufficiently high level, particularly from the standpoint of adequacy of the incubation media for leucocytes, as well as the lack of satisfactory methods for separation and analysis of the blood cells. Consequently, there are many confusing and contradictory data in the literature. In this review, many such studies have not been taken into consideration. It did not appear to be necessary to give an account of the entire history of the investigations into the chemistry and metabolism of leucocytes, since many fine, substantial reviews have been published in which this purpose has been accomplished and in which the results of the studies in this field up to 1959-1960 have been summarized. We refer the reader to the well-known reports of Fleischmann (1939), Valentine (1951), Beck and Valentine (1953), Briickel and Remmele (1954), Remmele (1955), Tullis (1953), Furth and Baldini (1959), Valentine (1960), Seitz and Luganova (1961a), Karnovsky (1962), as well as the monographs by Seitz (1961), Kugelmas (1959), Dameshek and Gunz (1958). Kassirskii and Alekseyev (1962), and others, where the problems of the biochemistry of normal and leukemia leucocytes also received some attention. Under these circumstances, we feel that our aim should be, first, to

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supplement the above sources with a review of the more recent papers; and second, to evaluate critically some of the results and trends in the investigations in the field of the biochemistry of cancer arid leukemia. First of all, it should be mentioned that (1) in this paper, as a rule, only biochemical studies of the blood cells are considered; even cytochemical data which are worthy of independent review are used only in a few special cases as supplemeiital material; ( 2 ) the contents of this paper are limited to a consideration of the chemical and metabolic characteristics of the blood cells of humans only, arid not of animals; (3) special attention is paid to aspects of the energy metabolism of blood cells, while other aspects are dealt with only indirectly and only so far as they are related t o this subject. While taking into account the data in the literature, we have based this paper mainly on the results of investigations on the biochemistry of the blood cells of normal subjects and patients wit,h leukemia, which were carried out over many years in the biochemistry laboratory of the Leningrad Institute for Blood Transfusion (Director: Docent A. D. Belyakov). Several of my colleagues from the Biochemist,ry Laboratory also took part in these studies: I. S. Luganova, A. D. Vladimirova, V. A. Yegorova, and morphologist V. I. Teodorovich, as well as L. M. Rozanova and A. I. Blinova from the Hematology Cliiiic. We are also indebted to Professor S.I. Sherman for his assistance in consultations on hematological problems. II. Leucocytes

One of the most fundamental questions, which was stated by Warburg and which also interested many other investigators, is: How unique and specific is the metabolism of malignaiit neoplasms with its aerobic glycolysis, low respiration, and high anaerohic glycolysis? The solution to this question could be of great import,aiice in understanding the development and characteristics of neoplasms, and possibly even for working out the principles of cancer t,herapy. Warburg himself stated that aerobic glycolysis is not confined to neoplasms. It also occurs in some noncancerous tissues: in benign tumors, lymph glands, emhryoiiic epithelium, retina. Warburg ascribed t,he occurrence of aerobic glycolysis in noncaiicerous tissue to two causes: in some cases he referred tJothe tissues as being in a nonstationary state, while in others he said that the tissues were damaged. According to Warburg, damage is a factor which irreversibly disrupts the Pasteur effect. h-evertheless, Warburg did see essent>ialdifferences in metabolism between cancerous tissues and those noncancerous tissues which carry out aerobic glycolysis. He noted that in malignant tumors the ratio aerobic glycolysis/ respiration is 3-4, while in normal tissues showing aerobic glycolysis this ratio is approximately 1. Even in 1956, Warburg still maintained that the

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lack of correspondence between the extremely high glycolysis and the low, or, as he believes, “damaged,” imperfect respiration is the most characteristic difference between malignant neoplasms and normal tissues. This view is seriously and justly criticized by a number of authors. Interesting thoughts on this subject were expressed by Weinhouse in the discussion in Science (1956) and later at the International Congress in London (1958). The problem of aerobic glycolysis and its relation to growth and cellular functions is one of those problems on the solution of which may depend, to a considerable degree, our understanding of the nature of malignant growth. Since it has been felt thus far that the absence of aerobic glycolysis is a characteristic feature of the majority of animal tissues, while malignant tumors deviate most strikingly from this rule, all cases of coexistence of respiration and glycolysis in noncancerous tissues deserve the most penetrating study. I n this connection, our attention mas drawn to the leucocytes which, according to many reports, are able to form lactic acid in the presence of air. A systematic investigation of leucocyte metabolism carried out in our laboratory demonstrated that they are distinguished by several peculiarities in their metabolism which deserve the serious attention of biochemists. The leucocytes are an exceptionally attractive subject for a study of the relationship between chemical processes and function, since some of them have phagocytic activity and practically all have ameboid motility. The value of leucocytes for experimental studies is determined to a considerable degree by the fact that they are single, freely moving, undamaged cells which have many advantages over tissues, the cells of which are difficult to supply with oxygen and nutrients during experiments. Moreover, the mound surface of tissue slices can substantially influence the character of the metabolism, modifying it to some extent. These factors-permeability and damage-should in no case be disregarded when working with tissue slices, and it is apparently never possible in practice to make a thorough evaluation of the above factors or to make any corrections.

A. GENERAL INDEXESOF METABOLIC ACTIVITYOF LEUCOCYTES Data in the literature on the intensity of respiration and glycolysis in leucocytes are contradictory. For the Qo, of these cells one can find values ranging from 0.4 to 22.8; for Q&, from 0 to 17.8; and for Q&, from 2.7 to 57.8. This lack of agreement can be explained in many ways: (I) The experiments were not performed on pure suspensions of leucocytes but on whole blood or insufficiently purified suspensions; (2) a n inadequate medium was used-salt solutions instead of blood serum; (3) the influence of the density of experimental suspensions on the metabolic indexes of cells was not always taken into account; (4) the methods used for quantita-

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tive evaluatiiig of the metabolism were not always sufficiently accurate, particularly in earlier investigations. In the investigations which were carried out in our laboratory over the course of several years, the study of leucocyte metabolism was carried out under optimal conditions, and the possible sources of error enumerated above were taken into account as much as possible. Leucocytes were isolated from the blood by sedimeritat,ion in gelatin-citrate solution (Teodorovich, 1958; Luganova et al., 1963a). The cell suspensions obtained were of a high degree of purity. Human blood swum was used as the basis of the experimental medium; in some cases it was diluted 1 : 1 or 1 :3 with KrebsRinger phosphate. In some experimeiits, glucose-free donor's serum was used (Luganova et al., 1957a). For isolating the leucocytes, we used the blood of patients in whom one or another cell form was absolutely predominant. Thus, in patients with chronic myeloid leukemia, the cells of the myeloid series reached 85-95%; in chronic lymphadenosis the lymphocyte count was above 90%; and in acute leukemia, the proportion of young, undifferentiated cells was 30-95y0. I t should be noted that some loss of lymphocytes occurred during separation of leucocytes from the blood of healthy donors; because of their lower density t,hey tended t o remain in the supernatant fluid, as a result of which tlhe lymphocyt,e count in the final suspensions was approximately 10% instead of the original 30%. Consequently, the granulocyt,e count was increased to about 90%. The physiological normality of t'he isolated cells was generally checked by their phagocytosis, ameboid motility, and formation of granules with neutral red as seen under the microscope. Two methodological factors are of critical importance in evaluating leucocyte metabolism in experiments in vitro, namely, the composition of the medium and t,he cell concentration in the suspension. Serum is unquestionably the best experimental medium for leucocytes. We have observed that in some cases, for instance, in experiments with lymphocytes (cells having purely oxidative metabolism), t,he use of Krebs-Ringer carbonate instead of serum may lead to noticeable aerobic glycolysis. Moreover, the absolute values for the respiration and glycolysis of the cells are higher in serum than in salt solutions. On the other hand, the respiration and glycolysis of leucocytes depend greatly on the number of cells per unit volume of the experimental suspension. This dependence was noted earlier by Sofier and Wintrobe (1932), Ponder and MacLeod (1936), Barron and Harrop (l929), and Hartmaii (1952). Our experiments demonstrated that the relative rates of respiration and glycolysis increase with aii increase in the dilution of the leucocyte suspension, reaching stable maximal values a t a cell concentration of 0.036 ml./ml. was used. I n the calculation of the Warburg coefficients Qo2,Q"&z, Q&, Q was expressed

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in m ~of gas . ~absorbed or evolved (for respiration, 0; for glycolysis, COz equivalent to the amount of lactic acid formed) per milligram dry weight of cells in 1 hour. Lactic acid was determined colorimetrically by the method of Barker and Summerson (1941) ; respiration, manometrically; ADP (adenosine diphosphate) by and ATP (adenosine triphosphate) adsorption on charcoal (Seitz, 1957). Table I shows a summary of the results of numerous experiments carried out to determine the levels of respiration and glycolysis in different types of human leucocytes under normal conditions and in certain hemopathological states, and for different cell concentrations in the experimental suspensions. The data given in Table I show that the values for oxygen utilization and glycolysis depend markedly on the density of the cell suspension; they also demonstrate one of the most important features of leucocyte metaboliem-the relatively low respiration and extremely high glycolytic activity. Moreover, certain types of leucocytes show a very high aerobic glycolysis; this group includes, along with leukemic leucocytes, the granulogcytes of healthy subjects as well as the leucocytes of patients with polycythemia, a disease which is apparently lionleukemic in nature. In this respect, all types of leucocytes could be classified into two metabolic groups: (1) cells having aerobic glycolysis; (2) cells lacking aerobic glycolysis. It will be shown below that this classification is not arbitrary, but has a profound meaning, reflecting the different biological, functional, and, apparently, genetic characteristics of these two categories of leucocytes.

+

1. Leucocytes with Aerobic Glycolysis

As seen from Table I, this group includes normal leucocytes, leucocytes from polycythemia patients, leucocytes from patients with chronic myeloid leukemia, and leucocytes from some patients with acute leukemia (metabolic type I). Features common t o all these cells are high aerobic and even higher anaerobic glycolysis; low or relatively low respiration; an inverse Pasteur effect (Crabtree effect) ; comparable aerobic and anaerobic resynthesis of ATP. The latter property is also shared by lymphocytes and some other cells, as will be detailed below. a. Glycolysis and Respiration of Normal Leucocytes. Normal leucocytes from healthy donors, according t o our data, contain lo9 cells in 1 ml. of freshly centrifuged leucocytes, corresponding to 187 mg. dry weight. When incubated in blood serum a t 37°C. in a concentration of 0.036 ml./ml. of the experimental suspension, donor leucocytes form lactic acid in an amount equivalent to q&2 = 18.8. Anaerobic glycolysis amounts to QZ& = 31.1 (Luganova and Seitz, 195813). Thus, the Pasteur effect in these cells is imperfect: respiration does not block glycolysis completely and both

T,ZBLE I RESPIRITION,GLYCOLYSIS, A N D THEIR RELATIONSHIP I N DIFFERENT KINDSOF HUMANLEUCOCYTEP No.

Leucocytes of

Q21ueose

Q0+2ucose

Qair GO2

QCO?

Qair

,I

+glucose

coz Qo,

Q ~ ~ ~ / Q O + ~ ~ ~ ~ ~ ~

7 . 5 f 0.5 (20) chronic myeloid leukemia 6 . 4 f 0 . 4 (20) polycythemia 7 . 1 f 0.5 (15) chronic lymphadenosis 7 . 9 f 0.5 (20) acute lenkemia, type I 10.4 f 0.6 (11) acute leukemia, type I1 9.8 f 0 . 9 (21)

1

Healthy donors

2

Patients with

3

Patients with

4

Patients with

5

Patieiit,s with

6

Patients with

1

Healthy donors

2

Patients with chronic myeloid leukemia

3

Patients with chronic lymphadenosis

4

Patients with acute leukemia, type I

5

Patients with acute leukemia, type I1

5.7 f 0.3 (20) 5.0 0.3 (20) 5.3 f 0.3 (15) 8 . 0 f 0.5 (20) 8.3 f 0.5 (11) 10.0 0.8 (21)

*

+

18.8 k 1 . 0 31.1 1.1 (20) (20) 13.2 f 0 . 7 23.3 f 0 . 8 (20) (20) 1 5 . 4 f 0 . 7 27.6 5 1 . 1 (15) (15) 0 27.2 f 1 . 0 (20) (20) 24.0 f 1.1 2‘3.0 f 1 . 2 (11) (11) 0 27.3 f 1 . 3 (21) (21)

3.3

5.5

2.6

4.7

2.9

5.2

0

3.4

2.9

3.5

0

2.7

0.1 ml. cells/ml. experimental suspension

3.4 f 0.2 (65) 4.1 f 0.2 (83) 6.5 f 0.5 (58) 8.4 f 0 . 5 (11) 7 . 0 f 0.6 (5)

The number of experiments is given in parentheses.

*

1.8

3.4

1.8

2.9

0

1.7

1.6

1.9

0

2

309

a

2 . 6 f 0 . 1 6 . 0 f 0 . 5 8.8 f 0 . 6 (65) (65) (65) 3 . 1 f 0 . 2 5 . 6 f 0 . 2 8.9 f 0 . 8 (83) (83) (83) 6 . 6 f 0.3 0 11.1 k 0 . 8 (55) (58) (58) 6 . 2 f 0 . 6 10.0 f 0 . 4 11.8 f 0 . 6 (11! (11) (11) 7.0 0.6 0 13.7 f 1 . 4 (5) (5) (5’

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0.036 ml. cells/ml. experimental suspension

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I. F. SEITZ

processes coexist. I n the presence of air, about 60% of the maximal glycolytic activity of the cell is preserved (Luganova et al., 1957b,e). T h e above data concerning the presence of aerobic glycolysis in the leucocytes of normal subjects are in accord with the results of many workers: Kempner (1939), Beck and Valentine (1952), MacKinney et al. (1953), Martin et al. (1955), Valentine (1955a), Remmele (1955), Larwence (1955), Beck (1955), MacKinney and Rundless (1956), Seelich et al. (1957), Beck (1958a,c), Valentine (1960). MacKinney and Rundless (1956) even found that aerobic glycolysis in the leucocytes of normal people is comparable to anaerobic. The only exception is the work of Warburg et al. (1958). These authors separated leucocytes by rather mild centrifugation from citrated blood and found that a suspension of these cells in serum did not carry out aerobic glycolysis. They concluded that the presence of aerobic glycolysis in leucocytes, reported by other investigators, is an artifact caused by mechanical or chemical injury to the cells. Warburg and his co-workers obtained the following metabolic coefficients for human leucocytes: Qo, = 1.7 - 3.3; Qo M 2 - 0; &Ego" = 13.0. Somewhat later, however, Zatti and Rossi (1961) demonstrated that Warburg's conclusion concerning the absence of aerobic glycolysis in human leucocytes was related to the use, in his experiments, of suspensions consisting of lymphocytes, which do not have aerobic glycolysis. Warburg's method for separating the leucocytes from the blood was apparently such that the polymorphonuclear leucocytes were removed while the examined suspension contained only lymphocytes. It is possible that the data of Burk and his co-workers (1959) concerning the weak aerobic glycolysis in normal human leucocytes can be attributed to the same cause. A review of all the data in the literature, with all the possible methodological errors and imperfections in technique taken into consideration, justifies the conclusion that normal human granulocytes exhibit high aerobic glycolysis. The respiration of normal human leucocytes is not high: in the presence of glucose it corresponds to Qo, = 5.7. However, without addition of glucose (endogenous respiration), the Qo, becomes as high as 7.5. This phenomenon of an inverse Pasteur effect (Crabtree effect) is quite characteristic for cells with aerobic glycolysis (Seitz, 1955, 1961). In normal human leucocytes, respiration is inhibited 24y0 by glycolysis. This depression of the O2 consumption in the presence of glucose is not a result of changes in pH in the course of glycolysis, nor of the accumulation of lactate. It also cannot be explained by a direct inhibitory effect of sugar per se, as was proved by us experimentally on cells of Ehrlich ascites carcinoma as well as on leucocytes (Yel'tsina and Seitz, 1961; Luganova et al., 1957c,d; Seitz, 1961). The effect of glucose on respiration in leucocytes is caused by its

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glycolytic utilization, i.e., by the appearance of glycolysis. Exhaustion of sugar in an experiment with a limited amount of glucose, or addition of 8 X lop4 M monobromoacetate to a sample containing glucose, restores respiration to the endogenous level (i.e., the sample without glucose). Another glycolytic poison-fluoride (2 X lop2M)-also stimulates respiration of leucocytes in the presence of glucose. The increase in oxygen consumption in this case often reaches 100% or even more. Apparently, along with abolition of the inverse Pasteur effect, N a F brings into play certain other mechanisms for activating respiration, particularly an increase in the coilcentration of ADP. It will be clear from the following discussion that fluoride depresses the concentration of ATP in leucocytes. Depression of oxygen consumption by leucocytes when glucose was added was also observed by Martin et al. (1955). Fructose did not preduce the same effect. Seelich et al. (1957) noted a difference in the respiration of normal leucocytes in the presence or absence of glucose, but this difference was not statistically significant. (’hernyak (1957) also noted a distinct inverse Pasteur effect in leucocytes. We must recognize, therefore, the existence in normal human leucocytes of a complex mutual regulation of energy metabolism-a bilateral interaction of respiration and glycolysis, Pasteur effect and inverse Pasteur effect. It would seem that the basis for the mutual inhibition of respiration and glycolysis in cells with aerobic glycolysis is competition for ADP. The values given in Table I for glycolysis and respiration in normal human leucocytes are very close to those reported by Warburg for human carcinoma and sarcoma (Warburg, 1926). Moreover, the ratio &“%,/Qo, = 3.3 or Q&/Qo, = 5.5 (average) observed in normal leucocytes are no lower than the corresponding ratios reported by Warburg for human neoplasms and postulated by him to be specific for malignant tumors. Since the morphological arid functional adequacy of the examined leucocytes was controlled, damage to the cells as a possible cause of the appearance of aerobic glycolysis can be excluded. The initial paradoxical facts obtained in our studies on leucocytes demand close attention and explanation. b. Glycolysis and Respiration o j Leucocytes in Polycythemia Patients. A study of leucocytes in polycythemia patients was of great interest since in this disease, along with an increase in the erythropoietic activity of the bone marrow, the Ieucopoietic tissue also becomes involved in the pathological process (Kassirskii and Alekseyev, 1962). I n the peripheral blood there is usually a shift to the left in polycythemia patients. I n the leucocytes of our patients, the metabolic coefficients were quite close to those of normal leucocytes: Q& = 15.4; Q& = 27.6; Qo, in the presence of glucose = 5.3;

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I. F. SEITZ

Qo2 endogenous = 7.1. The inverse Pasteur effect amounted to 26%; &&,/Qo, = 2.9; QZ&/&o, = 5.2. These values indicate a great similarity between the energy metabolism of leucocytes in polycythemia patients and that in healthy subjects, both quantitatively and qualitatively (aerobic glycolysis, inverse Pasteur effect). The values of the coefficients of respiration and glycolysis in these two groups of leucocytes were almost identical. c. Leucocytes of Patients with Chronic Myeloid Leukemia. It is well known that the leucocytes of the myeloid series predominate in chronic myeloid leukemia, while the lymphocyte and monocyte counts are very low. In our experiments 95y0of the cells in leucocyte suspensions from chronic myeloid leukemia patients were of the myeloid series with varying degrees of maturity. The myeloblast count did not exceed 3y0; 1 ml. of wet leucocytes contained 1.3 x lo9 cells, the corresponding dry weight being 210 mg. A study of the metabolism of leucocytes from patients with chronic myeloid leukemia demonstrated that there was no further increase in glycolytic activity in comparison with normal leucocytes, as might have been expected 011 the basis of the concept of so-called cancerous metabolism. On the contrary, both aerobic and anaerohic glycolysis were significantly lower in the leucocytes of chronic myeloid leukemia patients than in normal leucocytes (Table I ) : &“d& = 13.2; QZb, = 23.3. The respiration of these cells was characterized by Qo, = 5.0 in the presence of glucose and by Qo, = 6.4 in the absence of glucose. Inhibition of respiration by glycolysis (inverse Pasteur effect) amounted on the average to 22%; &&,/Qo, = 2.6 and Q&/&o, = 4.7. I t is thus possible to state that there is a distinct depression in the general level of energy metabolism in the leucocytes of chronic myeloid leukemia patients in comparison with the leucocytes of healthy subjects, due mainly to the decrease in glycolytic activity. Depression of the activity of the glycolytic system in the leucocytes of chronic myeloid leukemia patients (in the whole cells and in homogenates) in comparison with normal leucocytes was also pointed out by Beck and Valentine (1952)) Beck (1958a), Valentine (1955b), MacKinney and Rundless (1956), Martin et al. (1955)) Lawrence (1955)) Beck (1958c), and others. d . Leucocytes of Patients with Acute Leukemia (Metabolic T y p e I ) . The biochemistry of these leucocytes has been studied very little. The data in the literature characterize the young, uridiff erentiated leucocytes (myeloblasts, lymphoblasts) which predominate in the blood in this disease as cells with the oxidative type of metabolism (Kempner, 1939). The existence of two metabolic types of leucocytes in patients with acute leukemia was already noted several years ago by us (Luganova et al., 1957a,b,c,d; Luganova and Seitz) 1958a)b; Luganova and Seitz, 1959d,e,f; Seitz and

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Luganova, 1961a; Seitz, 1961). According to these data, all patients with acute leukemia can be classified into two groups according to the type of metabolism of the leucocytes in their blood. Out of 78 patients with acute leukemia examined, all with 80% or more young, undifferentiated, white blood cells, 38 were in the group with high aerobic glycolysis in their leucocytes; in the remaining 40 patients the leucocytes did not exhibit aerobic glycolysis. The latter group will be discussed later. As far as the leucocytes of the patients with acute leukemia of metabolic type I are concerned, these cells had a metabolism typical of cells of the myeloid series. In principle, the metabolism of the leucocytes of this group of patients with acute leukemia approximates the mebabolism of the leucocytes of patients with chronic myeloid leukemia with the distinction that the respiration arid glycolysis in these cells are much higher a i d the difference between aerobic and anaerobic glycolysis is smaller (see Table I). The leucocytes of patients with acute leukemia having type I metabolism, like all cells of the myeloid series, formed lactic acid in the presence of air and showed the inverse Pasteur effect. The average metabolic coefficients of these cells were: Q g , = 24.0; Q&, = 29.0. Aerobic glycolysis reached 83% of the anaerobic level. Qo, in the presence of glucose = 8.3; Qo, without glucose = 10.4. It is evident from the above values that the inhibition of respiration by glycolysis (inverse Pasteur effect) amouiit,ed to 20%. There are reasons to believe that patients with acute myeloid leukemia predominated in this group. According to morphological criteria, t,he basic cellular elements of the whit'e series in patients with metabolic type I acute leukemia are myeloblasts. We caiiiiot exclude the possibility that the metabolic type I group should also include acute leukemias represented by reticuloendothelial cells (this will be discussed in detail below). 2. Leucocytes Lacking Aerobic Glycolysis

This group of leucocytes is characterized by the inability of the cells to form lactic acid when iricubated in vitro in the presence of air. Consequently, respiration is not inhibited by glucose, and the inverse Pasteur effect is absent in these cells. This group includes lymphocytes and young, undifferentiated cells of the second large group of patients with acute leukemia (lymphoblasts). a. Lymphocytes in the Blood of Patients with Chronic Lymphadenosis. In chronic lymphadenosis the lymphocyte count can be as high as 95y0 and even higher. Since a considerable increase in the total leucocyte count in the blood frequently occurs in this disease, isolation of a practically pure suspension of lymphocytes in quantities sufficient for analysis does not present serious difficulties. However, their chemical composit,ion and metabolism have by far not been sufficiently studied. Contradictory views

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have been presented even with regard to the basic metabolic indexes for these cells. In earlier studies, the metabolism of the lymphocytes was characterized as being purely oxidative (Peschel, 1930). On the other hand, according to the data of MacKinney and Rundless (1956), lymphocytes have aerobic glycolysis. Almost nothing is known concerning the metabolism of phosphorus in the lymphocytes, and so on. However, the study of the lymphocytes is important since these cells, in contrast to the granulocytes, are mononuclear and have a completely different biology as well as different functions: they are devoid of phagocytic ability, but apparently participate actively in the synthesis of antibodies. Table I shows the results of determinations of the intensity of respiration and aerobic and anaerobic glycolysis in the lymphocytes of patients with chronic lymphadenosis during incubation in blood serum a t 37°C. in the presence and absence of glucose. According to our data, 1 ml. of wet lymphocytes contains 2.3 X lo9 cells, with a corresponding dry weight of 160 mg. Although the metabolic coefficients (particularly the level of anaerobic glycolysis) depend on the cell concentration in the experimental suspension, the absence of aerobic formation of lactic acid (Q& = 0) is characteristic. Respiration of the lymphocytes, corresponding to Qo, = 8.0, suppresses glycolysis completely. The Pasteur effect in these cells is absolute. The results of MacKinney and Rundless (1956), who found aerobic glycolysis in the lymphocytes, could be explained by the use of a salt solution instead of serum in their experiments. According to our data, lymphocytes show significant aerobic glycolysis in Krebs-Ringer carbonate solution. It is very characteristic that the inverse Pasteur effect is also absent in the lymphocytes-glucose does not depress the absorption of 02. This coincidence is not a chance occurrence: we have observed consistently that the inverse Pasteur effect occurs only in those cells which have sufficiently high aerobic glycolysis. In contrast, glucose never depresses respiration in cells with a purely oxidative metabolism (Seitz, 1955, 1961). This type of cells also includes the lymphocytes. At the same time, lymphocytes have high anaerobic glycolysis: QE& = 27.2. The intensity of this process is so great that it can be compared with the glycolytic activity of malignant tumors in man and of the cells of patients with acute leukemia. The potentially high glycolysis in the lymphocytes of patients with chronic lymphadenosis does not, however, prevent the existence in these cells of a complete Pasteur effect, and it is characteristic that the ratio &“&2/Qo, = 0. b. Leucocytes of Patients with Acute Leukemia (Metabolic Type I I ) . As was noted above, in 40 out of 78 cases of acute leukemia examined by us, the leucocytes did not have aerobic glycolysis. In the presence of air they had a purely oxidative metabolism: &“&z = 0 (see Table I), while under anaerobic conditions glycolysis was intense: QZ& = 27.3. I n this respect

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315

the leucocytes of patients with acute leukemia of metabolic type I1 greatly resemble the lymphocytes in patients with chronic lymphadenosis. Moreover, like the lymphocytes, this group of cells did not have the inverse Pasteur effect-absorption of 0 2 in the presence and in the absence of glucose was practically the same: Qo, in the presence of glucose = 10.0; Qo, without glucose = 9.8. The similarity of the basic metabolic indexes in the leucocytes of patients with acute leukemia of metabolic type 11, on the one hand, and lymphocytes, on the other, indicates a familial relationship between these two categories of white blood cells and, on the other hand, a fundamental difference between these cells and the cells of the myeloid series which are characterized by high aerobic glycolysis and a distinct inverse Pasteur effect. We are inclined to believe that the metabolic type I1 of acute leukemia is represented mainly by lymphoblasts and sometimes also by plasma cells, which also have an oxidative type of metabolism. The difficulty here lies in the fact that different hematologists interpret the same term differently; in particular, cells which are regarded by some as hemocytoblasts are characterized by others as lymphoblasts; some differentiate such forms as paramyeloblasts and micromyeloblasts, while others do not accept such a classification; there is a complicated discussion taking place concerning the nature of leukemias represented in the blood by reticuloendothelial cells or monocytes (monoblasts), arid so on. In this situation, a classification of the blood cells based on their chemical and metabolic properties would have very great importance. However, chemical hematology is only in the first stages of development. Our data concerning the existence of two metabolic types in patients with acute leukemia, as well as our explanation of this phenomenon, are in accord with the interesting paper by Fieschi et al. (1956). These authors, having examined the leucocytes of 29 patients with acute leukemia immunologically, microenzymatically, arid also in tissue cultures, classified the predominant leucocytes of the blood into two groups: (1) cells similar to those found in chronic myeloid leukemia; ( 2 ) cells of the lymphatic type. True, Burk et al. (1957) reported the existence of aerobic glycolysis in the lymphoblasts (QF= 10 - 20; QF= 20 - 30) in 3 cases of acute lymphatic leukemia. However, the cells were not incubated in serum, but in saline, which could cause a disturbance of the Pasteur effect. I n a later paper (Burk et al., 1961, 1962), aerobic glycolysis was not observed in the 1ymphoblasts.

3. T y p e s of Leucocyte Jletabolism and Blood Morphology I n some cases of leukemia, morphological identification of the blood cells is not difficult, and the cells themselves have distinct biochemical characteristics. I n polycythemia, the cells of the myeloid series predominate

316

I. F. SEITZ

with a shift to the left, mainly due to an increase in band cells; in chronic myeloid leukemia, cells of the myeloid series also predominate; however, the degree of immaturity is even more marked. In both cases, despite the different degree of maturity, the cells have the same nature and origin, as well as a qualitatively similar metabolism (aerobic glycolysis and inverse I’asteur effect). As far as the lymphocytes are concerned, the picture is no less distinct, although the opposite, both with respect to origin and function and in the type of metabolism (complete Pasteur effect, absence of the inverse Pasteur effect). The picture becomes significantly more complicated in acute leukemia. It is well known that there are several forms of acute leukemia in which the morphology of the blood is extremely diverse. Even among the youngest, undifferentiated, cellular elements, there are several different types, and there is not always a consensus in evaluating whether a particular cell belongs to one or another genetic category. Identification of the cellular elements in acute leukemia thus presents great difficulty (Kassirsltii and Aleltseyev, 1962; Dameshek and Gunz, 1958; Wintrobe, 1947). As far as the blood cells of patients with acute leukemia are concerned, we can state, according to our data, that myeloblasts and reticuloendothelial cells belong to the cells of metaholic type I (with aerobic glycolysis); lymphoblasts and plasma cells apparently belong to metabolic type 11. In the latter case, the number of experiments is still small, since cases of acute leukemia with a predominance of plasma cells are very rare. If we take into consideration the data presented above, namely, that granulocytes have a type I metabolism and lymphocytes a type 11, then it is possible from a biochemical point of view to divide all white blood cells provisionally into a group with the metabolism of myeloid cells and a group with the metabolism of lymphatic cells. Granulocytes and the young cells of this series (myeloblasts and reticuloendothelial cells) belong to the first group; lymphocytes, lymphoblasts, and plasma cells belong to the second group. One would expect that the genetically related groups of cells would possess an inhcrited enzymologic and metabolic make-up, in accordance with the general principles of phylogenetic and ontogenetic development. Consequently, enzymochemical similarity indicates a common origin of the cells. In the present case, one could speak of the development of two groups of leucocytes from two different roots-the myeloid and lymphatic, which are fundamentally different metabolically and apparently also genetically. I t could be argued that the metabolism and chemistry of leucocytes in the blood stream is not an innate property of these cells, but the results of aging, damage, and other influences. Anticipating the above, in many cases of acute leukemia we have examined not only the leucocytes of the

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

317

peripheral blood but also the cells of the bone marrow in the same patients. This investigation, which will be discussed in detail later, and on which a preliminary communication has already been published (Luganova et al. , 1962), demonstrated that acute leukemia patients can be classified into the same two metabolic groups, according to the character of the bone marrow cells, and that there is a strong correspondence between the type of metabolism of the leucocytes in the peripheral blood and that of the bone marrow cells : metabolic type I in the peripheral leucocytes always corresponds to type I in the bone marrow; type I1 in the peripheral blood corresponds to type I1 in the bone marrow. These experiments permit the conclusion that two different, metabolic types of undifferentiated leucocytes, in one case having the characteristics of myeloid cells and in the other those of lymphatic cells, occur not only in the blood but also in the bone marrow, that is, in the principal maternal tissue for the blood cells. Therefore, the characteristics of the metabolism of blood leucocytes have a primary and not a secondarily acquired character. The postulated principle of the biochemical classification of the white blood cells is evidently dependent on the genetic relationship and familial origin of different groups of cells. This principle is so fundamental that it overcomes even factors due to age differences. Independently of their agc and maturity, the white cells of the blood and bone marrow belong to one or the other metabolic category, depending on their genetic nature and origin. On the basis of the data obtained, it can be postulated that reticuloendothelial cells and granulocytes (including also the young forms of this series) originate from a single stem with an enzymochemical structure which is characteristic for it only; lymphocytes (and also lymphoblasts) as well as plasma cells originate from the other stem. These conclusions are in accord with the known data concerning the embryogenesis of these two systems. Myeloid tissue, as is well known, develops earlier than the lymphatic system, independently of it, and from another source, namely, from the cells of the mesenchyme or from the endothelium of the blood vessels, while the lymphocytes develop from the endothelium of the lymphatic capillaries. These two categories also differ morphologically, functionally (phagocytosis by the granulocytes; elaboration of antibodies by the lymphocytes and plasma cells) with respect to their behavior in tissue culture, and finally chemically. It should be mentioned that Maksimov, in the later years of his life, recognized the development of myeloid tissue from the histioblastscells having nothing in common with the lymphocytes. Since this point of view, explaining the presence of aerobic glycolysis i n one group and its absence in another group by fundamental differences in the nature and origin of the myeloid and lymphatic tissues, was not

318

I. F. SEITZ

shared by all of our colleagues and particularly not by the hematologists and clinicians adhering to the unitary theory, no final conclusions were drawn a t this time and further investigations were carried out.

4. Therapy and T y p e s of Leucocyte Metabolism: E$ect of A C T H and Cortisone o n Leucocyte Metabolism in Vitro We have attempted to find the explanation for the differences in metabolism of the youngest forms of leucocytes in acute leukemia, not on the cellular level, but on the level of the whole organism. Since, in some cases, we were studying patients who had been treated with various drugs, including ACTH (adrenocorticotropic hormone) and cortisone, it was natural to suspect that the appearance of aerobic glycolysis in the young, undifferentiated blood cells in some of these patients was the result of the action of the drugs used. Therefore, we examined the effect of ACTH and cortisone on the metabolism of the leucocytes in vitro. Different forms of leucocytes were studied, including leucocytes from patients with acute leukemia of metabolic type 11, as well as lymphocytes which, as was noted before, are absolutely devoid of aerobic glycolysis, and on which, therefore, the action of the above preparations, if any, could be seen in a particularly distinct way. These experiments demonstrated that ACTH and cortisone produce a marked disturbance of the Pasteur effect in these cells: the leucocytes began to accumulate lactic acid in the presence of air. It should be mentioned, however, that this effect was obtained a t considerably higher concentrations of the hormones than are attained therapeutically. A distinct effect of ACTH and cortisone (induction of aerobic glycolysis) manifested itself a t hormone concentrations corresponding to 0.3 units and 90 pg./ml. of the experimental medium (Luganova and Seitz, 1960a,b; Seitz, 1961). It is true that an analogous effect was also produced by considerably lower hormone concentrations, but only on the condition either that the experiment lasted more than 3 hours (180 minutes and longer) or that the cells were first incubated for some time with these hormones a t 0°C. or a t room temperature. It is important to note that aerobic glycolysis in the leucocytes of patients with acute leukemia of metabolic type 11, as well as in the lymphocytes, appeared under the influence of the hormones against the background of normal, and sometimes even stimulated, respiration. The ability of ACTH and cortisone to induce aerobic glycolysis in cells usually not having it, interesting as this may be by itself, cannot explain the existence of aerobic glycolysis in the leucocytes of a large group of patients with acute leukemia (type 11),for the following reason: the therapeutic hormone concentrations were many times lower than those which were effective in the in vitro experiments, even in those cases in which the

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

319

cells were incubated ~ 7 iththe hormone for several hours, as a result of which the threshold for active concentrations was decreased. Of course, it could be imagined that a very long, practically constant, contact between the leucocytes and hormones, as occurs in the blood stream of patients with acute leukemia, could produce a disturbance and changes in the metabolism which do not have time to appear in the experiments of short duration in vitro. I n this case, it would be necessary to admit that all of the youngest, undifferentiated leucocytes of patients with acute leukemia have an oxidative type of metabolism, but that under the influence of ACTH and cortisone, which were used systematically and for a long time by the patients, these cells become damaged, as a result of which they develop aerobic glycolysis. 5 . “Anti-Pasteur” Factors in the Blood of Patients with Leukemia

This assumption, however, is not confirmed by the facts. The leucocytes in a series of patients with acute leukemia who were receiving cortisone and ACTH systematically showed a purely oxidative metabolism, while the leucocytes of another group of patients with acute leukemia who were not receiving treatment with these hormones showed distinct aerobic glycolysis. Apparently, the differences in the metabolism of the leucocytes of patients with acute leukemia (types I and 11) are not induced by secondary causes (therapy), but are due to the very nature of the cells or organism in these patients. With this concept in mind, it would seem natural to look for some other factors in the blood of patients with acute leukemia which could determine the character of the metabolism of the blood cells and act, for instance, like the “endogenous anti-Pasteur factor” that we observed earlier (Yel’tsina and Seitz, 1950; Seitz and Yel’tsina, 1951; Seitz, 1961). In this case, this factor entering the blood stream (or formed in the blood itself) could be absorbed by the cells and could produce its effect in the isolated leucocytes after absorption by the cells. An examination of the sera of leukemia patients for their content of similar factors, which would be capable of transforming the metabolism, showed that the blood serum in some of the patients with acute leukemia had unusual properties. Typically aerobic cells-lymphocytes and leucocytes of patients with acute leultemia of metabolic type II-changed their metabolism fundamentally and begin to accumulate lactic acid in the presence of air (up to 10 mg. of lactate and even more per ml. cells per hour; see Luganova and Seitz, 1960a,b; Seitz, 1961), which was not observed in the sera of healthy donors. Such an ability to induce aerobic glycolysis in cells with a purely oxidative metabolism was manifested by the blood sera of patients with acute leukemia of metabolic type I, that is, those

320

I. F. SEITZ

patients in which the leucocytes manifested aerobic glycolysis. It is interesting that the blood serum of patients with chronic lymphatic leukemia was also active in inducing aerobic glycolysis in both the leucocytes of patients with type I1 acute leukemia and the lymphocytes, which do not form lactic acid in the presence of air in normal serum. Apparently, while in the blood stream of patients with lymphadenosis, the lymphocytes, which are aerobic by nature, behave like cells with a mixed metabolism in which respiration and glycolysis coexist, despite normal conditions of aeration. The serum of patients with type I1 acute leukemia, like the serum of healthy donors, did not induce aerobic glycolysis in either the lymphocytes or the leucocytes of patients with type I1 acute leukemia. The blood serum of patients mit,h chronic myeloid leukemia had similar properties. Hence, only the sera of patients with acute leukemia of type I, as well as of patients with chronic lymphadenosis, contain substances inhibiting the Pasteur effect in aerobic cells. Sera of healthy persons, of patients with chronic myeloid leukemia, as well as of patients with type I1 acute leukemia, do not contain these active components. Thus, the Pasteur reaction happens to be a sensitive indicator of the presence of metabolically active substances in the blood serum in some patients with acute leukemia or chronic lymphatic leukemia. I t would appear that these results provide a simple and convincing explanation for the existence of two types of metabolism in the leucocytes of acute leukemia patients. Aerobic glycolysis in the leucocytes of metabolic type I could be regarded as the result of the action of special substances present in the blood serum in these patients. We do not feel, however, that these experiments provide a final answer t o the question of the causes of the metabolic differences among the leucocytes of patients with acute leukemia. Thus, for example, the leucocytes of patients with type I acute leukemia form lactic acid aerobically even in the serum of healthy donors, which does not contain any specific inhibitors of the Pasteur effect. This requires the assumption that the action of the factors present in the blood of these patients produces an irreversible disruption of the Pasteur effect, or simple damage, with the result that these cells continue to ferment sugar in the presence of air even under the optimal conditions of serum from normal subjects. This assumption is contradicted by the results of analogous investigations on the lymphocytes of patients with chronic lymphadenosis. As noted above, the lymphocytes of these patients show a distinct aerobic glycolysis in their own serum (i.e., in the serum of patients with chronic lymphatic leukemia), while in the serum of healthy donors they demonstrate a purely oxidative metabolism with a complete Pasteur effect. Consequently, another assumption is required, that the factors present in the blood of patients with type I acute leukemia, which inhibit the Pasteur

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

32 1

effect irreversibly, differ from the analogous factors present in the blood of patients with chronic lymphadenosis, which inhibit the Pasteur effect reversibly. The above considerations do not permit US to regard the “anti-Pasteur” factors observed by us in the blood of some leukemia patients as the sole cause of the existence of metabolic type I in patients with acute leukemia. It is quite possible to explain the existence of aerobic glycolysis in this group of leucocytes, not on the basis of the influence of some substances in the blood plasma (consequently, as secondary changes), but on the basis of recognizing that this characteristic metabolism is an inherent property of these cells. In any case, so far there are no direct data which would contradict such an explanation. As was pointed out above, it would be most natural arid logical, in our opinion, to correlate the two different types of metabolism in the leucocytes in acute leukemia with the two types of cells which are different in their nature and origin, namely, the myeloid and lymphatic cells, recognizing the existence of two corresponding types of acute leukemia, each of which in turn iiicludcs several related forms. This point of view mas confirmed by our experiments carried out jointly with Prof. D. Burl<, M. Woods, and B. Stambuk in the Sational Cancer Institute in Bethesda in 1960, which showed that the leucocytes in cases of acute lymphatic leukemia (80-90% lymphoblasts) had a purely oxidative metabolism (our metabolic type 11) while the leucocytes of patients with acute myeloid leukemia (goycor more myeloblasts) had a high aerobic glycolysis (metabolic type I ) . A final solution to all of these problems belongs in the future, and further work in this direction is in progress. One unquestionable and fundamentally important fact remaiiis, however, that there are some substances of a so far unknown chemical nature which are absent in the blood of healthy persons, but which significantly affect the metabolism of the blood cells and possibly also other cells in the human body. The latter deserves an independent special study. 6 . Leuliemia and Energy Metabolzsna in the Leucocytes

A study of the fundamental biochemical principles related to the biology and functional activity of the leucocytes may not only be of theoretical interest, but may also have important practical value. At the present time, no active and well-directed influence on the metabolism of the leucovytes or on the cells of the bone marrow is yet possible. However, knowledge of the specific characteristics of the various biochemical processes in normal leucocytes and in the leucocytes of pathological blood can already be utilized today in clinical practice diagnostically and prognostically, and it promises even greater perspectives in the future.

322

I. F. SEITZ

The point of view exists that the leucocytes in the blood of patients with leukemia are malignant cells. Therefore, it is of interest to compare the severity of the disease with the character of the metabolism in the corresponding leucocytes, and particularly the evolution of leucocyte metabolism from the normal to chronic myeloid leukemia and finally to acute leukemia. Warburg (1956) regards depression of respiration and an increase in glycolytic activity as an indication of malignancy, thus affirming that the higher the degree of malignancy, the more distinctly these characteristics are manifested. In the light of these concepts, the metabolic coefficients acquire a special interest. Let us see what changes occur in respiration in the series : normal leucocytes-leucocytes of patients with chronic myeloid leukemia-leucocytes of patients with acute leukemia. The Qol values of these cells under conditions which are as close to physiological as possible (in the presence of glucose and a t a temperature of 37°C.) are as follows: 5.7-5.W8.3 (type 1)-10 (type 11). These values in no way support Warburg's concept that there is a depression of respiration in cells during their malignant transformation. From the above data, it is evident that respiration does not decrease with increasing malignancy, but increases, and is almost twice as great, in the leucocytes of patients with type I1 acute leukemia as in normal leucocytes. The level of anaerobic glycolysis-Qz& in the same order becomes: 31.1-23.3-29.0-27.3. The level of aerobic glycolysis-&"&, in the same cells and in the same order is: 18.8-13.2-24.0-0. Thus, there is no increase in aerobic or anaerobic glycolysis in the leucocytes of leukemia patients as compared with normal; rather there is an opposite trend. Moreover, in the lymphocytes and leucocytes of patients with acute leukemia of metabolic type 11, aerobic glycolysis is completely absent. Consequently, with an increase in malignancy of the pathological process, the respiration of the cells increases and glycolysis not only does not increase, but even becomes somewhat depressed. The Qco,:&o~ratio is maximal in normal leucocytes and lower in leukemia; in cases of type I1 acute leukemia it equals zero. According to these data, if Warburg's concept is accepted, it would be necessary to consider normal leucocytes as the most malignant and the cells of patients with acute leukemia as the healthiest. This simple comparison of the metabolism of normal leucocytes and those from cases of leukcmia underlines the unreasonableness of attempts to seek a specific connection between leukemia and the quantitative changes in the relative levels of respiration and glycolysis, and suggests that it is necessary to look for some other basis for an evaluation of the pathological process. I n the light of these data, the outcome of the discussion of Warburg's concept, which had been taking place for many years in an up-and-down manner and reached its high point in the pages of Science (1956), was clearly not in favor of this concept and indicated, on the contrary, that the views of Weinhouse are more convincing.

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

B. CONTENTAND RESYNTHESIS OF ATP LEUKEMIC HUMAN LEUCOCYTES

IN

323

NORMALAND

Respiration and glycolysis are the most important processes which mobilize the energy of the nutrient substrates for sustaining life. However, the true and most immediate motive force behind the most diverse physiological functions is ATP. Strange as it may be, very few studies have been devoted to the study of the content and turnover rate of this high-energy phosphorus compound in leucocytes, particularly in human leucocytes (Luganova et al., 1957a,b,c; Seitz and Luganova, 1961a; Seitz, 1961; Chernyak, 1957, 1958). A detailed study of this aspect of the chemistry of leucocytes carried out in our laboratory permitted a direct comparison of the energy potentials of different types of leucocytes in healthy subjects and in leukemia patients. Table I1 gives the results of quantitative determinations of the content and turnover rate of the labile phosphorus of ATP in human leucocytes. This table demonstrates the absence of any sharp variations in labile phosphorus, whether calculated per milliliter of freshly centrifuged cells or even per gram of dry weight. There was a noticeable difference between the myeloid elements (healthy subject, polycythemia, chronic myeloid leukemia) and the lymphocytes, when the calculations were based on the number of cells. However, this can be explained by the large differences in the dimensions of granulocytes and lymphocytes. It should, therefore, be accepted that the content of ATP phosphorus, when calculated on the basis of a comparable mass of living leucocyte tissue, does not manifest marked variations, although some increase (about 15%) was noted i n leukemia. As far as the turnover of the labile phosphorus of ATP is concerned, some increase in the turnover rate in the lymphocytes and a significant stimulation of leucocyte metabolism in patients with acute leukemia can be pointed out. The data in Table I1 show that the relative specific radioactivity per 30 minutes’ incubation in the above cells exceeded the corresponding values for all other leucocytes incubated for 60 minutes. It is noteworthy that oxidative phosphorylation and respiration are fully adequate from the standpoint of resynthesis of ATP in the leucocytes of patients with acute leukemia. This is particularly evident in the leucocytes of patients with type I1 acute leukemia in which glycolysis under aerobic conditions is absent, but the content arid metabolism of ATP phosphorus is no less than in other c~lls.These facts contradict Warburg’s concept that the respiration of neoplastic cells is qualitatively not fully adequate, being uncoupled from phosphorylation. The most interesting fact, and apparently one of great biological significance, is that resynthesis of ATP in the leuco(bytes is about the same under aerobic and anaerobic conditions. This applies equally to all kinds of white blood cells, independent of the character

CONTENT

AND

TURNOVER RATE OF

TABLE I1 THE L.4BILE PHOSPHORUS O F

Content of labile phosphorus of ATP

ATP

+ AUP

(ILK.)

Aerobically

Leucocytes of

In 1 ml. cells

;Inaerobically

In 1 g dry wt.

In 1ml. cells

In 1 g. dry wt.

IN

HUM.INLEUCOCYTES~

Specific radioactivity of intracellular mineral phosphorus (counts/ Aerobically ahaerobically minutelpg.) Relative specific radioactivity of ATP

7

r

m

Healthy donors (30) Patients with polycythemia (20) Patientswithchronicmyeloidleukemia (35) Patients with chronic lymphadenosis (25) Patients with acute leukemia (20)

119 f 4 . 1 636 rt 22 126 f 10.5 6 i 3 f 56 135 k i . 9 634 f 40

119 rt 4 . 5 636 f 41 137 f 6 . 3 743 f 33.8 140 rt 8.3 667 42

63 f 5.4 66 rt 4.9 i t f 7.9

62 f 4.7 68 k 5 . 7 70 rt 4 . 6

220 260

118 f 6 . 4

738 f 40

125 f 7 . 6 780 f 47.5

83

k 5.4

83 f 8.2

240

106 f 8 . 6

740

79 f 5.7

81 f 6 . 3

1175

k 61 110 f 7.3 785 f 51

394

Cells (0.1 ml./ml. of experimental silspension) were incubated in blood serum at 37°C. for 60 minutes with Pa?and glucose (2 mg./ml.). Leucocytes of patients with acute leukemia were incubated 30 minutes. The number of experiments is given in parentheses.

2

BIOCHEMISTRY O F NORMAL A N D L E U K EM I C CELLS

325

of their metabolism as well as of whether they belong to the first or second metabolic group. I t is clear from Table I1 that both the quantity and the turnover rate of the high-energy phosphorus in the leucocytes are practically the same under aerobic and anaerobic conditions. Leucocytes are a very rare type of animal cells in that they are capable of sustaining a fully adequate energy metabolism in the absence of atmospheric 02.As a rule, in animal tissues (brain, kidney, liver, etc.) the ATP decomposes catastrophically on transition to anaerobiosis, due to their weak glycolytic activity. Cancer cells are the only exception (Yel’tsina and Seitz, 1951; Seitz, 1955, 1961). This curious property of the leucocytes is apparently the result of evolutioiiary adaptation to their specific function over a long time. Along the same lines, one should consider still another property of the leucocytes. Experiments have shown that the absence of glucose for 30-60 minutes does not cause a particularly sharp depression in the content and turnover rate of the labile phosphorus of ATP. Thus, after 60 minutes’ incubation of the leucocytes in the absence of glucose under aerobic conditions, the following quantities and turnover rates were found for the easily hydrolyzable phosphorus of ATP (the values obtained in parallel experiments with glucose are given in parentheses). Normal leucocytes: 107 pg./ml. cells (119 pg.) and relative specific radioactivity (RSR) = 50% (63y0); leucocytes of patients with chronic myeloid leukemia: 107 pg. (135 pg.) and RSIt = 80% (71%). In the lymphocytes the decrease in the content of ATP and relative specific radioactivity was more noticeable: 79 pg./ml. cells (118 pg.) and RSR = 58% (83%). This is apparently related to the absence of sufficient glycogen in the lymphocytes, hydrolysis of which, under anaerobic conditions, maintains the energy metabolism of the granulocytes a t a proper level. The glycogen content iii the granulocytes is considerably higher. The fully adequate resynthesis of ATP under anaerobic conditions and the comparatively satisfactory energy supply in the absence of glucose are properties of the leucocytes which indicate the adaptability of these cells to unfavorable conditions of existence, whether in the sense of the oxygen supply or the supply of glucose. I t is quite natural to try t o correlate these characteristics of the energy metabolism of leucocytes with their functional activities in the organism. Apparently, under unfavorable conditions in a focus of inflammation with consequent deterioration in the oxygen and glucose supply, the leucocytes can survive for a certain period of time without losing their important physiological function in the body. Since resynthesis of ATP in leucocytes is possible under aerobic as well as anaerobic conditions, in the presence of glucose as well as in the absence of this important nutrient in the medium, it is justifiable to assume that in any such cases, phagocytosis, ameboid motility, and the synthesis of antibodies

326

I. F. SEITZ

can also proceed unhindered. In any case, the lack of energy would not be a limiting factor in these processes provided that the duration of these unfavorable conditions is not too long. However, even this circumstance apparently does not play a decisive role in fulfilling the important defensive functions of the leucocytes in the organism, since there is a constant replenishment of leucocytes by new ones in a focus of inflammation. The fact that it is possible for these cells to function for some time under conditions of a deteriorating supply of oxygen and sugar is of some theoretical significance. The connection between phagocyt,osis and the more stable glycolytic processes which guarantee sufficient resynthesis of ATP is apparently of great biological importance. The supply of energy necessary for the maintenance of phagocytic activity does not cease in the early stages of inflammation, when the supply of oxygen is diminished, nor in the subsequent stages of this process. NIoreover, glycolysis creates unfavorable conditions for pathogenic microorganisms. According to Dubos (1957), lactic acid has a high degree of bactericidal activity, considerably greater than that of other organic acids.

C. EFFECTOF METABOLIC POISONS ON LEUCOCYTE METABOLISM In order to determine the extent of the participation of respiration and glycolysis, each independent of the other, in the resynthesis of ATP, we utilized certain glycolytic, respiratory, and so-called uncoupling poisons. Monobromoacetate and fluoride were used as substances blocking glycolysis; cyanide, as a respiratory poison; and 2,4-dinitrophenol, trypaflavin, and methylene blue, as uncoupling agents. 1. E$ect of Monobromoacetate

It is generally accepted that it is possible by means of monobromoacetate (or monoiodoacetate) to block glycolysis without affecting respiration. I t is true that monobromoacetate in a concentration of 8 X lo-" $1 completely stopped the formation of lactic acid under aerobic as well as anaerobic conditions in leucocytes with aerobic glycolysis. In the lymphocytes, which lack aerobic glycolysis, bromoacetate effectively stopped anaerobic glycolysis. As was noted previously, inhibition of aerobic glycolysis in the cells of the myeloid series leads to the disappearance of the inverse Pasteur effect; in the presence of a glycolytic poison, glucose no longer inhibits the absorption of 02.Respiration is increased to the level of the samples without glucose. It could therefore be expected that the maintenance of respiration would be conducive to the maintenance of a normal level of ATP in the cells. Actually, however, this was not observed. There was a catastrophic decomposition of ATP in these samples (Table 111). This effect of monobromoacetate was observed under either aerobic

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

327

or anaerobic conditions and in granulocytes as well as in lymphocytes. A necessary condition for the manifestation of this paradoxical effect of bromoacetate was the presence of glucose in the medium. In the absence of glucose, this effect was considerably weaker. Such an effect of monobromoacetate is difficult to understand. If decomposition of ATP under the influence of a glycolytic poison could justifiably be expected under anaerobic conditions, where glycolysis is the sole source of energy for regeneration of ATP, then under aerobic conditions, with a normal level of 0 2 absorption by the leucocytes, there is no logical explanation for the hydrolysis of ATP. In fact, the Qo2of normal leucocytes is 7.5, which corresponds, assuming a P : O ratio (effectiveness of phosphorylation) of 3, t o 45.0 equivalents of resynthesized ATP, while the maximal level of glycolysis in these cells, namely, Q& = 31.1, would be adequate for the resynthesis of 31.1 equivalents of ATP. The effect of bromoacetate on the resynthesis of ATP in cells not having aerobic glycolysis, for instance, in lymphocytes, appears even stranger. In dd monobromoacetate produces no noticeable changes this case, 8 X in respiration-the absorption of O2 remains practically the same as without the poison. Nevertheless, the ATP in these cells is rapidly destroyed (Table 111). I n addition to the destruction of ATP, there is another characteristic feature of the action of bromoacetate on the leucocytes, namely, a sharp decrease in inorganic phosphate in the cells and an increase in the acid-soluble fraction of the phospho-organic compounds (not counting ATP). As is well known, in most cases the content of ATP and inorganic phosphate in the cells vary in opposite directions. Under the influence of bromoacetate, the drops in the concentrations of ATP and intracellular inorganic phosphate proceed in parallel. Apparently, the poison blocks oxidative phosphorylation without impairment of respiration. At the same time, reesterification must also be increased since there is no increase in inorganic phosphate. The assumption that there is suppression of oxidative phosphorylation is favored by the very low relative specific radioactivity of the labile phosphorus of ATP in the presence of monobromoacetate (Table 111).An influence of the poison on cellular permeability similar to the effect described by Shacter (1957) is also not excluded. There are no essential differences in the reaction of different kinds of leucocytes with respect to monobromoacetate. 2 . Ej’ect of Fluoride

The effect of another inhibitor of glycolysis-fluoride-resembles the action of monobromoacetate in many respects. It is true that the stimulation of O2absorption under the influence of 2 x 10V M K a F is considerably more marked than that observed with bromoacetate. However, a rapid

TBBLE 111 EFFECT OF BROMOACETATE AND UNCOUPLING AGENTSON RESPIRATION. GLYCOLYSIS.AND

Leacorytes, additions. and conditions of incubation

Absorption of O2 (PI. 1

Formation of lactic acid (pa.)

15 tQ

M PHOSPHORYLATION IN H U M A N

Labile P of ATP

Intracellrilar mineral P ~

~

pg.

RSRb

125 154 49 30 160 150 127

58 48 20

LEUCOCYTES"

pg.

Specific radioactivity

Pa.oc (pg.)

Healthy donor Aerobic without glucose Aerobic glucose Aerobic glucose Brac.d Aerobic glucose S a F ' Aerobic glucose DNPf Aerobic glucose MBg Anaerobic glucose

+ + + + +

+

+ + + +

1158 1020 1170 1908 1150 1300 -

1.1 7.1 0

0 12.0 12.3 11.7

50 60 50

77 67 29 36 75 70 67

126 145 178 120 150 160 144

104 50 80 25 54 92 43

90 40 56 12 62 90 34

423 533 392 1145 409 305 508

93 95

102 70

296 312

44

-

160 300 270 -

Patient, with chronic myeloid leukemia Aerobic without glucose Aerobic Bracd Aerobic glucose glucose Bracd ilerobic Aerobic glucose TPFh Anaerobic glucose Anaerobic glucose Bra@

+ + + +

+

+

+ +

1260 1200 1104 1275 1020

+

0 0 8.7 0

12.2 12.1 0.8

63 29 112 21 103 112 10

Patient with chronic lymphadenosis Aerobic Aerobic

+ glucose

1715 1700

0 0

117 133

102 232 160 314 167 251

!+

r

+ + +

+ + +

Aerobic glucose DNP’ Aerobic glucose RIBg Aerobic glucose KCN* Anaerobic glucose

+

2030 2510 -

-

Figures are given/ml. fresh leurocytes; medium: serum = relative specific radioactivitp. c Pa.o. = acid-soluble organic fraction (without ATP). d Brac. = monobromoacetate, 8 X 1 0 F M . c NaF = sodium fluoride, 2 x 10-2 M . f D N P = dinitrophenol, 8 X M. RIB = methyleiie blue, 5 X lo-* M . TPF = trypaflavin, 5 X lop5 M . KCN = potassium cyanide, 4.3 X l O P M . a

b

RSR

9.0 8.9 8.3 8.6

122 120 121 114

98 99 84 100

85 92 116 95

+ Krebs-Ringer phosphate 1:1; incubation a t 37%.

270 295 172 136 for 60 minutes.

-

-

sm

Q

z

E e s

e

0

%I

3K

> r

5U

330

I. F. SEITZ

destruction of ATP and an increase in the fraction of acid soluble phosphoorganic compounds (not including ATP) also take place in the cells in this case. The turnover rate of the labile phosphorus of ATP and of the acid soluble phospho-organic compounds varies but little. 3. Cyanide and Uncoupling Agents

The effect of these inhibitors on leucocyte metabolism is in agreement with expectations. The ability of all kinds of leucocytes to resynthesize ATP quite well under anaerobic conditions explains the absence of any noticeable effect of cyanide, 2,4-dinitrophenol, trypaflavin, or methylene blue on the content and turnover rate of the phosphorus of ATP (Table 111). Indeed, cyanide (4.0-4.3 X M ) blocks respiration but stimulates glycolysis in the leucocytes to maximal levels, thus making possible a rapid resynthesis of ATP under anaerobic conditions; dinitrophenol (8 X M ) , in conM ) , methylene blue (5 X M ) and trypaflavin ( 5 X trast to ryanide, do not suppress respiration; moreover, the first two even stimulate it noticeably. However, by disrupting the Pasteur effect, they increase the glycolytic activity of the cells to the maximal levels in anacrobic samples, and the vigorous glycolysis induced in this way is fully adequate for the resynthesis of ATP and a normal turnover rate of the labile phosphorus of ATP (Table 111). As is well known, glycolytic phosphorylation is insensitive to the uncoupling poisons. Thus, these compounds create the conditions of anaerobiosis despite the high level of 0 2 absorption. As a rule, while disrupting the Pasteur effect in the leucocytes, these poisons also eliminate the inverse Pasteur reaction, so that absorption of 0 2 increases. The only difference in the action of uncoupling agents on granulocytes and lymphocytes is the fact that in the former, dinitrophenol, trypaflavin, and methylene blue increase aerobic glycolysis to the level of anaerobic glycolysis, while in the latter it is induced de novo. 4. Some Results of the Use of Metabolic Poisons in Experiments o n Leucocytes

As can be been from the above discussion, the use of the respiratory, glycolytic, and uncoupling agents has furnished additional information on the characteristics of the metabolism of leucocytes, although it has not led t o the solution of the stated problem, i.e., the determination of the relative contribution of respiration and glycolysis in the general balance of ATP. All the inhibitors used, while blocking one of the alternative processes completely, also affected the other. Blocking of respiration produced an increase in glycolysis and, vice versa, inhibition of glycolysis led to increased respiration. The automatic compensation and interaction of the energy-supplying processes in metabolism complicate the analysis of

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

33 1

the contribution of respiration and glycolysis to the general resynthesis of cellular ,4TP, but also demonstrate the amazing adaptability, flexibility, and dynamism of the regulation of the energy metabolism of leucocytes. Furthermore, the results obtained indicate a manifold effect of the metabolic poisons on leucocyte metabolism. Their effect cannot be explained by the blocking of one isolated process, but must be ascribed to a whole series of secondarily induced reactions, such as, for example, in the case of phosphorus metabolism in leucocytes under the influence of fluoride and monobromoacetate. D. CHARACTERISTICS O F THE METABOLISM O F INORGAR'IC PHOSPH.4TE LEUCOCYTES OF NORMAL AND PATHOLOGICAL BLOOD

IN

Experimental data testify unequivocally in favor of the conclusion that the rate of penetration of inorganic phosphate from the surrounding medium into the leucocytes increases sharply in acute leukemia and in exacerbations of chronic myeloid leukemia. This manifests itself in a considerable increase in the specific radioactivity of intracellular inorganic phosphate (and consequently the specific activity of the labile phosphorus of ATP) in the leucocytes of patients with acute leukemia in comparison with the specific radioactivity of inorganic phosphate in normal leucocytes or the leucocytes of patients with chronic lymphadenosis, or even in comparison with the leucocytes in the blood of patients with chronic myeloid leukemia, in which some intensification of the metabolism of inorganic phosphate also occurs. Under identical experimental conditions, the specific radioactivity of the intracellular inorganic phosphate of the leurocytes of patients with acute leukemia, after 30 minutes' incubation in the blood serum with P32 at 37"C., is 5-10 times as high as that for normal leucocytes, and the specific radioactivity of that of leucocytes in chronic myeloid leukemia is about twice as high as in normal blood. €3. NUCLEICACIDS, PHOSPHOLIPIDS, AND PHOSPHOPROTEINS I N HUMAN

LEUCOCYTES There is much more information in the literature concerning the acidinsoluble phosphates in the leucocytes than there is on ATP. The DNA (deoxyribonucleic acid) content in the leucocytes of the blood and in the cells of the bone marrow has been determined in many investigations under normal as well as pathological conditions (Davidson et al., 1951 ; Mandel and Metais, 1952; Menten, 1952; Menten and Willms, 1953; Rigas and Osgood, 1955; Morita and Asada, 1956; Terent'yeva et al., 1957, 1959). A majority of the authors found that the DNA content, as computed per cell, remains about the same in both normal and leukemia leucocytes (Lawrence, 1955; Polli and Semenza, 1955; Rigas et al., 1956; Polli, 1959;

332

I. F. SEITZ

and others). However, other investigators found that the amount of DNA in lymphocytes and granulocytes is different (Menten, 1952; Petrakis, 1953). Polli and Semenza reported a lack of uniformity in the properties and composition of DNA in normal and leukemic leucocytes) Polli and Semenza, 1955; Polli, 1959). As far as RNA (ribonucleic acid) is concerned, many investigators agree that its level in the white blood cells declines with an increase in the degree of maturity, and that the RNA content in leukemic leucocytes is greater than in normal leucocytes (Kedrovskii, 1951a,b; Thorell, 1947; Davidson et al., 1951; Will et al., 1957). Menten and Willms (1953) found that the quantitative ratio of RNA to DNA in the leucocytes of patients with leukemia varies from 0.3 to 0.85. Claude (1944) reported that the RNA in leukemic cells is localized in the mitochondria and that microsomes are absent from the supernatant fluid. A considerable number of investigations have been carried out for the purpose of studying the phospholipids in the leucocytes of normal and leukemic blood (Byrom and Kay, 1928; Boyd, 1936; Boyd and Murray, 1937; Morita and Asada, 1956). Boyd (1936), Lawrence (1955), and Rigas et al. (195G) observed a decline in the content of phospholipid phosphorus in the lymphocytes of patients with chroiiic lymphadenosis, as well as an increase in content related to cellular maturation. Increases in phospholipids in cases of granulocytic leukemia were observed by Bloom and Wislocki (1950), who interpreted this finding as being due to the accumulation of cytoplasmic granules which, as is well known, contain a large quantity of phospholipids. Claude (1944) found that 7 5 4 5 % of the lipids in the mitochondria of leukemic cells is accounted for by phospholipids. Very little work has been done on the phosphoproteins in leucocytes. Some data on phosphoproteins are given in the paper by Lawrence (1955). From these data, it can be concluded that the quantity of phosphoproteins in lcucocytes is decreased in leukemia. No essential differences were found in the content of phosphoproteins in the leucocytes of patients with chronic myeloid leukemia and chronic lymphatic leukemia. These results were confirmed by Rigas et al. (1956). It is of interest that the turnover rate of the phosphorus of the acid-insoluble compounds has hardly been investigated a t all. A study of the content and metabolism of the acid-insoluble phosphates has also been carried out in the laboratory of the author of this review (Luganova aiid Seitz, 195%; Luganova, 1958). The purpose of this study was to obtain information on those facets of leucocyte metabolism which are related to the growth and function of the cells. According to our data, the percentage content of phosphorus in different kinds of human leucocytes, when computed on the basis of dry cell weight, was: DNA, 0.42-1.29; RNA, 0.12-0.40; phospholipids, 0.26-0.29; phosphoproteins, 0.01-0.018 (Table IV).

CONTENT

AND

TURNOVER RATE OF

Leukocytes of Healthy donors Aerobically .knaerobically Patients with chronic myeloid leukemia Aerobically Anaerobically Patients with polycythemia Aerobically Anaerobically Patients with chronic lymphadenosis Aerobically Anaerobically Patients with acute leukemia her obically Anaerobically

THE

TABLE I V NUCLEICACID, PHOSPHOLIPID, A N D

RN.1 phosphorus Pg.

RSRb

1950 k 179 3 . 0 k 0 . 6 1780 f 193 3.8 f 0 . 6

4043 k 210 2 . 3 f 0 . 3 3930 f 100 2 . 4 f 0 . 2

3590 f 453 1 . 8 f 0 . 2 3150 f 420 2 . 1 f 0 . 4

PHOSPHOPROTEIN PHOSPHORUS I N

DNA phosphorus Mg.

RSRb

PLPD phosphorusc rg.

RSRb

4750 f 299 0.17 f 0.02 2570 f 230 0 . 8 f 0 . 3 4900 f 336 0.14 f 0.01 2360 f 189 1 . 2 k 0 . 2

HUMAN LEVCOCYTES~

PP p h o s p h o r d rg.

RSRb

111 f 30 106 f 24

10.1 f I . 9 11.5 f 1.3

12940 f 595 0.25 f 0 . 0 3 2943 f 318 1 . 0 f 0.06 143 rf: 24 1 3 . 5 f 1 . 7 12080 f 920 0 . 3 4 f 0 . 1 2875 k 319 1 . 4 k 0.07 131 f 17 1 5 . 8 f 1 . 9 6750 f 440 0 . 1 3 rf: 0 . 0 2 6580 f 600 0.11 f 0 . 0 2

-

-

167 f 45 14.0 f 2 6 187 f 25 11.0 f 1 . 5

0 Figures are given in pg. phosphorus/g. of dry cell weight. Incubation time: 60 minutes except in the case of acute leukemia, when i t was 30 minutes; temperature: 37°C. ; cells suspended in donor serum. RSR = relative specific radioact,ivity. c PLPI) = phospholipids. d PP = phosphoproteins.

334

I. F. SEITZ

The smallest difference among the different kinds of leucocytes is noticeable in the content of phospholipids. The phosphoprotein content in the cells increases sharply in acute leukemia and in chronic lymphadenosis. The content of RNA phosphorus varies greatly; it is twice as high in the lymphocytes as in the circulating leucocytes of patients with chronic niyeloid leukemia, and it is almost 3 times as high in the circulating leucocytes of healthy donors. The content of DNA phosphorus in normal leucocytes is somewhat lower than in the circulating leucocytes of patients with chronic myeloid leukemia. The content of DNA phosphorus in the lymphocytes is very high-3 times as high as in normal leucocytes when computed on a dry weight basis. This fact is understandable if one considers the relationship between the nuclear mass and the mass of the whole cell in the lymphocytes. A rather considerable increase in RNA phosphorus as well as some increase in DNA phosphorus is noted in the leucocytes of the group of acute leukemias in comparison with the leucocytes of healthy donors. A computation of the ratios between the content of RNA and DNA phosphorus in different kinds of leucocytes gave the following results: healthy donors, 0.28; polycythemia patients, 0.23; chronic myeloid leukemia patients, 0.41; acute leukemia patients, 0.53; chronic lymphadenosis patients, 0.31. I n other words, with the development of leukemia, there is a relative increase in cellular RNA. All compounds of the acid-insoluble phosphorus-containing fraction of leucocytes are in a state of continuous metabolism, which is evident from the results of a study of the turnover rate of their phosphorus. The highest turnover rate in this group of compounds is shown by the phosphoproteins, particularly in acute leukemia. The turnover rate of the phosphorus in RNA and phospholipids is considerably lower. It was pointed out above that the metabolism of all kinds of human leucocytes under anaerobic conditions is equivalent, from the standpoint of energetics, to the metabolism under the conditions of aerobiosis. I t is of great interest that this is also true for RNA, DNA, phospholipids, and phosphoproteins. The rates of metabolism and the content of all these compounds in the leucocytes are practically identical under aerobic and anaerobic conditions. Apparently, the glycolytic breakdown of sugar in the absence of atmospheric oxygen can be quite sufficient for the resynthesis of even the most complicated high-molecular-weight compounds present> in the white cells of the blood. Effective resynthesis of ATP, nucleic acids, phospholipids, and phosphoprotein in leucocytes under anaerobic conditions testifies to the high adaptability of these cells to a deficient supply of oxygen and explains why they are able to carry out their specific functions under conditions of oxygen deficiency and even in the complete absence of atmospheric oxygen.

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

335

F. RELATIONSHIP BETWEEN ENERGY METABOLISM AND ANABOLISM IN LEUCOCYTES There is a widespread opinion that synthesis and growth are dependent on aerobic glycolysis (LettrB, 1954; Holzer et al., 1955a,b). It is assumed, in this connection, that aerobic glycolysis furnishes some intermediate products of glucose catabolism which can serve as a good building material for iritracellular syntheses. Since many kinds of leucocytes have aerobic glycolysis, it seemed of interest t o study the role of this process as a source of building material for the synthesis of the basic chemical components of the cells. The use of uniformly labeled C14-glucosehas enabled us to trace the utilization of C14 fragments for the synthesis of proteins, nucleic acids, and other compounds. Along with C14-glucose,we have studied the metabolic transformations of the end product of the glycolytic catabolism of sugar, namely, L ( + ) - C ~ ~ lactate, also uniformly labeled. Leucocytes were incubated for various times at 37°C. in human blood serum, while we measured O2 absorption, formation of lactic acid, utilization of glucose, and the presence of C14 in the expired COe, proteins, and nucleic acids. These experiments demonstrated that the breakdown of C14-glucose in the course of leucocyte metabolism is accompanied by incorporation of C14fragments from the initial substrate into the expired carbon dioxide as well as into the proteins, riucleic acids, and glycogen. Incorporation of C1* into these compounds occurs under both aerobic and anaerobic conditions (Seitz, 1959, 1961). Table V shows data on the incorporation of C14 fragments from the metabolized C14-glucoseinto the proteins in different kinds of leucocytes. The intensity of this process was characterized by two indexes: (1) the specific radioactivity of the isolated proteins, and (2) the absolute level of incorporation of C14 fragments. Both indexes indicate a considerable increase in the assimilation of C14 fragments into the proteins in leukemias, particularly in acute leukemia and chronic myeloid leukemia. It can be seen from the table that, when the cells are incubated with C14-glucose under identical conditions, the specific radioactivity of the protein is more than 4 times as high in the leucocytes of patients with chronic myeloid leukemia and more than 3 times as high in patients with acute leukemia as the corresponding value for the leucocytes of healthy donors. The absolute iricorporation of C14 and the absolute mass of CI4 incorporated into protein are also considerably higher in leukemia. For the sake of comparison, the results of a study of incorporation of CI4-glycine into protein are also given. The results in this case were about the same as in the experiments on incorporation of fragments of C14-gIucose:a considerable increase

336

I. F. SEITZ

INCO RP O RiTI O N OF

TABLE V C’4-FR.lGMENTS FROM GLuCOSE-C14 I N D GLYCINE-1-C14INTO PROTEINS OF HIJMANLEUCOCYTEV ~

THE

~~~

Incorporation into proteins From gl~cose-C1~* Leucorytes from the blood

From glycine-l-C14~

Absolute Absolute Specific incorporat,ion Specific incorporation radioactivity (pg.) radioactivity (fig.)

of

Hcalthy donors

59 h 4 (12)

0.76

Patients with chronic myeloid leukemia

76.0 f 6 . 8 (20)

1.8

121 f 11 (10)

1.68

Patients with chronic lymphadenosis

20.0 f 1 . 9 (18)

0.4

168 f 10 (10)

2.0

Patients with acute leukemia

62.0 f 0 . 6

1.2

334 42 (10)

3.6

(12)

*

The numbers in parentheses correspond to the number of experiments. 0.1 ml. cells, incubated a t 37°C. in a mixture of 1 I n experiments with g1r1cose-C~~: part donor serum and 2 parts Krebs-Ringer phosphate; gliicose-C14 added = 2 mg. or 600,000 counts/minute. In experiments with g1y~ ine -C~ 0.1 ~ :ml. cells, incubated a t 37°C. for 60 minutes in Krebs-Ringer phosphate lo(%donor serum; gly~ine-1-C’~ added = 0.4 mg. or 200,000 counts/minute. a

+

in the incorporation of CI4-glycine in leukemias (Luganova and Seitz, 1961a,c; Seitz and Luganova, 1961a,b; Seitz, 1961). The total radioactivity iiicorporated into the nucleic acids as a result of the incubation of leucocytes with C14-glucoseis noticeably higher than that appearing in the protein. For instance, in the leucocytes of healthy donors, the percentage of CI4 fragments incorporated into the nucleic acids in relation to the total amount of glucose used was 0.33, as compared with 0.09 for proteins. Assimilation of CI4 fragments from metabolized glucose into the nucleic acids was several times higher in the leucocytes of pathological blood than in those of normal blood. Maximal incorporation (1.62% of the glucose used) was noted in the leucocytes of patients with acute leukemia. It was only a little lower, however, in the leucocytes of patients with chronic myeloid leukemia (1.38%) and chronic lymphadenosis (1.14%). An analogous increase in the incorporation of C14into the nucleic acids in cases of leukemia occurred when CL4-glycinewas used. Thus, the matter apensholds for proteins and nucleic acids : Incorporation is minimal

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

337

in normal leucocytes but increases sharply in cases of leukemia. Apparently, increased utilization of C14 fragments for anabolic purposes can be related in some way to the degree of maturity of the leucocytes. The youngest cells (acute leukemia, chronic myeloid leukemia, chronic lymphadenosis) are still developing and growing, rapidly increasing their cellular mass. The more mature cells (normal granulocytes) are closer to a stationary metabolism. The complexity of the problem of leukemia and cancer cells is that it is difficult to define where the phenomena related to normal cellular immaturity end and where the determining role of the specific neoplastic changes begins. We shall return to this question below. It also seemed of interest to determine the absolute magnitude of the assimilation of the products of glucose catabolism by the leucocytes and to evaluate the importance of this process in the anabolic metabolism of the cells, considering this question in relation to the specific role of aerobic glycolysis which has been postulated by some investigators. According to our calculations, healthy human leucocytes utilize less than 0.1% of the glucose used in the processes of respiration and glycolysis for the synthesis of proteins; this is increased 6.5 times in the leucocytes of patients with chronic myeloid leukemia, 4.5 times in the leucocytes of patients with chronic lymphatic leukemia, and 5.5 times in the leucotytes of patients with acute leukemia. If one considers the highest value of incorporation obtained in experiments with the leucocytes of patients with chronic myeloid leukemia, which was 0.26 pg./mg. protein/hour, even in this case the relative contribution of CI4 fragments from glucose to the resynthesis of cellular protein was very slight, amounting to only 0.26% of all the cellular protein synthesized per hour. In view of the short life span of leucocytes, the role of this process must be practically negligible. I t would be possible to conclude from this fact that the anabolic utilization of the products of glucose metabolism in leucocytes, including the products of aerobic glycolysis, does not play any significant role, a t least quantitatively. It was pointed out above that all kinds of leucocytes have a n amazing ability to accomplish the full resynthesis of ATP and other phospho-organic compounds under anaerobic conditions. The high adaptability of leucocytes to a deficiency of O2 was manifested distinctly in experiments on the assimilation of Cl4 fragments from glucose and CI4-glycine.As a rule, incorporation of CI4 into the proteins and nucleic acids of the leucocytes was only a little lower under anaerobic than under aerobic conditions, provided that the utilizable substrate and glucose were present in the medium in sufficient amounts. In our earlier experiments, lower values were observed for incorporation of C14 fragments from metabolized C14-glucose into the protein and nucleic acids of leucocytes under anaerobic conditions, due probably t o the fact that the amount of glucose in the medium was limited

338

I. F. SEITZ

in expectation of a rapid completion of glycolysis and the appearance of additional oxidative assimilation of accumulating lactate (Seitz, 1961). If glucose is present in the medium in excess, glycolysis in the leucocytes is almost twice as rapid under anaerobic conditions as under aerobic conditions, which guarantees an assimilation of CI4 fragments under anaerobic conditions comparable to that in aerobic samples, where glycolysis is smaller but where in addition there is considerable respiration. It is important to note that there is no parallel between the magnitude of glycolysis and the assimilation of the products of C14-glucosecatabolism in leucocytes. Cells with less glycolytic activity (aerobic and anaerobic), such as myeloblasts, hemocytoblasts of patients with acute leukemia, and lymphoblasts of patients with chronic lymphadenosis, assimilate C14 fragments more rapidly than leucocytes with higher glycolytic activity (normal granulocytes). Our data showing a sharp increase in assimilation of CI4-glycine into the proteins of the leucocytes in cases of leukemia are in agreement with the results of Wirizler (1958), who also showed an increase in incorporation of C14-glycine into leucocytes in leukemias. Winder and his co-workers (Winder, 1958; Wirizler et al., 1957) found that there was considerably more incorporation of uracil-2-C14, adenine-8-C14, and formate-C14 into nucleic acids, and of formate-C14 into protein in leukemic leucocytes. As a rule, the rate of incorporation of the precursors of proteins and nucleic acids increased in the order: normal leucocytes, chronic lymphatic leukemia, chronic myeloid leukemia, and acute leukemia. Incorporation of C14formate into the thymine of DNA was considerably greater in the leucocytes in chronic myeloid leukemia than in the leucocytes of healthy donors or patients with chronic lymphadenosis (Wells and Winder, 1959). Investigations of the incorporation of C14 into the proteins and nucleic acids of the leucocytes a t the expense of the oxidation of ~(+)-lactate-C'~ show that oxidation of the end product of glycolysis can serve as a source of building material for the synthesis of these cellular compounds; however, the magnitude of this process is not large. Roughly speaking, approximately 1/4 to 1/2 of the total incorporation of C14 into leucocytes under aerobic conditions in the presence of glucose-C14is accounted for by utilization of the products of oxidation of newly formed lactate (Seitz, 1958).

G. SUBSTRATE OF RESPIRATION IN LEUCOCYTES I n experiments with C14-glucose,our attention was drawn to the fact that the amount of C1402 formed in leucocytes as a result of respiration was considerably smaller than would be expected on the basis of the amount of oxygen absorbed, assuming that respiration in white blood cells takes place a t the expense of glucose. Moreover, we repeatedly observed that

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

339

various types of leucocytes, incubated without glucose, can maintain respiration for some time without any noticeable decrease in 0 2 absorption. Apparently, leucocytes do not need a supply of glucose for respiration and oxidize some of their own endogenous substrates. Moreover, as was mentioned above, glucose even depresses respiration in leucocytes having aerobic glycolysis. All these facts can be interpreted only in the sense that glucose as such is a substrate for glycolysis in the leucocytes but not for respiration. Such a conclusion sounds somewhat paradoxical, since glucose is a necessary and normal physiological ingredient of the fluids and tissues of the animal organism and is one of the basic nutrient materials. Nevertheless, this is precisely the case and experiments with uniformly labeled C14-glucoseconfirm it. A simple calculation shows that during oxidation of glucose to C02 and water, 22.4 pl. of O2 should be absorbed for 30 pg. of sugar utilized in this way. If the amount and radioactivity of the C14glucose used in the sample are known, it is easy to calculate the amount of oxidized glucose from the quantity of CI4O2evolved. Comparison of the amount of glucose actually oxidized with the theoretical value computed on the basis of the amount of oxygen absorbed in the experiment (i.e., 22.4 p1. 0 2 = 30 pg. glucose) characterizes the degree of utilization of glucose in respiration. The greater the disparity between these two valuesthe theoretical and actual oxidation of glucose-the smaller the extent of participation of glucose in respiration and the greater the participation of substances other than glucose. Experiments have thus demonstrated that oxidation of glucose can explain only a small fraction of the amount of 0 2 absorbed by the cells. The percentage of respiration at the expense of glucose varies from 8.0 to 16.7y0 in healthy donors; from 3.9 to 12.470 in patients with chronic myeloid leukemia; from 3.5 to 12.6y0 in chronic lymphadenosis; and from 2.7 to 15.4oj, in acute leukemia (Luganova et al., 1959; Luganova and Seitz, 1959a,b,c; Luganova arid Seitz, 1960a,b; Seitz, 1961). The amount of glucose that is oxidized directly (by other than the Embden-Meyerhof pathway) is even less, since in the presence of monobromoacetate, which blocks the preliminary glycolytic breakdown of glucose, incorporation of CL4decreases even more. It is possible to conclude that glucose in the leucocytes is, essentially, a substrate for glycolysis but not for respiration. In any case, this is true for experiments of relatively short duration (30-60 minutes). The leucocytes also oxidize lactate. However, incorporation of C14 into COz via this reaction is very small (up to 5y0).Apparently, neither glucose nor lactate plays any significant part in the respiration of human leucocytes. At least 80% of the oxygen absorbed by the leucocytes is related to the oxidation of some endogenous and apparently noncarbohydrate substrates. There are indications in the literature that the respiratory coefficient in the

340

I. F. SEITZ

leucocytes is less than 1 (Seelich, 1957 and others) and that the respiratory substrate in the leucocytes of horses may be glycerophosphate (Wagner et al., 1956); a t the present time, however, the evidence is not sufficient t o identify the substances which determine the course of endogenous respiration in human leucocytes. The possibility that leucocytes utilize lactate was demonstrated by Beck (1958a). He also studied the oxidation of glucose in these cells, by the phosphogluconate pathway and established that the proportion via the phosphogluconate pathway is greater in leukemic leucocytes than in normal cells. However, the absolute significance of this pathway of glucose oxidation is small in all kinds of leucocytes-not more than 10%. Coxon and Robinson (1956) ascribe considerably more significance to the hexosemonophosphate shunt. According to Beck, regulation of this alternative pathway in the cells depends first of all on the concentration of glucose-6-phosphate and hexokinase, and secondarily on the concentration of triphosphopyridine nucleotide. Beck ascribes the greater participation of the phosphogluconate pathway in leukemic leucocytes to an insufficiency of hexoltinase.

H. GLYCOGEN METABOLISM IN LEUCOCYTES OF NORMAL PERSONS AND PATIENTS WITH LEUKEMIA AND POLYCYTHEMIA

No analysis of the energy metabolism of the leucocytes would be complete without a n examination of the problems related to the content and metabolism of glycogen in these cells. The importance of doing this is further emphasized by the existence of numerous data indicating the dependence of phagocytosis on glycogen (Wagner, 1946; Wislocki et al., 1949; Wachstein, 1949; Sherstneva, 1958; Pavlov, 1960; Puchltov, 1955; Cohn and Morse, 1960; Bazin and Avice, 1953; Fisher and Ginsburg, 1956; Karnovsky, 1962), as well as by the variations in glycogen content in various diseases, particularly those of the blood. 1. Content and Turnover Rate of Glycogen in H u m a n Leucocytes

I n 1939, A. M. Genltin, on the basis of observations on the glycogen in the blood of children with pneumonia, suggested that the glycogen of the blood is found only in the leucocytes. This hypothesis was basically confirmed by the results of subsequent investigations carried out by histochemical and chemical methods. Wagner (1946, 1947) found that the erythrocytes and thrombocytes do not contain glycogen and that the reducing substance found in the lymphocytes is not glycogen. According to Wagner, all the glycogen of the blood is concentrated in the granulocytes. However, Wachstein (1949) as well as Gibb and Stowell (1949) demonstrated the presence of glycogen in the thrombocytes by histochemical methods. It was shown that the distribution of glycogen in the granulocytes

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

341

is not homogeneous: eosinophils and basophils contain no glycogen a t all, and the glycogen content of the neutrophils increases as they mature. According to many investigators, there is just as much glycogen in the leucocytes of healthy donors as in striated muscles and its content is relatively stable, which is indirect evidence of a constant regeneration of glycogen (Sieracki, 1955; Valentine et al., 1955). Valentine (1951), Valentine and Beck (1951) and Wagner (1947) studied the glycogen content in leucocytes by chemical methods. They found that in the leucocytes of healthy donors the glycogen content represents 1/20-1/40 of the dry weight of the cells. The glycogen content in the white blood cells changes in certain pathological states. Thus, excessive accumulation of glycogen in the leucocytes has been noted in infectious leucocytosis (Valentine, 1951) , glycogen disease, and polycythemia (Wagner, 1946; Valentine, 1951 ; Bridge and Holt, 1945). Astaldi and Verga (1957) observed that glycogen appears in the lymphocytes. However, Valentine (1960) did not detect glycogen in the lymphocytes in this group of patients, even in those cases when the suspension contained 100% lymphocytes. I n contrast to infectious leucocytoses, polycythemia, and glycogen disease, the glycogen content decreases in chronic myeloid leukemia, and this polysaccharide is practically absent in acute leukemias (Valentine, 1951, 1960; Valentine et al., 1953; Valentine, 195513). These data indicate strongly that the content of glycogen in leucocytes varies considerably in various pathological states. There have been very few investigations devoted to the processes of synthesis and breakdown of glycogen in leucocytes. More than 20 years ago, Willstatter and Rohdewald (1937) established that the breakdown of glycogen in horse leucocytes proceeds via the phosphorolytic pathway. In analogous investigations on human leucocytes, Wagner and Yourke (1952) established the existence of the phosphorolytic pathway of glycogen catabolism in these cells as well. In addition, these authors identified the intermediates formed in the process of glycogenolysis. Rohdewald (1946, 1952) demonstrated the possibility of reproducing glycogen synthesis in vitro by the phosphorylase pathway when the cells were poisoned by toluene. Similar results were obtained by Wagner and Yourke (1952). There are no data in the literature on the intensity of glycogen metabolism in leucocytes and the rates of its breakdown and synthesis. The question of the transformations of this polysaccharide under anaerobic conditions, which is essential for an understanding of the biology and function of the leucocytes, has also not been investigated. The lack of such data makes it difficult to understand the chemical basis of the functional activity of the leucocytes and the nature of glycogen metabolism in leukemia.

342

I. F. SEITZ

Over the course of several years, we collaborated with I. S. Luganova in a study on the content, turnover rate, and mechanism of synthesis and breakdown of glycogen in the leucocytes of healthy persons and patients with various forms of leukemia and polycythemia. The cells were separated by the gelatin citrate method and incubated at 37°C. in a 1: 1 mixture of the blood serum of healthy donors and Krebs-Ringer phosphate (pH 7.4) under aerobic and anaerobic conditions. There was usually 0.1 ml. cells in 2.8 ml. of the experimental suspension. As anticoagulants, heparin was used in the early experiments, and later EDTA (ethylenediaminetetraacetic acid). After hydrolysis of the cells for 30 minutes in 30% KOH at 100°C., the glycogen was precipitated with ethanol (60%)) and purified by reprecipitation from aqueous solution and washing with 60% ethanol. The purified glycogen was dissolved in hot distilled water, and in this solution the quantity (from the glucose) and radioactivity were determined. Table VI shows the results of determinations of the glycogen content in leucocytes from healthy donors and patients with leukemia and pol ycythemia. TABLE VI GLYCOGEN CONTENT IN LEUCOCYTES OF HEALTHY SUBJECTS AND PATIENTS WITH LEUKEMIA AND POLYCYTHEMIA Glycogen (mg.) in Leucocytes of

Leucocytes (1010)

Healthy donors 61.0 f 5 . 5 Patients with polycythemia 96.1 f 4 . 5 Patients with chronic myeloid leukemia 45.4 2.6 Patients with chronic lymphadenosis 6.4 f 0 . 7 5.8 k 0.68 Patients with acute leukemia

Cells (1010) of myeloid series

Dry weight (1 9.)

73.0 f 6 . 5 108.0 f 5.1 49.9 2.9 -

35 60 23 8 5

*

The glycogen content of the leucocytes is given in mg. per 1O1O cells, including all the white cells in the suspension, and also in mg. per 1O'O cells of the myeloid series, since it is known that glycogen is present mainly in the latter. Therefore, a differential count was performed in the suspensions of the separated leucocytes. As was mentioned above, during the separation of the leucocytes from the blood, the ratio between the cells of the myeloid and lymphoid series changes, i.e., the suspension obtained is depleted of lymphocytes. This fact should be kept in mind while evaluating Table VI. It should be noted, however, th at the above is valid only for the leucocytes of normal blood. As far as the leucocytes isolated from the blood of patients with chronic myeloid leukemia and polycythemia is concerned, the ratio

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

343

between the granulocytes and lymphocytes hardly changes a t all, since the percentage of lymphocytes in these patients is very small to begin with. From Table VI it can be seen that 1O1O myeloid cells from healthy donors contain 73.0 mg. of glycogen (3.5% of the dry weight). In chronic myeloid leukemia the amount of glycogen decreases to 49.9 mg. (2.3%). On the other hand, in polycythemia a considerable increase is observed in the content of this polysaccharide-to 108.4 mg. ((3% of the dry weight). The increased glycogen content of leucocytes from patients with polycythemia, compared with normal leucocytes, cannot be explained only by the changes in the differential count, i.e., by the predominance of younger forms of granulocytes, since it is ltnown that young cells of the myeloid series contain less glycogen than mature cells. It might be supposed that the excess accumulation of glycogen in leucocytes from polycythemia patients results from impairment of the enzyme systems catalyzing glycogen breakdown, just as in the muscles in various diseases (Schmid and Mahler, 1959; Schmid et al., 1959; Illingworth et al., 1956). The experiments showed, however, that the rate of glycogenolysis in leucocytes from polycythemia patients was higher (instead of lower) than in normal leucocytes. This was found in experiments in which the cells were incuhated in the absence of glucose and in which the increment in lactic acid and decrease in glycogen were determined every 15 minutes. After 2 hours’ incubation there was a decrease in glycogen content amounting to about 70% (average) of the initial value in the leucocytes of healthy donors, about 60% in the leucocytes of patients with chronic myeloid leukemia, and about 50% in the leucocytes of polycythemia patients. It should be noted, however, that the absolute value of the decrease in glycogen content in the leucocytes of healthy donors and polycythemia patients was about the same-about 500 pg., since the glycogen content in these cells is different. Only about 400 pg. of glycogen disappeared in leucocytes from chronic myeloid leukemia patients under the same conditions of incubation. Incidentally, it is of interest to note that the rate of glycogen breakdown depends, in all types of human leucocytes, on the degree of aeration. Under anaerobic conditions, 20y0 more glycogen was metabolized than in the presence of air. This indicates that the Pasteur effect takes place in the leucocytes not only during glycolysis but also during glycogenolysis. It follows from the above that the increased glycogen content of the leucocytes from polycythemia patients, in comparison with normal leucocytes, is apparently not associated with disruption of glycogenolysis in these cells. Its cause must rather be sought in the assimilation phase, i.e., in the mechanism of polysaccharide resynthesis. It should be noted particularly that the glycogen content of lymphocytes and undifferentiated leucocytes from acute leukemia patients was

344

I. F. SEITZ

very low-0.8 and 0.5%, respectively. The almost complete absence of glycogen in the leucocytes from acute leukemia patients is remarkable in that it reflects the well-known general tendency to a diminution or even complete disappearance of glycogen in rapidly growing neoplastic cells. Resynthesis of glycogen in leucocytes was studied on cells which were first incubated in a glucose-free medium. Determinations of the glycogen content before and after incubation without glucose for 2 hours a t 37°C. showed that the glycogen content decreased sharply during incubation. After glycogen depletion the leucocytes were washed in the centrifuge with physiological saline and then incubated again, but in the presence of glucose (5 mg./2.8 ml. of experimental suspension). The glycogen content was determined during incubation at various time intervals. These experiments showed that incubation of leucocytes was depleted glycogen in a medium containing a sufficient amount of glucose results in a rapid restitution of the glycogen content to the initial level. It is of interest that the rate of resynthesis of glycogen in leucocytes from chronic myeloid leukemia patients was considerably higher than in leucocytes from healthy donors or from polycythemia patients. Thus, the glycogen content in leucocytes from chronic myeloid leukemia patients reached the initial level after 15-30 minutes’ incubation with glucose, while in the leucocytes from healthy donors and polycythemia patients, resynthesis proceeded slower and was complete only after 45-60 minutes. Thus, the increased glycogen content in polycythemic leucocytes cannot be explained by their ability to resynthesize glycogen a t a higher rate. Moreover, the greatest activity with respect to resynthesis of glycogen was shown by the leucocytes of patients with chronic myeloid leukemia, in which the glycogen content was noticeably below the normal, and was only about half that found in the leucocytes of polycythemia patients. I n these circumstances, it was reasonable to expect that additional data which might reveal the cause of the differences in the glycogen content of leucocytes under various pathological conditions could be obtained by determining the rate of glycogen turnover during incubation with C14glucose. Table VII summarizes the results of experiments in which, along with determinations of the glycogen content, the rates of glycogen metabolism in various kinds of leucocytes were studied. The data in Table VII indicate that the glycogen in the leucocytes is regenerated very rapidly, the rate of this process being considerably greater in leukemic blood cells than in normal cells. The lowest rate of glycogen metabolism was observed in the leucocytes of healthy donors (specific radioactivity under the selected experimental conditions equals 1098 counts/mg./minute). In the leucocytes of polycythemia patients, the rate of glycogen turnover, judging from the specific radioactivity, was 135

345

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

TABLE VII CONTENT A N D TURNOVER RATEOF GLYCOGEN IN LEUCOCYTES OF HEALTHY DONORS A N D PATIENTS WITH LEUKEMIA AND P O L Y C Y T H E M I A ~ ~ ~ Glycogen (mg./ml.)

Leucocytes of Healthy donors Patients with polycythemia Patients with chronic myeloid leukemia Patients with chronic lymphadenosis Patients with acute leukemia

____

-_

SRc of glycogen ~

Aerobically Anaerobically Aerobically Anaerobically 6 . 3 f 0.55 9.8 k 0 . 5 5.5 k 0 . 3

6.4 k 0.6 10.8 5 0 . 5 6 . 0 5 0.7

1.6 f 0.16

1.4 f 0.2

1359 f 241

2020

0.8 f 0 . 1

7234 f 831

6635 f 749

0.8

k 0.1

1098 5 168 1127 k 153 1646 185 1686 f 174 16128 k 852 15911 f 929

k 314

a Cells were incubated in a 1: 1mixture of blood serum and Icrebs-Ringer phosphate buffer, pH 7.4, for 40 minutes a t 37°C. in the presence of uniformly labeled glucose-Cl4 (1.55 mg. or 135,000 counts/minute/ml. of suspension) ; the anticoagulant was heparin. b Ten experiments. c SR = specific radioactivity (counts/minute/mg.).

times greater than normal; in the leucocytes of patients with chronic myeloid leukemia, it was 15 times greater. The specific radioactivity of the glycogen in the leucocytes of patients with chronic lymphadenosis was only a little higher than in normal leucocytes, which arouses a suspicion that part of the glycogen determined in the lymphocytes actually belonged to individual granulocytes from which the lymphocytes could not be separated completely, although in the suspensions of lymphocytes used in the above experiments the lymphocyte count was 96-10070. However, the high specific radioactivity of the glycogen from the leucocytes of acute leukemia patients testifies against the possibility of “contamination” of the suspensions by concomitant granulocytes. Evidently, the immature forms of leucocytes in acute leukemia contain small quantities of glycogen, or of some substance of a similar nature, which metabolizes at a high rate. It is interesting that the low content and high turnover rate of glycogen are characteristic for practically all cases of acute leukemia, not being independent of the predominant type of young cells. Thus, the figures obtained in an experiment in which reticuloendothelial cells predominated (87%) were very close to those obtained in another experiment in which the suspension contained 88% myeloblasts. The data presented in Table VII indicate that the conditions of aeration do not influence the content and metabolic activity of the glycogen in the leucocytes. Under conditions of anaerobiosis (in the presence of glucose in the medium), the glycogen content in each type of cell does not decrease and there is also no decrease in the turnover rate of the polysaccharide, in

346

I. F. SEITZ

comparison with the values found in cells under the conditions of a sufficient supply of atmospheric oxygen. It should be noted that the greater part of the glycogen of the leucocytes extracted with cold trichloroacetic acid. Three 10-minute extractions with a 5% solution of trichloroacetic acid a t 0°C. are sufficient to extract more than 90% of the glycogen from the cells. The remaining glycogen is bound rather firmly to the structural elements of the cells and even three subsequent extractions cannot remove it. Comparison of the specific radioactivity of individual fractions of the glycogen from cells previously incubated with C14-glucose permits some interesting conclusions. We found that the specific radioactivity of the glycogen as a whole was usually lower than the activity of the first three trichloroacetic acid extracts. On the other hand, glycogen that could not be extracted with trichloroacetic acid generally had a low specific radioactivity. This may indicate either structural and functional heterogeneity of the different glycogen fractions, or a different metabolic activity of individual parts of the glycogen micelle manifesting itself in a nonhomogeneous distribution of the radioactive label. The metabolic heterogeneity of the glycogen particles is also demonstrated by experiments on the fermentation of previously labeled C14-glycogenfrom leucocytes. If leucocytes are incubated with C14-glucose,then washed and suspended at 37°C. in a medium without sugar, determinations of the content and specific radioactivity of glycogen after short time intervals show a definite regularity in the loss of the label. The greatest loss of radioactivity occurs within the first few minutes, when the decrease in glycogen is very small. For instance, in one of the experiments, after 15 minutes’ incubation of leucocytes containing C14-glycogen,50% of the initial radioactivity and only 8% of the glycogen had disappeared; after 30 minutes’ incubation, the figures were 70 and 2y0,respectively; after 45 minutes, 87 and 24%; and after 60 minutes, 92 and 40%. Thus, glycogen particles are not homogeneous in their metabolism and the outer layers are considerably more labile than the deep layers. 2. E$ect of Insulin on Glycogen Metabolism in Leucocytes

Insulin plays an important role in regulating carbohydrate metabolism in various types of cells. We therefore studied the effect of insulin on the rate of glycogen metabolism in the leucocytes of healthy subjects and patients with leukemia and polycythemia under in vitro conditions. The experimental results are given in Table VIII. The data presented in Table VIII show that insulin (0.7 units/ml. of suspension) significantly increases the incorporation of C14-glucoseinto the glycogen of leucocytes under both aerobic and anaerobic conditions. Insulin increases the metabolic activity of glycogen, not only in the leucocytes

347

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

EFFECTO F

Leucocytes from the blood of

TABLE VIII GLYCOGEN MET.4BOLISM

IN

HUMAN LEUCOCYTESU

Conditions of Formation 0 2 of lactic incubation and absorption acid additions (PI.) (PP.1

Specific radioactivity of glycogen Amount of glycogen (counts/ (pg.) minute/mg.)

I N S U L I N ON

Healthy donors

Aerobirally Aerobically insulin Anaerobically Anaerobically insulin Patients with Aerobically polycythemia Aerobically insulin Patients with Aerobirally chronic myeloid Aerobically leukemia insulin Patients with Aerobically chronic lymph- Aerobically adenosis insulin

+

+

+ + +

840 1050

560 600

2520 5750

1120 1130

580 600

3650 6100

39 41

560 630

1300 1340

4560 7400

36 39

700 770

480 500

19200 25728

43 41

0 0

100 100

1420 1700

42 45

a The conditions of incubatiori were as in Table VII; 0.1 ml. cells in each vessel. Insulin added; 2 units/2.8 ml. suspension.

of normal donors, but also in the cells of pathological blood: the leucocytes of patients with polycythemia and chronic myeloid leukemia. The effect of insulin on glycogen metabolism in the lymphorytes of patients with chronic lymphatic leukemia was less pronounced. The carbohydrate metabolism of human leucocytes is thus sensitive to the action of insulin in vitro. We did not make a special study of the mechanism of the stimulatory effect of insulin. There are reasons to believe, however, that this effect is due to changes in cellular permeability and not to activation of the system responsible for synthesis. It will be shown below that insulin does not influence resynthesis of glycogen in the reconstructed, cell-free, enzyme system. 3. E$ect of Versene on Glycogen Metabolism in H u m a n Leucocytes The use of anticoagulants is indispensable in working with isolated leucocytes. We usually used a solution of heparin: 5 units/2.8 ml. of experimental suspension. While studying glycogen metabolism in leucocytes, we also tested the effect of other anticoagulants: sodium citrate (0.5%) and Versene (0.1yo).These experiments showed that the various anticoagulants do not differ in their effect on the glycogen content of the leucocytes. The

348

I. F. SEITZ

rate of metabolism of this polysaccharide (judging from its specific radioactivity) is about the same with the use of heparin or citrate. However, incorporation of the label from C14-glucose into glycogen was sharply increased in the presence of Versene. A particularly pronounced stimulation was observed in the leucocytes of healthy donors. The specific radioactivity of the glycogen from normal leucocytes, incubated with Versene, can be 3-5 times as high as the specific radioactivity in comparable experiments with heparin. The stimulation of glycogen metabolism in the leucocytes of patients with chronic myeloid leukemia is less pronounced; here, the specific radioactivity of the glycogen increases in the presence of Versene but is only 1.5-2.0 times as high as in the experiments in which heparin is used. 4. E$ect of Metabolic Poisons on Glycogen Metabolism in Leucocytes

Glycolysis plays a significant role in the energy supply of the leucocytes. The data cited above also indicate the energy-producing effectiveness of glycolysis in relation to the synthesis and metabolism of glycogen in the leucocytes. Furthermore, the rate of glycogen turnover in these cells is exactly the same under anaerobic conditions as under aerobic conditions. It appeared of interest, in this connection, to determine what the rate of glycogen resynthesis would be under conditions where glycolysis was prevented and only the oxidative mechanism for supplying energy to the cells was preserved. Two specific inhibitors of glycolysis, monobromoacetate (8 X lo-* M ) and sodium fluoride (3 X M ) were used to clarify this question. The experiments were performed on leucocytes with aerobic glycolysis (leucocytes from healthy donors, polycythemia patients, and chronic myeloid leukemia patients) in the presence of glucose. Fluoride and monobromoacetate in the concentrations indicated completely blocked glycolysis but did not impair respiration. As a matter of fact, a distinct stimulatory effect on oxygen consumption could be noted (elimination of the inverse Pasteur effect). Table IX shows the results of several typical experiments. It can be seen that under the influence of bromoacetate glycolysis is completely inhibited and respiration increases; a t the same time, however, the rate of glycogen turnover decreases, while the glycogen content either does not change or decreases insignificantly. We may assume that the disturbances in glycogen metabolism produced by bromoacetate are associated not only with inhibition of glycolysis but also with the harmful effect of this poison on phosphorus metabolism in the leucocytes, as mentioned above. The rapid breakdown of ATP and the almost complete disappearance of intracellular inorganic phosphorus in the leucocytes in the presence of bromoacetate evidently impair the primary esterification of glucose, and may cause a number of other metabolic

TABLE IX EFFECTOF MONOBROMOACETATE, FLUORIDE, BORATE,A N D PYROPHOSPHATE ON THE CONTENT AND TURNOVER RATEOF GLYCOGEN IN HUMAN LEUCOCPTES~ Glycogen Specific radioactivity consumption (counts/minute/ Coniposition of the medium (additions) (rl.) (PP.) Amount mg.1 Leucocytes of Healthy donors G1u~ose-C'~ 48 823 620 1234 M G1uc0se-C~~ Brac.,b 8 X 56 35 640 428 Glucose-C14 NaF, 3 X M 68 0 500 10 33 674 1100 2200 Patients with polycythemia G1u~ose-C'~ Glucose-Cl4 Brac.,b 8 X M 42 0 1100 610 Glucose-C14 NaF, 3 X M 52 0 860 228 Patients with chronic G1uco~e-C'~ 60 645 480 22000 myeloid leukemia G1~1cose-C~~ 8X M 68 0 380 6960 Glucose-C14 NaF, 3 X M 83 0 260 10 60 665 740 1353 Healthy donors G1u~ose-C~~ G 1 u ~ o s e - C ~ ~IC3B03,3 X 10P M 52 560 500 0 G1uc0se-C'~ NaF, 6 x A2 61 455 700 7 Glucose-Cl4 NaF, 3 X M 58 525 700 220 Patients with polycythemia G1uc0se-C~~ 56 560 725 990 Glucose-C14 L B O $ , 3 X lop2 M 52 490 400 0 G1uc0se-C~~ Na4PZ07, 1.1 X M 52 525 750 466 G1~icose-C~~NaF, 3 X M 57 455 700 7 62 700 580 8040 Patients with chronic Glucose-C14 myeloid leukemia G1uc0se-C~~ &B03, 3 x 211 55 810 520 178 G1uc0se-C~~ Na4P20,, 1.1 X M 60 630 540 3100 G11icose-C~~ NaF, 3 X M 64 560 480 248 a Cells (0.1 ml.) were incubated in a 1 : 1 mixture of Krebs-Ringer phosphate buffer, pH 7.4, and blood serum for 40 minutes a t 37°C. The anticoagulant was heparin. Uniformly labeled g 1 ~ c o s e - Cadded: ~ ~ 3.5 mg. with a total radioactivity of 300,000 counts/minute/2.8 ml. Brac. = monobromoacetate. 0 2

Lactic acid formation

+ + + +

0

?I

+ + + + +

+ + + + + +

+

c)

m

r r

u,

350

I. F. SEITZ

changes which hinder the normal resynthesis and breakdown of glycogen. Sodium fluoride caused even greater disturbances in the metabolism of glycogen in the leucocytes. These disturbances were manifested by a marked decrease in the glycogen content and, which is especially characteristic, complete inhibition of glycogen turnover. When fluoride was present practically no C14-glucosewas incorporated into the glycogen in the leucocytes. These changes in glycogen metabolism caused by sodium fluoride cannot be explained by inhibition of enolase. It is well known that the effect of this poison is complex and extends to a number of other enzymes, particularly phosphoglucomutase, myokinase, phosphorylase, phosphatase, and possibly a number of others. As was noted earlier, impairment of ATP formation may also play a role in fluoride inhibition of glycogen resynthesis in leucocytes. In view of the uncertainties regarding the effect of fluoride on glycogen resynthesis in leucocytes, additional experiments were carried out on this subject. These experiments showed that a t a fluoride concentration of 0.003-0.006 144, glycolysis and glycogenolysis in the leucocytes are practically unaffected (Table IX) ; nor is cellular respiration depressed by fluoride. It would seem that when the entire chain of enzymes of glycolysis and respiration is preserved, glycogen synthesis would also remain unchanged. Actually, however, this is not true. At low concentrations (3-6 X M ) fluoride has no effect at all on glycogenolysis, glycolysis, or respiration, but very effectively blocks glycogen resynthesis in leucocytes. These experiments with fluoride clearly indicate that the synthesis and breakdown of glycogen in leucocytes are accomplished by two different enzyme systems, and not by a single reversible phosphorylase reaction, as had been conventionally believed. A possible alternative to the phosphorylase mechanism of glycogen synthesis in leucocytes could be a pathway for the synthesis of the polysaccharide via uridine diphosphoglucose (UDPG) :

+ +

UTP glucose-1-phosphate UDPG UDPG glycogen(,, + UDP

+

+ pyrophosphate

(1) (2)

There is evidence in the literature that the second reaction in glycogen synthesis by the uridine diphosphoglucose mechanism is inhibited by borate (Leloir et al., 1959). On the other hand, it might be expected that the formation of UDPG in the reversible reaction ( l) , and consequently the synthesis of glycogen, would be inhibited by the excess of pyrophosphate in the medium. Experiments have shown that pyrophosphate in a concentration of 1.1 x M M and borate in a concentration of 3 x actually do disturb glycogen synthesis from C14-glucosein the leucocytes; this is evident from the decrease in specific radioactivity of the glycogen.

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

351

It is of interest that these inhibitors block only the synthesis of the polysaccharide, but not the breakdown, since in the absence of glucose the breakdown of glycogen in the leucocytes is not inhibited. Both inhibitors also fail to inhibit glycolysis significantly. Thus, the experiments with borate and pyrophosphate, as well as the fluoride experiments, indirectly indicate that glycogen synthesis in leucocytes is accomplished via the uridine diphosphoglucose pathway. 5 . Leukemia and the Glycogen Content in Leucocytes Summarizing the results of an examination of the differences in the glycogen content of normal and pathological leucocytes, we must admit that neither the investigation of the rate of glycogenolysis and resynthesis of the polysaccharide, nor the study of its turnover rate can answer the question as to what determines these differences. Indeed, considering the high level of the glycogen content in polycythemia, one could expect either a higher rate of resynthesis or a slower breakdown. However, this was not observed in the experiments. On the other hand, the lower glycogen content in the leucocytes of patients with chronic myeloid leukemia could be the result of either slower synthesis or accelerated breakdown. Actually, however, just the reverse is true: resynthesis of the polysaccharide in the leucocytes of these patients is accelerated while the breakdown is somewhat slower than normal. It is precisely these characteristics of glycogen metabolism in the cells of patients with chronic myeloid leukemia which are apparently responsible for the very high values of the specific radioactivity of glycogen obtained in experiments in which freshly isolated leucocytes are incubated with radioactive glucose. The leucocytes of patients with chronic myeloid leukemia are the only type of white blood cells in which the absolute content of glycogen is noticeably increased (by 10-20%) as a result of incubation with glucose. This absolute increase, a t the expense of the radioactive glucose of the surrounding medium, can explain the high specific radioactivity of the glycogen obtained in in vitro experiments when the leucocytes of patients with chronic myeloid leukemia are incubated with C14-glucose.In the other types of leucocytes the glycogen level was practically the same before and after incubation. I n the leucocytes of healthy donors, patients with polycythemia, and patients with lymphadenosis, the breakdown of glycogen is balanced by resynthesis, and the specific radioactivity reflects the true rate of polysaccharide turnover. In the leucocytes of patients with acute leukemia, which show a high specific radioactivity of glycogen incubation with C14-glucosein vitro, the amount of glycogen in the cells is so small that it is difficult to judge with any degree of certainty what the causes of this increased activity may be, especially

352

I. F. SEITZ

to what extent it may be the result of the absolute increase in glycogen in the course of incubation, similar to what takes place in the leucocytes of patients with chronic myeloid leukemia. Special attention should be paid to the fact that in any kind of experiment-be it incubation of freshly isolated cells, rich in glycogen, or of previously incubated leucocytes with a low polysaccharide content-in all cases resynthesis of glycogen at the expense of glucose from the medium proceeds only up t o a strictly determined level which is intrinsic and characteristic of each type of cell. There are various mechanisms that regulate the absolute levels of glycogen storage in the cells. It might be thought that the factor which limits the synthesis of glycogen in the leucocytes is the genetically determined number of primary granules of glycogen which contain the basic enzyme systems catalyzing its synthesis and breakdown. It is still possible that the increase and decrease in the amount of glycogen in the cells under different conditions and in different functional states are not due to changes in the number of polysaccharide granules, but to changes in their dimensions. This supposition naturally requires experimental proof. If it is true, then the differences in the glycogen content between normal leucocytes and the leucocytes of pathological blood are determined not so much by the activity or deficiency of the enzyme systems effecting breakdown and synthesis of glycogen, as by genetic factors, in the first place by the number of primary glycogen granules-the centers of biosynthesis of the polysaccharide. I n connection with the results obtained in determinations of the glycogen content in various types of leucocytes, particularly its significant decrease in the leucocytes of patients with chronic myeloid leukemia and very sharp decrease in acute leukemia, it is appropriate t o consider the significance of this fact in the light of the data on the low glycogen content in tumors (Greenstein, 1954; Weber, 1961; Weinhouse, 1962). The leucocytes in chronic myeloid and acute leukemia reflect a tendency, which they have in common with malignant neoplasms, to a decrease in the glycogen content. Since there are numerous indications that the basic defensive function of the leucocytes of the myeloid series-phagocytosisdepends on the energy of glycogen breakdown, a decrease in the content of this polysaccharide in the cells is naturally reflected in a decrease in their phagocytic activity; this is supported by the data in the literature. The increased glycogen content found in the leucocytes of patients with polycythemia speaks against the malignancy of these cells. As far as the low level of glycogen in the leucocytes of patients with chronic lymphadenosis is concerned, this fact does not prove malignant transformation of these cells, although it also certainly does not disprove it. Lymphocytes are cells which differ from the granulocytes in their function, metabolic charac-

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

353

teristics, and origin, and can therefore not be compared in this respect with the cells of the myeloid series. It should also be remembered that, according to cytochemical data, the glycogen content is also very low in normal lymphocytes. In evaluating the data on the determination of glycogen in leukemic leucocytes, one should keep in mind one very important fact: that in these diseases there is a marked rejuvenation of the leucocytes in the peripheral blood, with a strong shift to the left, and that a low glycogen content is characteristic of young cells. It is well known that in normal bone marrow, the appearance of significant amounts of glycogen occurs only after the myelocyte stage. In younger, Undifferentiated cells, arid in the cellular forms preceding the myelocytes, the polysaccharide content is very low. Consequently, the decrease in the glycogen content of the leucocytes in chronic myeloid and acute leukemia could be ascribed not only to a leukemic transformation, but also to an unspecific effect of the rejuvenation of the cells. At the present time, it is difficult to decide to what extent the low glycogen content, in leukemia is due to the specific malignant pathology, on the one hand, or simple cellular immaturity, on the other. It is important, however, to note that one of the essential moments in the development of the leukemic process is precisely the arrest of the differentiation of the leucocytes and the appearance of undifferentiated or insufficiently mature cellular forms in the blood and bone marrow. 6. T h e U D P G System in Glycogen Synthesis in Normal Leucocytes The facts cited above, which are not in accord with the principles of the phosphorylase mechanism of glycogen synthesis in leucocytes, prompted us to test another alternative-glycogen formation in the blood cells with the participation of UDPG. As is well known, the following reactions leading to the formation of glycogen are possible in the liver, muscle, brain, and some other tissues (Leloir and Cardini, 1957; Leloir and Goldemberg, 1960; Leloir et al., 1959; Robbiiis et al., 1959; Villar-Palasi and Larner, 1958; Breckenridge and Crawford, 1960; Stetten and Stetten, 1960; Strominger, 1960). Glucose

hexokinase + ATP __ glucose-&phosphate + ADP

Glucose-&phosphate Glucose-1-phosphate UDPG

(3)

--f

.

phosphoglucomu tase

' glricose-1-phosphate

+ UTP .

UDPG-pyrophosphorylase

UDPG

(4)

+ pyrophosphate

+ glycogen(,) UDPG-glycogen synthetase UDP + glycogen(,+l) --f

(5)

(6)

Our aim was to study all these reactions in the preparations of human leucocytes in order to discover evidence for the presence of the intermediate

354

I. F. SEITZ

products as well as of the enzymes participating in the process. The problem was, first of all, to establish the presence and then measure the activity of the following enzymes: phosphoglucomutase, UDPG-pyrophosphorylase, UDPG-glycogen transferase (synthetase). The presence of hexokinase in the leucocytes was shown earlier by other authors (Beck, 1958a). It was also necessary to prove the presence in the leucocytes of the coenzyme which plays the central part in the biosynthesis of glycogen-UDPG. In experiments with extracts of acetone powders and with leucocyte homogenates, we succeeded in demonstrating the presence in these cells of all enzymes concerned with the synthesis of glycogen via the uridine diphosphoglucose pathway. Along with hexokinase and glucose-6-phosphate dehydrogenase, which have been studied in detail by other authors, we have found active phosphoglucomutase, UDPG-pyrophosphorylase and UDPGglycogen transferase in the leucocytes. Determination of the activity of hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, and UDPG-pyrophosphorylase was carried out spectrophotometrically in 0.05 M Tris buffer (pH 7.4) a t room temperature on the basis of light absorption a t 340 mp due to reduction of T P N t o TPNH. Enzyme extracts were obtained from acetone powders of the leucocytes by 20 minutes’ extraction a t 0°C. with 0.1% NaHCOa in the ratio 1: 100. Acetone powders were prepared by treating the leucocytes with 20 volumes of acetone precooled to - 12°C. Leukocyte homogenates were prepared by quadruple freezing and thawing of the cells and subsequent homogenization in 0.05 M Tris buffer with Versene. a. Glucose-6-Phosphate Dehydrogenase. The very first experiments demonstrated that the leucocytes do contain a highly active glucose-6phosphate dehydrogenase in a concentration surpassing that of all other enzymes investigated, making addition of this enzyme to the system unnecessary. Under the conditions of our experiments, dehydrogenation of glucose-6-phosphate in the extracts of acetone powders of normal leucocytes proceeded a t a rate of 196 pmoles/g. protein of the preparation/minute. b. Hexokinase. The hexokinase activity was determined by the following series of reactions:

+ ATP hexokinase - glucose-6-phosphate + ADP glucose-&phosphate Glucose-6-phosphate + TPN 6-phosphogluconate + TPNH dehydrogenase

Glucose

--f

---f

(7 1 (8)

The hexokinase activity found in the homogenates of leucocytes from healthy donors was 27 pmoles of glucose phosphorylated/g. of protein/ minute. The corresponding figure obtained by Beck (1958a) was about 36 pmoles/g. protein/minute.

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

355

c. Phosphoglucomutase. The test system for the determination of phosphoglucomutase activity was as follows: glucose-l-phosphate S glucose-6-phosphate

(9)

followed by oxidation of glucose-Bphosphate to 6-phosphogluconate according to reaction (8). The phosphoglucomutase activity of extracts from acetone powders of leucocytes from healthy donors was 80 pmoles of glucose-l-phosphate transformed/g. of protein/minute. d. UDPG-Pyrophosphorglase. For the determination of UDPG-pyrophosphorylase, the same system was used as for the determination of phosphoglucomutase, since the product of UDPG-pyrophosphorylase activity is glucose-l-phosphate. In this case, however, glucose-l-phosphate was not added to the medium, but an excess of phosphoglucomutase was introduced. Thus, the first reaction catalyzed by UDPG-pyrophosphorylase: UDPG

+ pyrophosphate

UTP

+ glricose-l-phosphate

(10)

is followed by reactions (9) and (8). In the presence of excess phosphoglucomutase and glucose-&phosphate dehydrogenase, the rate of the process is determined, on the whole, by the activity of the UDPG-pyrophosphorylase present in the leucocyte preparations. It is well known that the UDPG-pyrophosphorylase reaction is reversible and can proceed in either direction, depending on the concentration of the reagents. Under our conditions, one of the reaction products (glucose-l-phosphate) was continuously removed from the reaction by being transformed into glucose-6phosphate and thereafter into 6-phosphogluconat e. Thanks to this, it was possible not only to determine the UDPGpyrophosphorylase activity, but also to “titrate” the UDPG content of the sample. We made use of this technique later for the quantitative determination of UDPG in extracts from chromatogram spots. We have tested the ability of enzyme preparations from leucocytes to catalyze the UDPG-pyrophosphorylase reaction in both directions : in the direction of the condensation of U T P with glucose-l-phosphate to form UDPG, and in the direction of the pyrophosphorolysis of UDPG into these two components. We found that leucocytes are rich in UDPG-pyrophosphorylase and that their extracts rapidly hydrolize UDPG, but that they synthesize it just as effectively if the conditions are changed. According to our data, the pyrophosphorolytic cleavage of UDPG to UT P and glucose1-phosphate in extracts of the acetone powders of normal leucocytes (0.05M Tris buffer a t 20°C.) takes place at a rate of 52 pmoles/g. of protein/ minute. The reverse reaction (the condensation of U T P and glucose-lphosphate to yield UDPG) was followed in a system containing, in addition

356

I. F. SEITZ

to the UDPG-pyrophosphorylase extracts, UDPG-dehydrogenase from calf liver (prepared by the method of Strominger, 1960) and DPN, which is required for UDPG-dehydrogenase activity. I n this system, the UDPG formed by the UDPG-pyrophosphorylase reaction is oxidized to UDPglucuronic acid at the expense of DPN, and the course of the reaction can be followed from the increase in absorption a t 340 mp resulting from the reduction of DPN to DPNH. UDPG

+ 2 DPN UDPG-dehydrogenase UDP-glucuronic acid + 2 DPNH -+

These experiments also showed that the leucocytes contain a n active UDPG-pyrophosphorylase. e. UDPG-Glycogen Transferase Activity in Normal Leucocytes. The finding that the leucocytes contain a highly active UDPG-pyrophosphorylase which catalyzes the formation and breakdown of UDPG (a coenzyme with a broad spectrum of metabolic activity) still did not suffice to prove the possibility of glycogen synthesis via UDPG, although such a synthetic pathway would be impossible without the participation of UDPG-pyrophosphorylase. The only direct proof of the possible synthesis of glycogen in the leucocytes by the uridine diphosphoglucose pathway would be the demonstration of the direct transfer of glucose from UDPG to glycogen. This was made possible by experiments with UDPG labeled with C14in the glucose moiety. The enzymatic transfer of a glucose residue from UDPG to a glycogen primer represents the final link in a chain of reactions beginning with the phosphorylation of glucose to glucose-6-phosphate and terminating in the formation of a polysaccharide. This transfer is catalyzed by the enzyme UDPG-glycogen glucosyl transferase, which was first discovered in the liver by Leloir and Cardini (1957). Our experiments with leucocytes have shown that there is an active UDPG-glycogen transferase, catalyzing the transfer of a glucose moiety from UDPG to glycogen, in both the extracts of acetone powders and the homogenates of these cells. On incubation of leucocyte extracts with UDPG-CI4 and a glycogen primer, the radioactive label was transferred from the hexose-containing nucleotide to the polysaccharide (for the preparation of UDPG-CI4 and the details of these experiments, see Seitz et al., 1963). This transfer was accompanied by the liberation of a n equivalent amount of UDP. In extracts from the leucocytes of normal subjects, the rate of transfer of a glucose moiety from UDPG to glycogen was 6.8 pmoleslg. of protein/minute. In extracts of acetone powders of leucocytes, as well as in freshly centrifuged homogenates, it is possible to synthesize glycogen not only from UDPG but also from its more remote precursor, glucose. T o make the UDPG-glycogen transferase of the leucocytes extracts the ratelimiting

357

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

enzyme in the system, excess hexokinase, phosphoglucomutase, and UDPGpyrophosphorylase were added to the sample, along with ATP, UTP, MgC12, cysteine, and glycogen. In this case, the rate of formation of glycogen-CI4 is determined solely by the leucocyte synthetase (UDPG-glycogen glucosyltransferase) artivity, while the UDFG is continuously regenerated and made available for the synthesis of more glycogen. These experiments showed that the full system catalyzes the rapid incorporation of g1uc0se-C~~ into glycogen, the reaction depending on the presence of UTP and UDPGpyrophosphorylase (see Table X). This is specific evidence for the uridine TABLE X UDPG-GLYCOGEN GLUCOSYL TRANSFERAGE ACTI VI TY I N THE EXTR.\CTS POWDERS OF H U M A N LEUCOCYTES~~~ Radioactivity of glycogen (counts/minute)

Amount of glucose-C14 transferred

+

670 9783 2688 1727

2.2 32.5 9.0 5.8

+

704 7665 2650 2118

3.3 25.6 8.8 7.1

Composition of the sample and conditions of incubation Complete system, 0°C. Complete system, 37°C. Complete system, 37°C. NaF (3 X 10P M ) UTP and UDPG-pyrophosphorylase excliided from the system Complete system, 0°C. Complete system, 37°C. Complete system, 37°C. NaF (3 X 10-2 M ) UTP excluded from the system 0

O F ACETONE

Experiments with glucose-C14.

* The complete system contained

the following: 25 pM Tris buffer, pH 7.4; 2.5 pM cysteine; 2.8 phl g1uc0se-C~~ (with a total radioactivity of 150,000 counts/minute) ; 1.5 p M ATP; 2.0 pM UTP; 250 pg. crystalline phosphoglucomutase; 250 pg. crystalline hexokinase; 0.07 ml. UDPG-pyrophosphorglase (120 pg. protein) ; 0.025 ml. inorganic pyrophosphatase (50 pg. protein); 0.5 mg. glycogen; 0.1 ml. of an acetone powder extract of leucocytes. The total volume was 0.6 ml. Incubation time: 60 minutes.

diphosphoglucose mechanism of polysaccharide synthesis. The omission of UTP from the medium, and still more the omission of U T P and UDPGpyrophosphorylase, led to a sharp decrease in the incorporation of glu~ 0 s e - C 'into ~ glycogen. When only UTP was omitted, the incorporation remained approximately 20% of maximal, which can be explained by the use of nondialyzed leucocyte extracts. There is no theoretical reason for discarding the possibility that the system may also synthesize glycogen in some other way not involving UDPG, for example, via the phosphorylase pathway. However, it should not be forgotten that our experiments were carried out, under conditions which are particularly favorable for the

358

I. F. SEITZ

synthesis of glycogen by the phosphorolytic pathway, since no inorganic phosphate was added and glucose-1-phosphate was continuously being regenerated by the hexokinase-phosphoglucomutase system, so that the inorganic phosphate/glucose-1-phosphate ratio was kept at artificially low levels such as would never be encountered in intact living cells. Despite this objection, as can be seen from Table X, the amount of glycogen-CI4 synthesis proceeding indepently of UTP and UDPG-pyrophosphorylase was slight. Consequently, the principal pathway for glycogen synthesis from g1uc0se-C'~in leucocyte preparations is as follows :

+

g1~1cose-C~~.4TP

hexokinase

+ ADP

C14-glucose-6-phosphate

phosphoglucomutase

C14-glucose-6-phosphate, C14-glucose-l-phosphate UDPG-C14

' C14-glucose-l-phosphate

+ UTP ,

+ glycogen(,,

UDPG-pyrophos-

UDPG-glycogen synthetase

-+ UDP

UDPG-C14

+ pyrophosphate

+ C14-glycogen(,+l)

(12) (13) (14) (15)

It is interesting to note that fluoride (0.03 M ) sharply inhibits incorporation of glucose-C14into glycogen in this system, just as in experiments with intact cells (see Section II,H,4). Yet, the UDPG-glycogen synthetase reaction, which transfers a glucose moiety from UDPG-C14 to glycogen, is not directly inhibited by fluoride. This indicates that the inhibition of glycogen synthesis by fluoride in intact leucocytes is due not to a direct specific depression of the polysaccharide-synthesizing system, but to a secondary effect of the inhibitor on either the phosphorylation of glucose to glucose-6-phosphate or the transformation of the latter to glucose-lphosphate. f. Detection of UDPG in the Leucocytes. The data presented above indicate the presence in the leucocytes of enzymatic activity connected with the synthesis of glycogen from glucose via UDPG. This analysis of the mechanism of glycogen synthesis in the leucocytes would be quite unsatisfactory, if the most important coenzyme for this process, UDPG, could not be detected in these cells. For this reason, we isolated a pure suspension of leucocytes, practically free of erythrocytes and thrombocytes, from human blood and determined the UDFG content. The procedure for the isolation of UDPG from leucocytes is as follows: the nucleotides were adsorbed from an HCl extract of the cells on activated charcoal and eluted with aqueous ethanol (50%) containing 0.05 M NH40H. The eluate was evaporated to a small volume at room temperature in a vacuum, and the concentrated solution was applied to a chromatogram (Whatman No. 1 or V F 11). The nucleotides were separated by descending chromatography in a solvent system consisting of alcohol and ammonium acetate (70:30),

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

359

pH 7.5 (technique of Paladini and Leloir, 1952). The spots were developed in an ul trachemiscope, and those corresponding in position to the indicator UDPG (R, on Whatman No. 1 paper = 0.23) were cut out and extracted with water. In the extracts, the adsorption a t 260 and 280 mp was determined and then specific enzymatic tests were carried out with UDPGpyrophosphorylase and UDPG-dehydrogenase. The ratio of the adsorption at 280 mp to that at 260 mp was 0.35-0.40. I n the enzymatic tests with UDPG-pyrophosphorylase in the presence of MgC12, cystcine, and TPN, in 0.05 M Tris buffer (pH 7.4), the extracts of the chromatogram spots produced an increase in adsorption at 340 mp which depended specifically on the pyrophosphate and was proportional to the UDPG content. For 1 pmole of UDPG, AE340= 1.33. Analysis of the UDPG spots with UDPGdehydrogeiiase and DPN also produced an increase in adsorption a t 340 mp due to the formation of DPKH. The appearance of UDP-glucuronic acid in this reaction was demonstrated chromatographically. In experiments in which the leucocytes were incubated with g1uco~e-C~~ at 37°C. before analysis, the isolated UDPG was highly radioactive. Our data show that donor leucocytes contain approximately 0.05 pmole of UDPG per ml. of fresh cells. We are aware of only one paper (Ondarza, 1960) in which the UDPG content was studied in the blood of normal and leukemic subjects. In this study, the leucocytes were not isolated and the UDPG was determined in the entire cell mass after centrifugation of the blood a t 2000 r.p.m. for 10 minutes. The content of UDPG arid other free nucleotides was determined in the cells of 5 normal subjects arid 2 patients with granulocytic leukemia. The average UDPG content was found to be 1.17 pmoles per 100 ml. blood in the normals and 3.00 pmoles in leukemia. However, the author’s data showed that there were 7,440 leuc~cytes/mm.~ in the first case and 268,000 in the second. Thus, tthe UDPG content in the leucocytes of patients with myeloid leukemia would be only about >f4 that in normal human leucocytes, if one considers the leucocytes to be the principal site of nucleotides in the blood. Another possible explanation would be that the UDPG content in leukemia does not increase proportionally in the thrombocytes and erythrocytes. Our determinations of the UDPG content in various types of leucocytes showed that the content increases significantly in chronic myeloid leukemia (by approximately 20070); in polycythemia, it is about normal; and in chronic lymphadenosis it is below normal. 7. The UDPG System in Glycogen Synthesis in the Leucocytes of Patients with Leukemia and Polycythemia The large differences in the turnover rate and content of glycogen in the leucocytes of normal subjects and patients with leukemia and poly-

w

ACTIVITYO F

TABLE XI PHOSPHOGLUCOMUTASE, UDPG-PYROPHOSPHORYLASE AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE I N ACETONE POWDER EXTRACTS OF LEUCOCYTES FROM HEALTHY DONORSAND P l T I E N T S WITH POLYCYTHEMIA A N D LEUKEMIA=

m

0

Acetone powder extracts of leucocytes from patients with Chronic leukemia Activity expressed in

Donors

Polycythemia

Myeloid

Lymphatic

Phosphoglucomutaseb pmoles of glucose-1-PO4 transferred/minute/g. of protein 80 k 6.9 Per cent of corresponding activity of donor leucocytes 100

96 f 11 120

64 80

4.2

10 12

* 1.5

Acute leukemia

11 14

* 1.1

s

UDPG-p yrophosphorylasec pmoles of UDPG transferred/minute/g. of protein Per cent of corresponding activity of donor leucocytes

52 k 4.9 100

72 k 4 . 3 138

rn

k 4.3

10 f 1 . 0 19

9 f 0.9 17

164 f 8 . 5 84

30 k 2 . 3 15

18 f 2 . 7 9

48 92

Glucose-&phosphate dehydrogenased pmoles of glucose-GPO4 transferred/minute/g. of protein 196 k 12 100 Per cent of corresponding activity of donor leucocytes

236 f 11 120

~

Enzymatic activities determined at 20°C. The figures represent the average of 15 experiments. During the determination of phosphoglucomutase, the vessels contained: 2 pM glucose-l-PO4; 5 pM cysteine; 20 p M MgC12; 1 p M TPN; 125 pM Tris buffer, pH 7.4; 0.1 ml. of the extract being tested (250 pg. protein). During the determination of UDPG-pyrophosphorylase, the vessels contained: 0.15 pM UDPG; 5 p M cysteine; 20 pM MgC12; 1 pM TPN; 125 pM Tris buffer, pH 7.4; 500 rg. crystalline phosphoglucomutase; 0.1 ml. acetone powder extract (250 pg. protein). During the determination of glucose-6-phosphate dehydrogenase, the vessels contained : 2 p M glucose-6-phosphate; 1 p M T P N ; 20 phl MgC12; 125 pM Tris buffer, pH 7.4; 0.05 ml. of the extract being tested (125 pg. protein). a

?

E

2

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

361

cythemia might be explained if it were possible to detect differences in the activities of individual links in the enzyme systems for the biosynthesis and degradation of glycogen between pathological and normal leucocytes. In this connection, it appeared of interest to compare the activity of the enzymes controlling the rate of glycogen synthesis by the uridine diphosphoglucose pathway in various groups of leucocytes. It seemed particularly important to study the activities of phosphoglucomutase, UDPG-pyrophosphorylase, and UDPG-glycogen glucosyltransferase. Hexoltinase also takes part in glycogen synthesis, but its effect is less specific since the reaction product (glucose-&phosphate) is metabolized via not just one but several pathways, and the channeling of glucose-&phosphate in the direction of glycogen synthesis is completely controlled by phosphoglucomutase, the activity of which in the leucocytes is relatively low. It is precisely this step, the transformation of glucose-&phosphate into glucose-1-phosphate, which is apparently the first control point in the leucocytes along the pathway from glucose to glycogen, as well as in the opposite direction: from glycogen to hexose under the influence of phosphorylase. An equally important roIe is played by UDPG-pyrophosphorylase, involving a coenzyme which is the transport form of glucose and the substrate of a number of reactions leading to the formation of a group of substances with important metabolic functions: UDP-glucuronic acid, UDP-galactose, etc. Finally, a decisive role in glycogen synthesis is played by UDFG-glycogen transferase, which completes a whole chain of reactions in the biosynthesis of glycogen by transferring a glucosyl residue from UDPG to the polysaccharide primer. Tables X I and XI1 give the results of the determination of phosphoglucomutase, UDPG-pyrophosphorylase and UDPG-glycogen transferase activity in various types of human leucocytes. The low activity of all three enzymes (phosphoglucomutase, UDPGpyrophosphorylase and UDPG-glycogen transferase) in the lymphocytes of patients with chronic lymphatic leukemia and in the undifferentiated leucocytes of patients with acute leukemia is striking. Based on 1 g. of protein in the acetone powder extracts, the activity of the first enzyme in the lymphocytes of patients with chronic lymphadenosis was 12% of the activity in donor cells, while the UDPG-pyrophosphorylase activity was 19% of normal, and the UDPG-glycogen transferase activity was 31% of normal. In the undiflerentiated leucocytes of patients with acute leukemia, the enzymatic activities in the same order were 14, 17, and 4oY0 of normal. It is still difficult to say to what extent these significant differences are the expression of the leukemic nature of the cells or the result of their varying maturity and the fact that they belonged to different biological categories (mature polymorphs vs. undifferentiated young blasts; granulocytes vs. lymphocytes). In any event, the differences are real and are most marked in the cells of patients with acute leukemia.

362

I. F. SEITZ

TABLE XI1 SYNTHESIS OF GLYCOGEN FROM UDPG-W IN ACETONE POWDER EXTRACTSOF HUMANLEUCOCYTES" Acetone powder extracts of leucocytes from patients with Activity expressed in

Healthy donors

Polycythemia

Chronic myeloid leukemia

Chronic lymphatic leukemia

Acute leukemia

pmolesof glucose 6 . 8 f 0.62 7 . 5 f 0.69 5 . 2 0 . 5 2 . 1 f 0 . 5 2 . 7 f 0.4 transferred/min(14) (12) (14) (10) (5) ute/g. of protein Per cent of the corre100 110 77 31 40 sponding activity of normal leukocytes The vessels contained 25 pM Tris buffer, pH 7.4; 10 pM MgC12;0.12 r M UDPG-CI4 with a total radioactivity of 6500 counts/minute; 2.0 p M glucose-6-phosphate; 0.5 mg. glycogen; 0.05 ml. extract of leucocyte acetone powder (125 pg. protein). Total volume = 0.6 ml. Incubation a t 37°C. for 30 minutes. The number of experiments is given in parentheses.

A decrease in leucocyte enzymatic activity is also observed in chronic myeloid leukemia, but to a lesser extent than in acute leukemia. In the chronic case, the phosphoglucomutase, UDPG-pyrophosphorylase, and transferase activities are 80, 92, and 770/, of normal, respectively. In polycythemia, the activities of all three enzymes were higher than in donor cells (120, 138, and 110% of normal, respectively). The activity of glucose6-phosphate dehydrogenase was also above normal (120%). Particular attention should be paid to the fact that the synthetase activity was generally higher in polycythemic leucocytes than in normal leucocytes and especially in leukemic leucocytes. In general, there was a definite parallelism, even though no strict proportionality, between the glycogen content in the leucocytes and the UDPG-glycogen transferase activity. This is easy to understand if it is accepted that the enzyme is adsorbed on micelles of polysaccharide, as indicated by data in the literature on the glycogen content and synthetase activity of the liver (Leloir and Goldemberg, 1960; Luck, 1961). However, this concept of the structural and chemical unity between the polysaccharide and the enzyme which synthesizes it in the leucocytes gives no indication as to the mechanism which bring about this high concentration of the polysaccharide in the cells. If there is a genetically determined number of glycogen granules in the leucocytes, then this may also determine the concentration of the necessary enzyme, since the latter is apparently insoluble and is bound up with the

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

363

structure of the glycogen particle. It must be pointed out that this would also limit the content of enzyme in the cell and hence the rate of polysaccharide resynthesis. 8. Phosphorylase Activity in the Leucocytes

Phosphorylase plays a direct role in glycogen metabolism; thus, the activity of this enzyme may determine, to a significant extent, both the content and the turnover rate of glycogen in leucocytes. The presence of phosphorylase in leucocytes was first demonstrated by Willstatter and Rohdewald (1937) and confirmed by Wagner aiid Yourke (1952). Later, however, little attention was paid to the study of leucocyte phosphorylase, aiid a t the present time we have 110 idea how the activity of this enzyme changes in leulcemia. As far as normal human leucocytes are concerned, Williams and Field (1961) recently showed that the phosphorylase activity in homogenates a t 37"C., judging from the transformation of glucose-lphosphate into glycogen, amounts to 20.6-37.4 pg. of inorganic phosphate liherated/107 leucocytes/30 minutes. According to Hulsmanii et al. (1961), the phosphorylase activity in leucocyte homogenates, recorded spectrophotometrically at 340 mp, amounts to 75-179 pmoles of glucose-l-phosphate transformed/g. of protein/hour at 20°C. Our determinations of the phosphorylase activity in leucocyte homogenates yielded the results shown in Table XIII. The activity was determined from the light absorption at 340 mp in a system in which glucose-lphosphate, formed by the phosphorolytic cleavage of glycogen in the presence of phosphate buffer, AMP, and cysteiiie, was transformed into glucose-6-phosphate, which was then oxidized to 6-phosphogluconate by glucose-6-phosphate hydrogenase with the simultaneous reduction T P N to TPNH. Based on 1 g. of protein in the leucocyte preparations, the phosphorylase activity was significantly depressed in the cells of patients with chronic myeloid and especially chronic lymphatic leukemia. The phosphorylase activity in extracts of fresh leucocytes from patients with acute leukemia was not determined. 9. Activity of Enzymes in Extracts o j Fresh Leucocytes

Along with the phosphorylase values, Table XI11 gives the results of the determination of the activity of other enzymes in the sediment of homogenates of freshly isolated leucocytes: hexokinase, phosphoglucomutase, UDPG-glycogen glucosyl transferase (both from data on the synthesis of glycogen-C"' from g1uc0se-C~~ by the reconstituted enzyme system, and from the direct transfer of a glucose-C14 moiety from UDPG-CI4 to the glycogen primer), and glucose-&phosphate dehydrogenase. Comparison of these data with the results presented in Tables XI and XI1 for

ACTIVITYOF

CERTAIN

TABLE XI11 ENZYMES I N THE EXTRACTS OF FRESH LEUCOCYTES FROM

NORMAL,

LEUKEMIC, AND

POLYCYTHEMIC SUBJECTSa*'

UDPG-glycogen transferase according t o Glucose-6phosphate dehydrogenase

Hexokinase

Healthy donors (10) Patients with polycythemia (10)

164 135

27 22

96 58

Patients with chronic myeloid leukemia (10)

125

17

58

54

26

13.5

Leucocytes of

Patients with chronic lymphadenosis (10)

Phosphoglucomutase

Phosphorylase

Transfer from UDPG-C14

Incorporation of g1uc0se-C~~ as initial substrate

3.3

15.0

26.2 (5)

1.1

10.0

10.0 (8)

a The number of experiments is given in parentheses. The activity is expressed as pM of transformed (or transferred) substrate per gram of protein in the centrifuged homogenates of fresh leucocytes per minute. The determinations were carried out at 20"C., except in the case of UDPG-glycogen transferase, which was studied at 37°C. For t,he details of the method, see text and the articles by Seita et al. (1963) and Luganova et al. (1963b).

H

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

365

extracts of acetone powders of leucocytes reveals satisfactory agreement for these various enzyme preparations. It is true that the homogenates showed significantly lower glucose-6-phosphate dehydrogenase and phosphoglucomutase activity, and significantly higher UDPG-glycogen glucosyl transferase activity; the reasons for these differences are now being clarified. It should be pointed out, however, that approximately the same ratios between normal and pathological cells are maintained in the sediment of fresh leucocyte homogenates as in the extracts of acetone powders, and that there is a decrease in the activity of almost all the enzymes studied in the leucocytes of patients with chronic myeloid and especially with chronic lymphatic leukemia. In polycythemia, there is a significant increase in UDPG-glycogen glucosyl transferase activity. The enzymatic activity of leucocytes from patients with acute leukemia was not studied in homogenates. 10. Some Conclusions Experiments with either extracts of leucocyte acetone powders or homogenates of fresh leucocytes have thus shown that there is a decrease, in leukemia, in the activity of all enzymes taking part in glycogen synthesis: phosphoglucomutase, UDPG-pyrophosphorylase, and UDPGglycogen glucosyl transferase. There is also a significant decrease in the glycogen content and an increase in the turnover rate in these diseases. A comparable effect is seen in all types of leucocytes, but the tendencies are most pronounced in acute leukemia. It therefore seemed to us that it would be most interesting to evaluate these rather characteristic shifts in the metabolism of leukemic leucocytes on a broader scale, particularly by comparing them with the corresponding data for a classical malignant tumor. For this purpose, we used Ehrlich ascites carcinoma. This was especially interesting because the Ehrlich ascites cells are theoretically very similar, metabolically speaking, to granulocytes, and especially to the young, undifferentiated cells of this (myeloid) series found in acute leukemia: they have a rather low respiration, high aerobic and anaerobic glycolysis, a pronounced inverse Pasteur effect, and comparable resynthesis of ATP and other organophosphorus compounds under aerobic and anaerobic conditions (Yel’tsina and Seitz, 1951; Siyanitskaya and Seitz, 1958, 1962; Seitz, 1961). In collaboration with M. F. Kharchenko, we found that ascites carcinoma cells show the same defects in the enzyme system for glycogen synthesis as are encountered in the cells of patients with acute leukemia, only unfortunately in an even more pronounced form. Table XIV shows results of a study of the activity of a number of enzymes in extracts of acetone powders of Ehrlich ascites carcinoma cells.

366

I. F. SEITZ

TABLE XIV AcrrvITY O F THE ENZYMES INVOLVED I N THE SYNTHESIS A N D CATABOLISM O F GLYCOGEN, A S WELL A S GLUCOSE-6-PHOSPHATE DEHYDROGENaSE I N EHRLICH AscITEs CARCINOMA" UDPG-glycogen transferase according to Glucose-6PhosphoPOc dehyglucodrogenase Hexokinase mutase

a

UDPGpyrophosphorylase

Synthesis Synthesis of glycogen of glycogen from from Phosphorglucose-Cl4 UDPG-CI4 ylase

The number of experiments is given in parentheses. The activity is expressed in

phl substrate per gram of protein in an extract of ascites carcinoma cells per minute. The

enzymatic activity was determined in 0.05 M Tris buffer, pH 7.4 a t 20°C. (37°C. for UDPG-glycogen transferase). The details of the method were as in experiments with leucocytes (see Section 11,I).

It is clear from Table XIV that the phosphoglucomutase, UDPG-pyrophosphorylase, and UDPG-glycogen glycosyl transferase activities are very low in cancer cells; the phosphorylase activity is also low. I n other words, all of the enzymes connected with glycogen metabolism are present in Ehrlich ascites carcinoma cells in amounts which are significantly below those in normal cells. Low values for phosphoglucomutase, UDPG-pyrophosphorylase, and UDPG-glycogen glycosyl transferase activity were also obtained in solid tumors by Nigam et al. (1962). It is also interesting that the content of glycogen (or its analogs) in ascites carcinoma cells is very low (approximately 150 pg./ml. fresh cells), while its turnover rate in experiments with g 1 ~ c o s e -Cis~ ~ quite high (the specific radioactivity under experimental conditions comparable to those with leucocytes was 19,000 counts/minute/mg.) . As we shall see, this similarity between the basic aspects of the metabolism of cancerous and leukemic cells goes so far that it can hardly be looked upon as a simple coincidence. Apparently deeper theoretical relationships should be looked for here. I. OTHERENZYMATIC ACTIVITIESIN NORMAL AND LEUKEMIC HUMANLEUCOCYTES There are interesting data in the literature on the content of a whole series of enzymes in the leucocytes of normal human subjects and patients with various types of leukemia. Particularly detailed studies in this direc-

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

367

tion have been carried out by Valentine and Beck (Beck and Valentine, 1952; Beck, 1955, 1958a; Valentine, 1955b, 1960). They found that the rates of glycolysis in homogenates of normal leucocytes, leucocytes from patients with chronic myeloid leukemia, and leucocytes from patients with chronic lymphatic leukemia were 45, 13.2, and 4.8, respectively (in pmoles lactic acid/hour/lOM cells), while the oxygen absorption was 4.0, 1.2, and 1.1, respectively. Initially, they found that there is no enzyme in the leucocytes which could limit the rate of glycolysis, since the maximal rates per unit of tissue for each of the enzymes studied were higher than the maximal rate of glycolysis as a whole. However, although the maximal rates of lactic dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase are much higher than the rate of glycolysis, their activity levels are strictly proportional to the rates of glycolysis in the corresponding types of leucocytes. According to Beck, the activity of hexokinase, phosphofructokinase, and pyruvic oxidase is also decreased in leukemic leucocytes. On the other hand, aldolase, triosephosphate isomerase and phosphohexoisomerase are somewhat more active in the leucocytes of patients with chronic myeloid leukemia than in normal cells. There were no differences between the enzymes of leukemic and normal leucocytes with respect to the Michaelis constant (in relation to the substrate and the coenzyme) or the optimum pH. Later Beck showed that the rate-limiting step in glycolysis in all three types of leucocytes is hexokinase. Addition of hexokinase to leucocyte preparations from patients with chronic myeloid leukemia increased the rate of glycolysis by approximately 300%, while in normal leucocytes the rate was only increased 50%. It was also found that leukemic leucocytes are poor in ADP, and that addition of this compound to homogenates of leukemic leucocytes stimulates glycolysis more strongly than in homogenates of normal leucocytes. Furthermore, the A T P concentration in the leucocytes depends to a significant degree on the hexokinase (and phosphofructokinase) activity; since the hexokinase activity is depressed in leukemic leucocytes, the regeneration of ADP in the cells is impaired. A study of the phosphogluconate pathway showed that it plays a larger role in the metabolism of leukemic leucocytes than in normal leucocytes; in both cases, however, the fraction metabolized by this pathway did not exceed 10% of all the glucose utilized (Heck, 1958b,c). Interesting work has also been done with the phosphatases. The very fact that the leucocytes contain phosphatase was first discovered histochemically by Wachstein (1946). Haight and Rossiter (1950) found that in man, acid phosphatase is localized mainly in the lymphocytes while alkaline phosphatase is localized in the granulocytes. I t can now be considered proved, on the basis of a great deal of evidence, that the activity of these

368

I. F. SEITZ

enzymes is significantly changed in leukemia. A particularly large amount of work in this area has been done by Valentine and Beck (Beck and Valentine, 1951; Valentine and Beck, 1951; Valentine et al., 1954; Valentine, 1960). They showed that the alkaline phosphatase activity is sharply decreased in the leucocytes of patients with chronic myeloid, chronic lymphatic, and acute leukemia, while the content of this enzyme is increased in the granulocytes of patients with polycythemia, infectious diseases, and some other conditions. The fact that the alkaline phosphtase activity of the leucocytes is increased in polycythemia was confirmed by Sauer and RauschStroomann (1955). According to Moloney and Lange (1954), low levels of this enzyme were observed in the leucocytes of victims of the atomic bomb in Japan during the year before the development of clear signs of chronic myeloid leukemia. In this connection, Dameshek feels that the changes in the enzymatic activity of the leucocytes may occur very early in the preleukemic period (Dameshek and Gunz, 1958). In the morphologically homogeneous, undifferentiated leucocytes from patients with acute leukemia, Fieschi et al. (1956) as well as Salvidio (1958) detected two groups of cells: one with increased and the other with decreased alkaline phosphatase activity. Lajtha (1957) showed that the undifferentiated cells of the bone marrow (blasts) and lymphocytes do not contain alkaline phosphatase activity. This is an indication of the existence of two different alkaline phosphatases in the leucocytes (Trubowitz et al., 1957). Among the phosphatase, a special place is occupied by adenosine triphosphatase (ATPase). A special role is ascribed to this enzyme in cells having motor and some other functions. For this reason, the study of this enzyme in leucocytes, with their marked capacity for ameboid movement and phagocytosis, was especially interesting. A study which we carried out showed that all types of human leucocytes, both normal and leukemic, have a highly active ATPase which is localized on the surface of the cells and liberated into the medium by their activity (Luganova et al., 1957f; Seitz, 1961). The amount of this enzyme in 1 ml. of fresh cells splits 3.2-10.1 mg. of labile phosphorus from ATP per hour. In other words, during 4-5 hours of indubation at 37"C., the leucocytes are able to hydrolize a n amount of ATP equaling their natural dry weight. The optimum pH for this enzyme is 9; the ATPase, which is localized to the surface of the leucocytes, is inhibited by C a w and activated by Mgw. The enzyme is thermolabile and specific. Fructose-6-phosphate, fructose-1, 6-diphosphate, 3-phosphoglyceric acid, adenylic acid, DPN, and inorganic pyrophosphate are either not hydrolized a t all when they are added to intact undamaged leucocytes, or hydrolized a t a rate which is extremely low compared to the rate of hydrolysis of ATP. The biological significance of the enzyme is unclear. A weak ATPase activity was detected by Chernyak (1957) in the leucocytes

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

369

of rabbit pus. According to our data, 1 ml. of these cells hydrolizes approximately 4 mg. of labile phosphorus from ATP in 1 hour. The erythrocytes are completely devoid of this enzyme. The effect of the ATPase, which is localized to the surface of the leucocytes, can hardly be looked upon as the result of the direct hydrolytic action of the enzyme on the labile phosphorus of ATP. It is more likely that the cleavage of ATP is the net result of more complex positive metabolic processes, taking place on or near t)he surface of the leucocytes, associated with the high functional activity of the cells. The mobility, phagocytosis, and permeability of the leucocytes are undoubtedly connected with complex processes of energy metabolism in the membrane. The question arises whether the ATPase activity of the leucocytes with respect to exogenous ATP is not an indication that the white blood cells may be able to utilize not only intracellular ATP but also the energy sources in the medium, either directly on the outer surface of the cell or after preliminary pinocytosis. Experiments with P32have shown that the label does not appear in the extracellular ATP, which is good evidence for the unidirectional and dissimilatory nature of the process. The leucocytes are apparently able to utilize high-energy compounds from the medium on their surface, but are unable to accomplish their regeneration on the cell membrane. Elucidation of the ATPase effect on the surface of the leucocytes would apparently be a major step forward in our understanding of the biology and functional activity of these cells. This problem demands further detailed study. Ill. Thrombocytes

Only a few years ago, very little was known about the chemistry and metabolism of human thrombocytes. However, the literature on this subject is growing rapidly and our understanding of the biochemistry of the platelets is constantly expanding. Initially, the absence of a nucleus and the comparatively simple structure seemed to support the hypothesis that the thrombocytes are metabolically inert protoplasmic fragments from more complex cells; later studies, however, led to the detection in the platelets of practically all of the principal enzymochemical and metabolic indicators of a living functioning cell, with one significant exception : the thrombocytes do not contain DNA, which is essential for reproduction. Nevertheless, during the course of the few days which constitute their normal life span, the platelets are fully able to provide the chemical and energetic basis for those complex physiological functions, so important to life, for which they are responsible. The thrombocytes carry on both respiration and glycolysis, the energy of which is stored in ATP; they contain RNA, which is an indirect indication of their potential role in protein

370

I. F. SEITZ

synthesis; they are richly endowed with a great variety of enzyme systems which guarantee the maintenance of their vital functions. As in the case of the leucocytes, we shall limit the processes under consideration mainly to the energy metabolism of the thrombocytes and the problems directly related thereto, by comparing the platelets from healthy donors with those from patients with leukemia and polycythemia. The personal results which will be presented were obtained in collaboration with I . S. Luganova and V. A. Yegorova, with the participation of the morphologist V. I. Teodorovich and the clinician A. I. Blinova.

A. RESPIRATION, GLYCOLYSIS, AND RESYNTHESIS OF ADENOSINE TRIPHOSPHATE IN HUMAN THROMBOCYTES I n his studies, Tullis (1953) observed neither glucose consumption nor lactic acid formation in the thrombocytes. He therefore considered the platelets to be metabolically inert. However, Maupin (1954), Campbell et al. (1956, 1957), Morita and Asada (1956), Notario and Nespoli (1961), Chernyak et al. (1960a,b), N. B. Chernyak (1961), and others have detected definite respiration and glycolysis in human platelets. Even in their time, Endres and Kubowitz (1927) observed relatively intense respiration in the thrombocytes. I n a review published in 1959, Kugelmas came to the conclusion that the thrombocytes are metabolically active. These data on the metabolic activity of the platelets are confirmed by the reports of a number of investigators who have detected ATP in these cells (Born, 1956a,b; Born and Esnouf, 1959; Lohr et al., 19Gl; Gross et al., 1958; Morita and Asada, 1956; Maupin et al., 19G2; Born, 1962). As a matter of fact, there is a contradiction between the moderate metabolic activity of the platelets and their high levels of ATP. A study of the ATP content in the thrombocytes, as well as of the sources for the formation of this important compound, is especially interesting in view of the large amount of mechanical work performed by the platelets during the processes of blood coagulation and hemostasis. A whole series of important physiological functions performed by the blood stream in the living organism are connected with biochemical processes in the thrombocytes which are based on the transformation of ATP. For this reason, all questions relating to the generation and transformation of energy in the platelets acquire great importance. Studies on the thrombocytes have been carried out in our laboratory over the course of several years, both from the point of view of some of the over-all metabolic indexes, and in the sense of studying individual enzyme systems having a direct relationship to the energy metabolism of the platelets. Table XV shows the results of our studies on respiration, glycolysis,

RESPIRATION, GLYCOLYSIS, ATP,

AND

RNA

I N THE

TABLE XV THROMBOCYTES OF HEALTHY DONORS .4ND

P A T I E N T S XVITH PO L Y CY T H EM IA .4ND

Labile phosphorus of ATP

Respiration

(Qo~)

~-

Thrombocytea of

Gly colysis

~

(QCO~)

In the In the absence presence of glucose of glucose Aerobic

bic

~

Micrograms

-

bic

bic

141 f 2.2

140 f 9.5

bic

a

E Y

RNA phosphorus

Relative specific radioactivity

~

bic

Micrograms hic

-

bic

Relative specific radioactivity

-

bic

3.66 f 0.24

13.1 f 20.3 C! 1.1 1.28

Patientswithpolycythemia (16)

6.21 f 0.43

4.39 & 0.35

1 2 . 5 k 1 8 . 3 t 143.1 f 140.9 f 68.2 f 65.0 f 71.5 f 74.9 f 23.7 f 24.1 0.70 1.12 7.0 7.0 2.4 1.8 4.6 4.8 1.3 0.8

Patients with 6.94 f chronic myeloid 0.60 leukemia (8)

5.09 & 0.54

1 5 . 9 f 20.7 k 0.78 0.71

Q

The number of experiments is given in parentheses.

98.6 8.2

3

hj

z3 R

bic

Healthy donors (15) 5 . 0 1 f 0.36

97.4 f 8.0

!

0 d

CHRONIC MYELOIDLEUKEMI.\O

43.2 k 42.5 f 66.0 k 66.0 f 27.9 f 26.3 & 4.1 3.9 5.9 3.3 1.5 0.8

*

k

5 r 3

k 4 9 . 6 f 55.1 t 70.0 f 61.0 k 12.2 f 1 0 . 1 &

(3

3.1

4.0

5.4

5.9

0.9

0.7

' 2

d M

E

372

I. F. SEITZ

ATP content, and the turnover rate of ATP phosphorus in the thrombocytes from healthy donors and those from patients with chronic myeloid leukemia and polycythemia. The reason for selecting these two diseases was, first of all, that such patients are the most available source of thrombocytes in numbers large enough for biochemical study and analysis, and second, because their comparison is particularly interesting in view of the existence of two different opinions as to the nature of these pathological processes. One side assumes that these diseases are completely different in origin, while the other assumes that polycythemia can develop into myeloid leukemia, so that it represents a sort of initial stage of the leukemic process. In a series of experiments in which we compared the metabolism of normal and pathological thrombocytes, we obtained values for the respiration and glycolysis of normal thrombocytes which were very close to those reported in a n earlier paper (Luganova el al., 1958a,b). I n 1958, we reported the following values for the respiration and glycolysis of normal platelets : Qo, in the absence of glucose = 4.4; Qo, in the presence of glucose = 3.56; Qco2 under aerobic conditions = 12.6; &cot under anaerobic conditions = 19.3. As shown in Table XV, the metabolic indexes obtained in the new series of experiments were very similar (5.0, 3.7, 13.1, and 20.3, respectively). The data in Table XV show that the platelets from patients with polycythemia and chronic myeloid leukemia differ little from normal platelets in the intensity of glycolysis. There was some increase in respiration, especially in the thrombocytes from patients with chronic myeloid leukemia. Attention should be called to some metabolic peculiarities which are characteristic of all three types of thrombocytes, regardless of the quantitative differences in their metabolic indexes. The most important characteristic is the presence of a rather high level of aerobic glycolysis, amounting to approximately 2/3 (donor cells) to 3/4 (chronic myeloid leukemia) of the maximum possible under anaerobic conditions. The second characteristic of the platelets is the marked inverse Pasteur effect (Crabtree effect): respiration was approximately 33% lower in the presence than in the absence of glucose. Finally, there is a high QG, :Qo, ratio, which is characteristic, according to Warburg, of neoplastic tissues. It can be seen in Table XV that this ratio also remains high (above 3) in normal platelets. A very curious property of the thrombocytes is their ability to maintain identical ATP levels and rates of turnover of the phosphorus of this compound under aerobic and anaerobic conditions. This peculiar metabolic trait was also observed in the leucocytes. The thrombocytes thus resemble the leucocytes in all the basic qualitative characteristics of their energy

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373

metabolism: the existence of aerobic glycolysis, the presence of an inverse Pasteur effect, and the equivalent resynthesis of ATP under aerobic and anaerobic conditions. It is true that the absolute values for oxygen absorption and the formation of lactic acid are lower in the thrombocytes than in granulocytes. However, the ATP content in the platelets is no lower than in the leucocytes, perhaps even higher. This characteristic of the thrombocytes (a high level of ATP) has also been pointed out by various other authors (Born, 1956a,b; Gross et al., 1958; Luganova et al., 1958a)b; Lohr et al., 1961; Maupin et al., 1962). As can be seen from Table XV, 1 ml. of fresh normal platelets contains approximately 140 pg. of labile ATP phosphorus. In an earlier paper (Luganova et al., 1958a), we reported a somewhat higher value (164 pg.). Our data on the A T P content in the thrombocytes are close to those of Maupin et al. (1962) and Born (1956a). However, our values are several times lower than that reported by Lohr et al. (1961): 21.5 pM/lO" platelets. The ATP content in the thrombocytes of patients with polycythemia is no different from that in normal platelets. In chronic myeloid leukemia, however, there is a significant decrease in the content of this high-energy compound (by almost 33%). At the same time, the turnover rate of the labile phosphorus of ATP is sigiiificantly higher in the thrombocytes from polycythemia patients than in thrombocytes from healthy donors or patients with chronic myeloid leukemia. The over-all turnover rate of the A T P phosphorus is somewhat lower in the platelets than in the leucocytes. The relative specific radioactivity of the labile phosphorus after 40 minutes' incubation at 37°C. in a mixture of serum and physiological saline (1: 1) is 43-68y0. The problem of the role of respiration in the energy metabolism of the platelets is quite interesting. Since the respiration of normal thrombocytes shows a definite Pasteur effect (glycolysis is inhibited by about 33%)) it would be expected that this would also show up in phosphorylation. After all, the relationship between the Pasteur effect and oxidative phosphorylation is a universal law of general biology (Seitz, 1955, 1961). Coupled oxidative phosphorylation is generally disrupted only when the Pasteur effect is eliminated. Therefore, there was some reason to believe that the respiration of the platelets plays a role in the resynthesis of ATP. Under aerobic conditions, thanks to the Pasteur effect, glycolysis in the thrombocytes is depressed 33%) but the amount and turnover rate of the labile phosphorus of ATP are no lower than under anaerobic conditions (see Table XV). It follows from this that the deficit in phosphorylation when glycolysis is depressed under aerobic conditions must be compensated for by respiration. The work of Chernyak et al. is very interesting in this connection, however, since they obtained data which indicate that respiration in platelets is not coupled with phosphorylation. The mitochondria isolated

374

I. F. SEITZ

from thrombocytes absorbed 02, but did not esterify inorganic phosphate (Chernyak and Guseinov, 1960; Chernyak and Totskaya, 1963). It is possible that improved methods for the isolation of mitochondria from platelets and the use of more suitable media will still permit oxidative phosphorylation to be detected in these granules. Further work in this direction will be of interest. Chernyak et al. also noted the unusually high stability of AT P in the platelets. Incubation of thrombocytes under various conditions and storage for long periods of time did not lead to destruction of their ATP (Chernyak et al., 1960a,b). On the other hand, Maupin et al. (1962) found that ATP is rapidly destroyed when thrombocytes are stored in a medium containing glycerol. I n our experiments with thrombocytes, monobromoacetate (8 X M ) in the presence of glucose decreased both the content and the turnover rate of the labile phosphorus of ATP, while stimulating O2 absorption. It should be noted, however, that the disruption in phosphorus metabolism produced by monobromoacetate was much less pronounced in the platelets than in the leucocytes, with which the platelets have much in common as far as their energy metabolism is concerned. In human granulocytes in the presence of glucose in the medium, bromoacetate inhibited not only glycolysis and the phosphorylation coupled to glycolysis, but also the esterification coupled to respiration. In human platelets, this glycolytic poison decreased the ATP content and the turnover rate of its labile phosphorus only t o the level of the glucose-free sample, apparently without affecting oxidative phosphorylation. Thus, the ultimate effect of monobromoacetate is different in different cells. Under conditions of maintained respiration, its principal effect (inhibition of glycolysis and the phosphorylation coupled to glycolysis) may be accompanied by a series of secondary phenomena due, on the one hand, to the presence of sugar in the medium and possibly the glycogen reserves in the cells and, on the other hand, to the relative activities of the individual enzyme systems, among which the most important role is apparently played by the enzymes which catalyze the transesterification and mineralization of organic phosphates, especially ATP. I n agreement with this, the effect of bromoacetate on energy metabolism varies even within a group of cells which are so close to each other with respect to the nature of their energy metabolism as the leucocytes and thrombocytes. M ) increased the The uncoupling agent 2,4-dinitrophenol (8 X intensity of aerobic glycolysis in the platelets while having a relatively weak effect on the ATP content and the turnover rate of its labile phosphorus. The inability of dinitrophenol to produce marked changes in the resynthesis of ATP in the thrombocytes may be explained by the fact that, by disrupting oxidative phosphorylation, it completely abolished the Pasteur effect.

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375

By the same token, the uncoupling agent increases aerobic glycolysis to the level of anaerobic, thus, as reported above, guaranteeing a level of esterification equivalent t o that under conditions of good aeration. The response of normal platelets and platelets from patients with chronic myeloid leukemia and polycythemia to monobromoacetate and dinitrophenol is the same. B. NUCLEICACIDS,PHOSPHOLIPIDS, A N D PHOSPHOPROTEINS IN THROMBOCPTES A test for the presence of DNA in the thrombocytes gave negative results. This is connected with the absence of a nucleus or its biological equivalent in the platelets and agrees with the results of other authors who either failed to detect or detected only traces of this important compound in the platelets, although they did report the presence of RNA (Wagner, 1946; Morita and Asada, 1956, 1957; Wagner et al., 1956; Fantl and Ward, 1956; Lawkowicz et al., 1956; Green, 1954; Maupin et al., 1954). It can apparently be considered as definitely confirmed that only one of the nucleic acids, RNA, is present in the thrombocytes in recoverable amounts. Table XV shows the results of a determination of the content and turnover rate of RNA phosphorus in the thrombocytes of healthy donors and patients with polycythemia and chronic myeloid leukemia. The content of RNA phosphorus in the thrombocytes in these pathological states is practically no different from that in normal thrombocytes, fluctuating between 61 and 74.9 pg/ml. fresh platelets (396486 pg./g. dry weight). This is significantly less than in the leucocytes (Luganova and Seitz, 195813; Seitz, 1961). At the same time, the turnover rate of RNA phosphorus is significantly higher in the thrombocytes than in the leucocytes. There is also a definite decrease in the relative specific radioactivity of the RNA phosphorus in platelets from patients with polycythemia and especially from patients with chronic myeloid leukemia. When platelets were incubated for 40 minutes in a mixture of physiological saline and serum a t 37"C., the relative specific radioactivity was 27.9 in normal thrombocytes, 23.7 in patients with polycythemia, and 12.2 in patients with chronic myeloid leukemia. We are still unable to say how concretely the ATP deficiency and the sharply decreased turnover rate of RNA in patients with chronic myeloid leukemia affect the functional activity of the platelets. We can only hypothesize that the biochemical defect detected in the thrombocytes in this disease may be related to their morphological and functional imperfection. As far as the general role of RNA in the thrombocytes is concerned, its significance remains completely unclear. At the same time, the high turnover rate of the phosphorus in this compound indicates that it must play some important functional role.

376

I.

F. SEITZ

We have studied the phosphoproteins and phospholipids only in normal thrombocytes (Luganova et al., 1958a,b). The content of phosphoprotein phosphorus in the platelets is very small: 9.2 f 1.5 pg./ml. fresh thrombocytes (approximately 60 pg./g. dry weight). However, the phosphorus in this fraction is turned over quite rapidly, although the turnover rate is only approximately 50% of that of RNA phosphorus. The phospholipids in the platelets differ little from the corresponding fraction of normal leucocytes with respect to either the content (676 pg. phosphorus/ml. fresh thrombocytes or 4390 pg./g. dry weight) or the turnover rate of the phosphorus. According t o Morita and Asada (1956), the phospholipid content is the same in the thrombocytes as in the leucocytes. Our determinations, however, gave platelet values which were about 1.5 times as high as in the leucocytes. Ericltson et al. (1939) found that both the content and the ratios between the individual types of lipids were essentially the same in leucocytes and thrombocytes, cephalin accounting for 68% of all the lipids. According to these same authors, the lipid composition of the platelets does not change during diseases of the blood. Maupin (1954) found that lipids accounted for about 19% of the dry weight of the thrombocytes, and that 13.8% of this was phosphatides. Zilversmit et al. (1961) reported that the phospholipid content in human thrombocytes amounted to 155 pmoles/g. dry weight. Firkin and Williams (1961) studied the incorporation of P32into the thrombocytes of patients with leukemia and found that the platelets, like the leucocytes, contain phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Incorporation of P32into the lipids took place under both i n vivo and in vitro conditions.

c. EXPERIMENTS WITH

GLUCOSE-C'4: ASSIMILATION OF LABELED FRAGMENTS FROM GLuCOSE-C'~BY THROMBOCYTES Experiments with g1~cose-C'~ are of theoretical importance since the use of uniformly labeled glucose permits one to follow, not only the energy transformations of this substrate, but also the part played by its metabolic products in the anabolism of the thrombocytes.

1. Utilization of C14-Metabolites of Glucose for Growth The addition of uniformly labeled g1~cose-C'~ to platelets leads t o the appearance of labeling in the proteins and RNA, as well as t o its transformation to lactic acid and C1402. In one of our experiments, following the incubation of 0.2 ml. of fresh donor thrombocytes (20 mg. protein) in a 1 : l mixture of serum and physiological saline at 37"C., in the presence of 2.25 mg. of glucose-C14having a n over-all radioactivity of 96,000 counts/ minute, the platelet proteins showed 309 counts/minute and the RNA

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

377

had 400 counts/minute. In the case of protein, this corresponds to about 0.3% of the radioactivity of all the g1uc0se-C'~utilized. Consequently, the degradation of labeled sugar during glycolysis and respiration in the thrombocytes is accompanied by the effective incorporation of labeled particles into the most complex and vitally important structural and chemical components of the platelets, thanks to the formation of intermediate products of carbohydrate catabolism. The fact that there is turnover of the carbon skeleton of protein and RNA in the thrombocytes may serve as a valuable criterion for the existence of vital functions and metabolic activity and may, in combination with data on the adequacy of their energy metabolism, indicate that the platelets are indeed independent and autonomous entities from the points of view of structural chemistry, metabolism and physiology.

2. Oxidation of Glucose in Thrombocytes Since the ATP content of the platelets decreases when they are incubated in the absence of glucose (Seitz, 1961), it might be supposed that the inability of respiration to maintain a normal level of ATP under these conditions is due to a lack of the nutritive substrate (glucose). We were able to obtain a n answer to the question of the oxidative utilization of glucose by the thrombocytes by means of experiments with glucose-C14. We studied the formation of CI4O2during the respiration of platelets in the presence of uniformly labeled g l u c o ~ e - c and ~ ~ , found that the appearance of the label in the expired COZ was far inferior to the expected activity calculated on the basis of the amount of oxygen absorbed in the experiment. The values were too low to enable glucose to be looked upon as a principal and direct substrate for respiration in the thrombocytes, or to ascribe any real significance to this process in the energy metabolism of the thrombocytes. In our experiments, the oxidation of glucose was able to account for only 6-10% of the absorbed oxygen. The greater part of the respiration proceeded independently of the added sugar. In this respect, the thrombocytes appear t o be very similar to the leucocytes.

D. GLYCOGEN AND ITS METABOLISM IN HUMANTHROMBOCYTES Wagner (1946) failed to detect glycogen in the thrombocytes. Somewhat later, however, Wachstein (1949) demonstrated the presence of glycogen cytochemically. Storti (1953) detected polysaccharide granularity in the cytoplasm of the platelets, and in 1956 Gude et al. as well as Heckner demonstrated the preeence of glycogen and mucopolysaccharides in the thrombocytes cytochemically. Woodside and Kocholaty (1960) made a detailed study of the carbohydrate composition of human platelets and showed that this group of compounds accounts for 8.4% of the dry weight.

378

I. F. SEITZ

They also confirmed the presence glycogen and acid mucopolysaccharides. The significant mucopolysaccharide content in the platelets was pointed out by Odell and Anderson (1957) and by Anderson and Odell (1958), who divided them into two fractions : the first fraction included chondroitin sulfate and heparin, and contained galactosamine and glucuronic acid, while the second fraction contained glucosamine. Analogous results were obtained by Kerby and Langley (1959). 1. Content and Turnover Rate of Glycogen in the Thrombocytes of Normal Subiects and Patients with Leukemia and Polycythemia

Studies carried out in our laboratory showed that human thrombocytes contain significant amounts of glycogen (see Table XVI). TABLE XVI CONTENT AND TURNOVER RATE O F GLYCOGEN I N THE THROMBOCYTES O F HE.4LTHY DONORS AND PATIENTS WITH POLYCYTHEMI.~ A N D CHRONIC MYELOIDLEUKEMIA^ Glycogen content (% dry wt.) Thrombocytes of

After aerobic incubation

After anaerobic incubation

Specific radioactivity of glycogen (counts/minute/mg.) After aerobic incubation

After anaerobic incubation

1.49 f 0 . 0 8 1.58 rt 0.05 1682 f 143 1675 f 127 Healthy donors Patients with polycythemia 1.42 f 0.07 1.34 f 0 . 0 4 2174 f 110 2208 f 95 Patients with chronic myeloid 1.26 f 0 . 0 9 I . 15 f 0 . 1 1 3855 f 190 3850 5 175 leukemia (18)

The number of experiments is given in parentheses. Thrombocytes were incubated

for 40 minutes at 37°C. in a 1: 1 mixture of serum and physiological saline, with heparin (5 units/ml.) and Versene (0.080/,) as the anticoagulants. The vessels each contained 3.5 mg. of uniformly labeled glucose-C14 with a total radioactivity of 300,000 counts/

minute.

I n normal human platelets, this polysaccharide accounts for a n average of about 1.5% of the dry weight. It is interesting that the values which we obtained on the basis of 10" platelets agreed very well with the results of Lohr et al. (1961): 5.1 mg. or 28.4 pmoles on the basis of glucose. It is clear from Table XVI that the thrombocytes of patients with polycythemia have about the same amount of glycogen as normal thrombocytes. In the platelets of patients with chronic myeloid leukemia, however, the glycogen content is significantly decreased, to 1.26%. It is a curious fact that the glycogen content does not decrease under anaerobic conditions if there is glucose in the medium. This stability of the polysaccharide in the platelets

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

379

might be interpreted as evidence for its metabolic inertness, but such a conclusion would not correspond with the facts. When thrombocytes are incubated in the presence of uniformly labeled g l u ~ o s e - C ~ the ~ , label appears in the glycogen very rapidly. Thus, when platelets were incubated in a 1: 1 mixture of serum and physiological saline for 40 minutes a t 37"C., with 3.5 mg. of glucose-CI4 in the medium having a total radioactivity of 300,000 counts/minute, the glycogen isolated had a high specific radioactivity: 1682 counts/minute/mg. in the case of donor thrombocytes. This value is not only no lower than the analogous value for donor leucocytes, but even surpasses it significantly. This fact is a n indication of the high metabolic activity of glycogen in the thrombocytes and of its apparent role in the functional activity of these cells. Under analogous experimental conditions, the turnover rate of glycogen in the thrombocytes of patients with polycythemia was almost 1/3 higher than normal. Under aerobic conditions, the specific radioactivity of this polysaccharide in the platelets of patients with polycythemia amounted to 2 174 counts/minute/mg. Glycogen metabolism in the thrombocytes was found to be stimulated to a particularly significant degree in chronic myeloid leukemia. In the platelets of this type of patients, the specific radioactivity was found to be 3855 counts/minute/mg., or 2.3 times as high as in the normal. The meaning of this finding is unclear, but it certainly indicates that thrombocyte metabolism is a t a higher level in chronic myeloid leukemia. This is also indicated by the higher values for respiration and aerobic glycolysis in the thrombocytes of myeloid leukemia patients (see Table XV). This increased metabolism in the platelets during chronic myeloid leukemia is apparently less effective than under normal conditions, since the ATP content is significantly depressed. Particular attention should also be paid to the fact that the specific radioactivity of glycogen in platelets incubated with g1u~ose-C'~ under anaerobic Conditions was equal to that in a well-aerated sample. The comparable rate of glycogen resynthesis under aerobic and anaerobic conditions is in agreement with our finding that other important and complex compounds, such as ATP, are also resynthesized at normal rates under anaerobic conditions. This makes the chemistry of the thrombocytes resemble that of the leucocytes, and indicates the successful adaptation of the platelets to existence and physiological activity under the unfavorable conditions of an inadequate supply of oxygen. Alongside the similarity between thrombocytes and granulocytes mentioned above with respect to the basic indexes of energy metabolism (respiration, glycolysis, the relationship between the two, and the resynthesis of ATP), we can now mention their similarity with respect to the content and metabolism of glycogen. Although the granulo-

380

I. F. SEITZ

cytes contain almost 2.3 times as much glycogen, 1.5% of the dry weight is still a large amount. The turnover rate of this polysaccharide in the thrombocytes is also high. Moreover, the significant stimulation of glycogen metabolism in the leucocytes of patients with chronic myeloid leukemia is accompanied by an analogous increase in the turnover rate of this polysaccharide in the thrombocytes; the decreased glycogen content of the leucocytes in this disease is paralleled by a decreased content in the thrombocytes as well, although this decrease is not as pronounced. I n other words, the thrombocytes show all the traits of being derived from cells which are quite close to the granulocytes in their chemistry and metabolism. This conclusion is in agreement with the findings of hematologists and cytologists. Unfortunately, the thrombocytes of patients with acute leukemia are still practically unobtainable in amounts sufficient for biochemical investigation and analysis. This limits the possibility of a biochemical comparison between normal and leukemia thrombocytes. Nevertheless, the results obtained have revealed some significant differences between the biochemical indexes of platelets from patients with chronic myeloid leukemia and those from normal subjects. Among these are a higher respiration, a lower content of ATP, a lower turnover rate of RNA phosphorus, a decreased glycogen content, and a significant increase in its turnover rate. It is interesting that in polycythemia, a hematological disease which is apparently not leukemic in nature even though it is also characterized by stimulation of the leucopoietic series in the bone marrow, all of these biochemical indexes are either completely identical with the corresponding indexes for normal platelets, or differ from them only insignificantly. 2. Role of Uridine Diphosphoglucose and Related Enzymes in Glycogen Synthesis by Thrombocytes

The significant abnormalities in the amount and turnover rate of glycogen in the thrombocytes of patients with chronic myeloid leukemia suggested that these shifts might be due to some type of alteration in the system for polysaccharide resynthesis. In the section devoted to the leucocytes, publications have already been cited which favor the uridine diphosphoglucose mechanism of glycogen synthesis in plant and animal tissues. At the present time, there is a sufficient basis for believing that the principal role of phosphorylase is the breakdown of glycogen, since polysaccharide synthesis is accomplished mainly by a system linked to UDPG (Stetten and Stetten, 1960; Strominger, 1960). In a study which was analogous to that carried out on the leucocytes, we determined the activity of the principal enzymes of the uridine diphosphoglucose pathway for glycogen synthesis in the thrombocytes: phospho-

381

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

glucomutase, UDPG-pyrophosphorylase, UDPG-glycogen transferase, and the relationship of hexokinase to the process. Glucose-6-phosphate dehydrogenase also interested us since the high activity of this enzyme, surpassing the activity of all other enzymes on a molar basis, permitted us to avoid its exogenous addition even though it was required for the enzymatic spectrophotometric analyses. Along with the enzymes mentioned above, we studied the phosphorylase activity of the thrombocytes, since this enzyme may determine both the content and the turnover rate of glycogen to a significant extent. The principles and procedures for determining the enzymatic activities in the platelets were the same as with the leucocytes. The enzyme preparations used were the extracts of platelet acetone powders (see Section 11, on leucocytes). Table XVII shows the results of our determinations of the activities of all of these enzymes, with the exception of UDPG-glycogen glycosyl transferase, in the extracts of thrombocytes from both heakhy donors and patients with polycythemia and chronic myeloid leukemia. TABLE XVII ACTIVITY OF CERTAINENZYMES IN ACETONE POWDER EXTRACTS OF THROMBOCYTES FROM HEALTHY DONORSAND PATIENTS WITH POLYCYTHEMIA A N D CHRONIC MYELOIDLEUKEMIA" Thrombocytes of

Glucose-6PO4 dehydrogenase

Hekokinase

Phosphogluconiiitase

UDPGpyrophosphorylase

61 49

16 20

50 48

11

13 13

55

16

44

15

12

Phosphorylase

~~

Healthy donors (11) Patients with polycythemia (8) Patients with chronic myeloid leukemia ( 5 )

13

a The enzymatic activity was determined a t 20°C. in the acetone powder extracts of thrombocytes in 0.05 M Tris buffer, pH 7.4. The activity is expressed in pmoles of substrate transformed per gram of extract protein per minute. The number of experiments is given in parentheses.

An examination of the data in Table XVII does not reveal any such shifts in enzymatic activity as might clarify the differences between normal and leukemic thrombocytes in regard to the content and metabolism of glycogen. For each single enzyme, the activities in the thrombocytes from normal and pathological blood are quite similar. Unfortunately, these differences between normal and leukemic thrombocytes in regard to the content and metabolism of glycogen were also not explained by the results of a study of UDPG-glycogen glycosyl transferase [see Eq. (15), Section

382

I. F. SEITZ

II,H,6,e], although it is precisely in the activity of this enzyme that there are significant differences between normal leucocytes and those from patients with chronic myeloid leukemia. It is true that the decreased UDPG-glycogen glycosyl transferase activity in the thrombocytes of patients with chronic myeloid leukemia is in agreement with the data on the lower glycogen content of the platelets in this disease, but this cannot explain the high turnover rate of glycogen. Table XVIII shows the average results from 12 experiments in which we determined the rate of transfer of radioactive glucose from UDPG-C14 to the primer glycogen in extracts of thrombocytes from various sources. TABLE XVIII TRANSFERASE AciwITY IN VARIOUSTYPESOF HUMAN UDPG-GLYCOGEN THROMBOCYTES ACCORDING TO D.4TA ON INCORPORATION O F GLUCOSE-C'~ FROM UDPG-C14 INTO GLYCOGEN^^^ Thrombocytes of

Activity of g1uc0se-C~~ transferred (pmoles/g. protein/minute)

Healthy donors (12) Patients with polycythemia (12) Patients with chronic myeloid leukemia (12)

8 . 1 k 0.6 7 . 0 k 0.6 5 . 6 f 0.4

a The number of experiments is given in parentheses. The activity is expressed in pmoles glucose transferred from UDPG-C14 to the glycogen primer. The activity was determined in acetone powder extracts of thrombocytes in 0.05 M Tris buffer, p H 7.4, a t 37°C. The medium also contained MgC12, cysteine, glucose-6-phosphate, glycogen primer, and 70 pg UDPG-C14with an activity of 6300 counts/minute. I, For details of the method, see Luganova and Seitz (1963a).

The activity data, expressed in bmoles of g1uc0se-C~~ transferred per minute per gram of protein in the extracts of the thrombocyte acetone powders, indicate approximately a 33% decrease in the transferase activity in the platelets of patients with chronic myeloid leukemia: from 8.1 to 5.6. The corresponding value for the thrombocytes of patients with polycythemia (7.0) occupies an intermediate position between the normal values and those for chronic myeloid leukemia. The synthesis of g1~cogen-C~~ from a more remote precursor, gluco~e-C~~, also took place in the extracts of thrombocyte acetone powders. In a reconstructed enzyme system containing extracts of thrombocyte acetone powders as a source of UDPG-glycogen transferase, glycogen synthesis proceeded according to Eqs. (12-15). The chain of enzymes transforming g1uc0se-C'~to UDPG-C14 was represented by yeast hexokinase, muscle phosphoglucomutase, yeast UDPG-pyrophosphorylase, and yeast inorganic pyrophosphatase. The latter played an indirect role in the formation

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

383

of UDPG by hydrolyzing the inorganic pyrophosphate formed as a n intermediate in the UDPG-pyrophosphorylase reaction, thus facilitating the accumulation of UDPG [see Eq. (14)]. ATP and UT P were also necessary components in the reaction (for more details, see the articles by Seitz et al., 1963, and by Luganova arid Seitz, 1963a,b). Under these conditions, there was rapid incorporation of glucose-(Y into glycogen via the intermediate formation of tJDPG-CI4. The rate of this process was comparable to that of the direct transfer of glucose residues in the experiments with UDPG-C'* described above. In samples lacking both UTP and UDPGpyrophosphorylase, the incorporation of g 1 ~ c o s e - Cinto ~ ~ glycogen was only 33-67y0 of that in the full enzyme system. This fact indicates that glycogen synthesis in thrombocyte extracts proceeds via UDPG. It should be pointed out, however, that the analogous synthesis of glycogen from g1uc0se-C~~ in extracts of leucocyte acetone powders (Seitz et al., 1963) was significantly more sensitive to the absence of TJTP and UDPG-pyrophosphorylase. The demonstration of UDPG-pyrophosphorylase and UDPG-glycogen transferase activity in the platelets favored the presence of UDPG in the thrombocytes as an essential chemical and metabolic component. This was also indicated by experiments on the synthesis of glycogen from UDPG-CI4. Nevertheless, it seemed desirable t o obtain direct evidence for the presence of this important coenzyme in the platelets, and if possible to determine it quantitatively. UDFG has now been isolated from thrombocytes which were, in turn, isolated from the blood of healthy donors as well as that from patients with chronic myeloid leukemia and polycythemia. The procedure was the same as in our work with leucocytes (see Section 11,I). Three comparable experiments were carried out with thrombocytes from healthy donors, patients with polycythemia, and patients with chronic myeloid leukemia. The results showed no significant differences in the UDPG content between normal platelets and those from patients with polycythemia, the value in these cases being approximately 20-25 p g . (i.e., much less than 0.05 pmole) of UDPG per ml. of fresh platelets. In chronic myeloid leukemia, the UDPG content in the thrombocytes was approximately 1.5 times as high. As we shall see, the UDPG content of the thrombocytes is close to that of the leucocytes.

E. OTHERENZYMATIC ACTIVITIES IN HUMAN THHOMBOCYTES During the last few years, human thrombocytes have been studied for the presence or absence of a great variety of enzymes, and particular attention has been paid to the enzymes of glycolysis and respiration, various phosphatases, transaminases, etc. Lohr et al. (1961) found that the

384

I. F. SEITZ

thrombocytes contain very high levels of triosephosphate isomerase, hexosephosphate isomerase, pyruvic kinase, lactic dehydrogenase, S-phosphoglycerate-l-kinase, and various other glycolytic enzymes. On the other hand, the activity of hexokinase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, alkaline phosphatase, and some other enzymes was low. The enzyme systems of oxidative metabolism generally showed low activity. The possible significance of the very high activities of pyruvate kinase (6540 Bucher units) and triosephosphate isomerase (15,080 units), for example, remains unclear in view of the very low activity of hexoltinase (244 units), the enzyme which leads the sugar into the metabolic cycle and thus limits the actual activity of all the enzymes taking part in the later stages of the metabolism of phosphorylated carbohydrate. Arroba and Lagunilla (1961) found that the thrombocytes contain glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactic dehydrogenase, p-glucuronidase, a very active acid phosphatase, and other enzymes. Italian authors have published a whole series of papers on the enzymes in the thrombocytes, especially in children. Schettini et al. (1960) detected rhodanase in the platelets and determined the activity of 6-phosphogluconate dehydrogenase (Schettini et al., 1961); Di Francesco et al. (1960a) studied isocitric dehydrogenase, triosephosphate dehydrogenase (Di Francesco et al., 1960b) and hexokinase (1961) in the lysates of thrombocytes from children; Berni et al. (1960) studied the phosphorylase activity in the platelets of children and obtained a value of 463.7 pmoles of glucose-lphosphate metabolized per 100 mg. of protein per minute; in 1961, the same authors found the pyruvate lrinase activity in the thrombocytes of children up to 10 years of age to be 27.7 Bucher units per lo9lysed platelets (1961a); they also studied the acid phosphatase and ATPase activity in lysed thrombocytes (1961b). Dale (1960) demonstrated significant aldolase activity in human thrombocytes. Morita and Asada (1956) detected monophosphatases, acid and alkaline pyrophosphatases, ATPase, ADPase, AMP-5-nucleotidase, esterases, and a specific cholinesterase in the thrombocytes. They were unable to detect proteolytic or peroxidase activity in the thrombocytes, and the catalase activity was weak In 1960, while studying cytolyzates of erythrocytes, leucocytes, and thrombocytes from patients with chronic lymphatic, paramyeloblastic, and acute leukemia, Griginani and Lohr found that all of these cells have the same type of hexokinase and that there are no differences between normal and pathological cellular elements. Notario and Nespoli (1961) and Notario et al. (1961) also studied the thrombocytes in chronic lymphatic,

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

385

chronic myeloid, and acute leukemia (hemocytoblastosis). These authors noted no abnormalities in the entire cycle of respiration and glycolysis, nor in the levels of ATP, ADP, AMP, lactic dehydrogenase, malic dehydrogenase, glucose-6-phosphate dehydrogenase, aminoferase, glutathione reductase, DPNH-cytochrome c reductase, and TPNH-cytochrome c reductase. Wurzel et al. (1961) found that the glucose-6-phosphate dehydrogenase activity in the thrombocytes is depressed in acute leukemia (from 150-350 units/lOg platelets normally to 25-85 units). Decreases in enzymatic activity in this disease were also observed in the erythrocytes and leucocytes. All of these data indicate the great variety and richness of the enzymatic composition of the platelets, which serve as indirect evidence for the metabolic integrity and functional complexity of the platelets in the animal organism. We have not dealt with unique enzyme systems, related to the specific physiological function of the platelets, but have examined only the most general metabolic processes and reactions, making up the chemical and energetic basis for all vital activities and functions. Even within the framework of such a limited acquaintance with the chemical dynamics of the platelets, we have obtained the impression that the thrombocytes are complex systems, from the structurochemical and enzymological points of view, which are highly dynamic in their functional and metabolic activity. We have become convinced, from what has been said above, that thc metabolism and chemistry of the thrombocytes are normally very similar, in many respects, t o the characteristic properties of leucocytes of the granulocytic type. However, the changes in specific processes and reactions during leukemic degeneration in the thrombocytes do not always correspond to those in the leucocytes. Thus, for example, respiration and glycolysis are depressed in the leucocytes during chronic myeloid leukemia, but are stimulated in the thrombocytes; the ATP content in the leucocytes increases somewhat during this disease, while that in the thrombocytes falls; the activity of the enzyme systems for glycogen synthesis (phosphoglucomutase and UDPG-pyrophosphorylase) in the leucocytes drops significantly during chronic myeloid leukemia, but this is not observed in the platelets. On the other hand, the glycogen content and the rate of its synthesis from glucose-Cl4 change in the same direction in the leucocytes and thrombocytes during chronic myeloid leukemia : the polysaccharide content decreases, but its turnover rate rises; the UDPG-glycogen transferase activity also drops in both the leucocytes and the platelets. All of this goes to show that no fundamental shifts in the chemistry or metabolism of the thrombocytes, which might shed light on the nature of the pathology in chronic myeloid leukemia or pol ycythemia, have yet been discovered.

386

I. F. SEITZ

IV. Bone Marrow

The biochemical literature on the human bone marrow is relatively sparse, and even in such a valuable review as the article by Lajtha (1957), a number of aspects of the chemical composition and metabolism of the myelokaryocytes are not touched on a t all due to the lack of the corresponding factual information. There are also many contradictions in the results of individual investigators, due probably to the heterogeneity of the cellular composition of the bone marrow, especially when it is obtained by aspiration. Nevertheless, it can be concluded from sources in the literature that one of the characteristics of normal human bone marrow is the presence of a high level of aerobic glycolysis and a low level of respiration (RamonovaTskhovrebova, 1948; Beck and Valentine, 1953; and others). As far as alterations in the metabolism of the bone marrow during leukemia are concerned, there are also contradictory data on this subject. RamonovaTskhovrebova (1948) found that the O2 absorption and aerobic glycolysis in the cells of the bone marrow are increased significantly during chronic myeloid leukemia. On the other hand, during a cytochemical study of the bone marrow during both acute and chronic leukemia, Terent’yeva et al. (1960) observed a sharp decrease in the activity of the enzymes of the oxidative pathway (cytochrome oxidase and o thers) in the hemocytoblasts and other cellular elements during acute leukemia and in all types of cells during severe forms of chronic, myeloid leukemia. At the same time, it is well known that young blast cells absorb oxygen quite rapidly and have hardly any glycolysis (Bird et al., 1950; Kempner, 1939). Finally, Lajtha (1957) states in summary that the younger elements of the bone marrow have a high Qo2and a glycolysis which increases with increasing differentiation. It should apparently be recognized that the type of metabolism of the bone marrow reflects its cellular composition, and that since the latter fluctuates sharply in normal bone marrow depending on the method and site of aspiration, and in leukemic bone marrow depending on the nature and severity of the disease, contradictory results in biochemical studies are unavoidable in the absence of strict standardization of the conditions of sampling and of the precise morphology of the samples under investigation.

A. ENERGY METABOLISM OF BONEMARROW IN NORMAL SUBJECTS AND LEUKEMIC PATIENTS Our primary interest in studying the bone marrow was to clarify why the leucocytes of the blood are divided into two metabolic types during acute leukemia. Since the opinion has been expressed that the leucocytes of the peripheral blood are degenerate, “dying” cells and t h a t the aerobic

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

387

glycolysis of the granulocytes is an expression of these processes, it seemed quite important to study the cells of the bone marrow-the ancestral substance for the cells of the peripheral blood. This study was carried out together with I. S. Luganova and with the assistance of hematologist L. M. Rozanova. Bone marrow samples (1.5-3 ml.) were taken from the sternum of healthy donors and patients with leulremia by aspiration, after which suspension was transferred from the syringe to a test tube containing a gelatine-citrate solution (Teodorovich, 1'358). In 20-30 minutes, the erythrocytes settled out, and the myelokaryocytes were then isolated from the supernatant plasma by fractional centrifugation. All of the manipulations were carried out in a siliconized vessel. The isolated cells were then suspended in a 1: 1 mixture of donor serum and Krebs-Ringer phosphate, pH 7.4, using Versene in a final concentration of 0.1% as the anticoagulant. Respiration, glycolysis, and glycogen metabolism were studied in intact fresh cells; the enzymatic reactions mere studied in extracts of acetone powders of the myelokaryocytes. The acetone powders were obtained by the same method as in the work on leucocytes (see Section 11,I). The methods of investigation were the same as those used in the study of leucocytes and thrombocytes. 1. Some Metabolic Characteristics of Normal Bone Marrow

I t should be pointed out first of all that the nucleated cells of the normal bone marrow are characterized by a relatively low rate of respiration, which decreases by approximately 20y0on addition of glucose (an inverse Pasteur reaction, or Crabtree effect), arid by high aerobic glycolysis. The anaerobic glycolysis of the myeloliaryocytcs is still higher. As shown in Table XIX, the respiration of the nucleated cells of the bone marrow (in the presence of glucose) corresponds to a Qu, of 4.7, while the aerobic glycolysis is given by Wdb2 = 13.6. Both of these values are somewhat lower than the corresponding indexes for donor leucocytes (Qol = 5.7; &"&,= 18.8). Nevertheless, the main qualitative characteristics of the metabolism of bone marrow and leucocytes (granulocytes) are the same: low respiration, high aerobic glycolysis, a distinct inverse Pasteur effect, and an incomplete Pasteur effect. I t is interesting that M. N. Blinov of our laboratory obtained the following metabolic indexes for the myelokaryocytes of cadaver bone marrow ( 2 4 hours after death): Qo, (in the presence of glucose) = 4.4; C&2 = 17.4; Q& = 33.5. These values indicate the relatively high state of preservation of the enzyme systems of respiration and glycolysis, as well as of the mechanisms of their coordination (the Pasteur effect and inverse Pasteur effect), in bone marrow several hours after death.

388

I. F. SEITZ

TABLE XIX RESPIRATION, GLYCOLYSIS, CONTENT, AND TURNOVER RATE OF GLYCOGEN IN NUCLEATED CELLSOF BONEMARROW A N D IN PERIPHERAL LEUCOCYTES OF HEALTHY DONORP Glycogen

QftCose Myelokaryocytes 4.7

Gair COI

Per ml. cells (mg.)

0.39 13.6 rt 1.05 3.36

(8)

(8)

(%)

k 0.13 1.7 f 0.07 (8)

+

Donorleucocytes 5.7 k 0.28 18.8 rt 1.04 6.1 0.55 (35) (20) (35) a

Dry wt.

(8) 3.5 f 0.31 (20)

Specific radioactivity (counts/ minute/mg.) 11170

(8) 4778 (10)

The number of experiments is given in parentheses. The anticoagulant was Versene

(O.lyo). The values for the specific radioactivity of the leucocyte glycogen were also

obtained in experiments with this anticoagulant.

Table XIX also shows the results of a study of the amount and turnover rate of glycogen in the cells of donor bone marrow. The lower polysaccharide content (by approximately SOq;’,) in the bone marrow but the significantly higher turnover rate in experiments with uniformly labeled g1uc0se-C~~ (specific radioactivity of 11,170 counts/minute/mg. compared to 4778 in donor leucocytes) is noteworthy. According to the data of M. N. Blinov, the glycogen content in cadaver bone marrow ( 2 4 hours after death) amounts t o 3481 pg./lOg cells, which is no lower than in donor bone marrow. The turnover rate of the glucose component of glycogen is significantly lower in cadaver bone marrow, however, than in the normal (specific radioactivity of 4148 and 11,170 counts/minute/mg., respectively). 2. Energy Metabolism of Bone Marrow in Patients with Acute Leukemia

Interpretation of the results of biochemical studies of the bone marrow, especially in healthy subjects, is fraught with a number of difficulties, since the cellular composition of the normal bone marrow, like that of the peripheral blood but to an even greater degree, is nonhomogeneous. It is therefore not always possible to decide what type of cells was responsible for the observed metabolic or chemical indexes. At the same time, we do not yet have any methods for the individual isolation of all, or even the principal, cellular elements which go to make u p the bone marrow. Our task is made significantly easier when the bone marrow is more homogeneous, and especially when it is dominated by a single morphological type. This is often precisely the case in acute leukemia. In these cases, the majority of the cells in the bone marrow, as well as in the peripheral blood,

T.1BLE XX RESPIRATION, GLYCOLYSIS, CONTENTA N D METIBOLISMO F GLTCOGEN IN NUCLE~TE CELLS D OF BONEMIRROWAND LEUCOCYTES OF PATIENTS W T H ACTTTELETTKEMI \a Glycolysis of lactic acid in myelokaryocytes

No. of experiment

O? absorption (~1.)

h a .J

hnaerohic

435 525 770 560

540 630 910 840

PERIPHERAL

Glycolysis of lactic acid in peripheral leucocytes Glycogen

~

Aerobic

IN

PP.

SRb

O? absorption (PI.)

(PP.1

Aerobic

Glycogen Anaero- -~ bic Pg. SRb

Acute leukemia, metabolic type I 1 C

2= 3d

4d

30 40 62 44

60 40 80 60

13113 8000 24962 31466

56 52 38 42

490 455 665 700

59<5 875 875 945

60 60 140 60

10717 7500 9800 15700 P

Acute leukemia, metabolic type I1 5 6* 7/

28 30 41

0 0 0

680 480 620

40 40 60

4800 6350 2250

34 36 -

0 0

630 400

-

-

40 80 -

5300 6540 -

a Cells (0.1 ml.) in each vessel incubated for 30 minutes a t 37°C. in a 1: 1mixture of serum and Krebs-Ringer phosphate in the presence of 5 mg. g1uc0se-C~~ (30,000 counts/minute). SR = specific radioactivity (counts/minute/mg.). c In experiments 1 and 2, myeloblasts predominated among the leucocytes and myelokaryocytes (acute myeloblastic leukemia). d I n experiments 3 and 4, reticuloendothelial cells predominated (acute reticuloendotheliosis). In experiment 5 , lymphoblasts and lymphocytes predominated (acute lymphoblastic leukemia). f In experiment 6, plasma cells predominated (acute plasma cell leukemia). In experiment 7, hemocytoblasts predominated.

3

cu

00 (D

390

I. F. SEITZ

are representatives of a single type which, since it is in a n absolute majority, determines the type of metabolism or the perculiarities in the chemical composition. Table XX shows the results of a study of the leucocytes in the peripheral blood and the nucleated cells in the bone marrow of several patients with acute leukemia, the blood and bone marrow of whom were subjected to a detailed morphological study. The special feature of these experiments was that the leucocytes and myelokaryocytes used in each individual experiment were taken from the same patient. The preliminary results of this work were published earlier (Luganova et al., 1962), but now the experimental data are on a significantly broader base. We studied a total of 18 cases of acute leukemia (myeloblastosis, reticulosis, reticuloendotheliosis, plasma cell leukemia, and acute lymphatic leukemia). Unfortunately, the most surprising result of our study of the respiration and glycolysis of cells of the blood and bone marrow in the same patient was the detection of the two metabolic types with which we were already familiar and about which we wrote earlier, with obligatory correspondence between the leucocytes of the peripheral blood and the myelokaryocytes in the predominance of the first or second type of metabolism. In one group of patients (see Table XX), characterized by a predominance of myeloblasts and reticuloendothelial cells (acute myeloblastosis, reticuloendotheliosis), both the leucocytes isolated from the blood and myelokaryocytes isolated from the bone marrow accumulated lactic acid in the presence of atmospheric oxygen, despite the high level of respiration, On the other hand, Table XX shows the results of three experiments in which the nucleated elements of the bone marrow and the leucocytes of the peripheral blood both showed another type of metabolism. When the cells of these patients (acute lymphatic leukemia and acute plasma cell leukemia) were incubated in vitro in a 1: 1 mixture of serum and Krebs-Ringer phosphate in the presence of glucose, there was no aerobic glycolysis and the cells were characterized by a purely oxidative metabolism. The dominant cells in the blood and bone marrow of these patients were lymphocytes, lymphoblasts, or plasma cells (the latter in the case of plasma cell leukemia). The results obtained in the study of normal leucocytes and normal bone marrow, as well as the agreement between the metabolism of the peripheral leucocytes and that of the myelokaryocytes in patients with acute leukemia, enable us to draw several important conclusions. First of all, the aerobic glycolysis of the leucocytes of the myeloid series is not the result of their “aging,” degenerating, or “death” in the blood stream, but is a n original property of these cells, characteristic of their nature, by which analogous cells can also be distinguished in the bone marrow; second, the metabolic characteristics of the bone marrow are not invariable, but depend on the

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

391

nature of those cellular elements which acquire a dominant position as the result of the nature and peculiarities of the pathological process. There are two possible metabolic types among both the cells of the bone marrow and the peripheral leucocytes, in agreement with the two categories (myeloid and lymphoid) of cells in the hematopoietic system, which differ according to their origin. Each of these groups is internally heterogeneous, morphologically speaking, and includes various cellular elements. Metabolically speaking, however, all of these cells can be divided into two clearly distinguishable types : type I-cells with a mixed metabolism (coexistence of respiration and glycolysis); and type II-cells with a purely oxidative metabolism. These data also enable us t o explain the contradictions in the literature concerning the type of metabolism of the leucocytes and mylokaryocytes, especially the young forms, in various pathological conditions. I n those cases in which the authors studied acute leukemia of the lymphatic type, no aerobic glycolysis was found, but when they used cells from patients with acute myeloid leukemia, or undifferentiated, young cells of related origin, high aerobic glycolysis was detected. Finally, it may be possible to conclude that the low respiration, high aerobic glycolysis, and very high anaerobic glycolysis are not a property of leukemic cells as such, but are simply an expression of their origin and of the fact that they belong to a particular type of tissue. Thus, completely normal leucocytes or myeloltaryocytes have high glycolytic activity under both aerobic and anaerobic conditions, along with comparatively low respiration. On the other hand, the typically neoplastic cells of a number of patients with acute leukemia have a purely oxidative metabolism, without any aerobic glycolysis and with a high oxygen consumption. This property is valid for the entire ontogenetic series, from the youngest primeval cells to the oldest, and depends more on the genesis of the cell than on its age. Tables XIX and XX also show data on the glycogen content in the myeloltaryocytes and on its rate of turnover at the expense of g1uc0se-C~~ in the medium. It is obvious from the results presented that acute leukemia, whenever it is represented by a homogeneous undifferentiated cellular population in either the bone marrow or the periphery, is characterized by a sharp drop in the glycogen content regardless of the type of leukemia involved. The amount of this polysaccharide in the myelokaryocytes in acute leukemia is so small that doubt even arises whether the young, undifferentiated cells of the bone marrow really contain any glycogen at all, or whether we are determining the glycogen which has been introduced into the suspension along with the small number of more mature cells. However, the above-normal specific radioactivity of the polysaccharide in the myelokaryocytes of patients with acute leukemia of metabolic type I, and

392

I. F. SEITZ

the subnormal specific radioactivity of glycogen in the myelokaryocytes of patients with acute leukemia of metabolic type I1 indicate that we are dealing with the glycogen of highly specific cells with altered metabolic activity, rather than with the glycogen of the usual cell. The average specific radioactivity of glycogen (10 experiments) in the cells of group I (with aerobic glycolysis) was 27,700 counts/minute/mg. ;the corresponding value for 8 experiments with nucleated myelokaryocytes of type I1 was 5,200 (as shown in Table XIX, the normal is 11,170). These values are another indication of the significant difference between cells of different genetic series. It should be noted that the specific radioactivity of leucocyte glycogen in experiments with glucose-C14 reached 16,128 counts/minute/ mg. in chronic myeloid leukemia, compared to 1,359 in chronic lymphatic leukemia (see Table VII). It should be pointed out that the morphological identification of the young, undifferentiated cells was extremely difficult in a number of cases, and sometimes even questionable. Consequently, only the fact that all of the undifferentiated myelokaryocytes in acute leukemia can be divided into two metabolic types should be considered absolutely reliable, and not the classification of a particular morphological species as belonging to one type or the other. This classification is fraught with possible error and requires further precision. It is difficult to interpret the results of the study of glycogen in the bone marrow in acute leukemia consistently, or to ascribe the decreased polysaccharide content or change in its turnover rate specifically to the leukemic process, since analyses of the corresponding normal cells are lacking. The bone marrow in leukemia, especially acute leukemia, is completely different from that in the normal subject. Nevertheless, if we look upon the cellular composition of the bone marrow and peripheral blood in this disease as a n expression of leukemic degeneration, for which there is considerable basis, and if we consider the cells themselves as one of the concrete carriers of the pathology, then the decreased glycogen content and change in its turnover rate can be included among the symptoms of leukemia. It is true that these changes in the glycogen of the leucocytes and myelokaryocytes during leukemia can always be ascribed to a nonspecific rejuvenation of the cellular composition. It has been confirmed by numerous cytochemical studies that the younger cells of the white series contain less glycogen than the mature leucocytes. In the light of these data, the question of the specificity of the changes in the glycogen of the blood cells during leukemia is still impossible to answer with finality, although such specificity is favored, for example, by the changes in the turnover rate of glycogen in the leucocytes and myelokaryocytes during acute leukemia.

393

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

B. ENZYME SYSTEMS PARTICIPATING IN GLYCOGEN SYNTHESIS IN NORMAL AND LEUKEMIC BONEMARROW The nucleated cells of the bone marrow of healthy donors and patients with acute leukemia were used for a study of the activity of the enzymes which take part in glycogen synthesis : phosphoglucomutase, UDPGpyrophosphorylase and UDPG-glycogen glycosyl transferase. The same methods and preparations were used in this work as in the analogous study of leucocytes and thrombocytes (see above). The enzymatic activity was determined in extracts of acetone powders of the isolated myelokaryocytes. Table X X I shows the over-all results of this study. Along with the enzymes listed above, the table shows the results of a determination of the activity of glucose-6-phosphate dehydrogenase, in which we were also interested. The values in Table XXI indicate a significant drop in enzymatic activity in the myelokaryocytes of patients with acute leukemia, especially those of metabolic type 11. It is also interesting to note that in normal man, the activity of all of the investigated enzymes involved in glycogen synTABLE XXI PHOSPHOGLUCOMUTASE, UDPG-PYROPIIOSPHORYLASE, UDPG-GLYCOGEN GLYCOSYL TR4NSFERASE .ZND GLUCOSE-6-PltOSPH.\TE L)EHYI)ROGEN 4SE ACTIVITY~ IN NUCLEATED CELLSOF BONEMARROW OF H E ~ L T I IDONORS Y AND PATIENTS WITH ACUTE I J E V K E M I . ~ ~ ~

~~

Myelokaryocytes from

Glucose-6-POc Phosphogluco- UDFG-pyro- UDFG-glycogen dehydrogenase mutase phosphorylase transferase

Healthy donors (4) Patients with acute leukemia Type I(4) Type II(4)

96

78

31

3.0

02 20

45 11

27 7

2.0 1.1

~~

Activity is expressed in pmoles/g. of myelokaryocyte extract protein/minute. b Details of the method were as in the work with leucocytes. The number of experiments are given in parentheses. 0

thesis is lower in the myelokaryocytes than in the leucocytes. At the same time, the amount of glycogen in the leucocytes is approximately twice as high as in the nucleated cells of the bone marrow. These facts are apparently related to the varying degree of maturity of the cellular elements in the peripheral blood and in the bone marrow.

394

I. F. SEITZ

OF THE BONEMARROW C. SOMEOTHERDATA ON THE BIOCHEMISTRY OF THE LEUKEMIC PROCESS WHICHARE CHARACTERISTIC

According to the data of Davidson et al. (1948, 1951) a normal myelokaryocyte contains 9 x mg. of DNA phosphorus and 7 X loF7mg. of RNA phosphorus (see also Leslie, 1955). Menten and Willms (1953) found an average of 0.68 x mg. of DNA phosphorus per myelokaryocyte in lymphatic leukemia and 0.82 x loF9 mg. in myeloid leukemia. The RNA :DNA ratio fluctuated between 0.3 and 0.85. Somewhat earlier, mg. of nucleic acid Meiiten (1952) reported a value of 0.424.5 X phosphorus per cell for leukemic bone marrow. White et al. (1953) found that, within the limits of each series studied, the average content of DNA phosphorus per cell was highly constant for a broad range of different types of bone marrow cells. Using cadaver bone marrow, Libinzon (1961) showed that the cells contain 2.21 mg./g. of total phosphorus, 0.481 mg./g. DNA phosphorus and 0.258 mg./g. RNA phosphorus for 2-20 hours after death, with a DNA:RNA ratio of 1.95. Lajtha (1957) noted that the RNA content of the bone marrow decreases during cellular differentiation. Gurbaiiov (1961) studied the dynamics of the quantitative changes in RNA and free nucleotides in human myeloltaryocytes during the process of their differentiation and maturation. He showed that during maturation of the granulocytes from myeloblasts to daughter myelocytes, the RNA content decreases approximately 8-fold (from 7.9 to 1.048 X 10-l2 g.) and the nucleotide content decreases 7-fold. However, the metamyelocytes showed approximately 54% more RNA and 15% more nucleotides than the daughter myelocytes. Lajtha et al. (1954) showed that when bone marrow is incubated with P32and adenine-C14, the highest labeling is found in the nuclear RNA and there is much less incorporation into the cytoplasm. According to their data, the activity in RNA is especially high in young forms and is practically absent in mature granulocytes. The cells of the bone marrow actively incorporated methionine4Y5 into protein; the younger the cell, the more vigorously. The blasts of the human bone marrow incorporated S%ulfate less actively than the promyelocytes or myelocytes (cited by Lajtha, 1957). The incorporation of labeled amino acids into healthy human bone marrow decreases with increasing maturity of the granulocytes (Gavosto et al., 1960). Autoradiographic studies reported by Boll in 1957 showed that on incubation with P32,the cells of the normal human bone marrow or bone marrow from patients with chronic myeloid leukemia have a more active phosphorus metabolism than the bone marrow from patients with chronic lymphatic leukemia or plasmocytoma. The proliferative activity of the bone

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395

marrow was confirmed by incorporation of P32into the nuclei of young immature cells in vitro. Kozinets (1962) as well as Kazanova and Kozinets (1963) observed that when myelokaryocytes from patients with acute leukemia (hemocytoblastosis) were incubated with glycine-C14,intensive labeling appeared only in the hemocytoblasts. No label could be detected in the older cells, indicating a n absence of maturation. In addition, the labeling in the hemocytoblasts was usually subnormal and the cells died more rapidly than usual. I n chronic myeloid leukemia, on the other hand, the percentage of labeled hematopoietic cells differed little from the normal. These authors therefore concluded that nucleoprotein metabolism is disrupted in leukemic cells. It must be admitted that our understanding of the chemistry and metabolism of the human bone marrow is very limited, even in the normal subject, not to mention the various pathological states. To a great degree, this is apparently due to the difficulty in obtaining the material, especially in some forms of leukemia in which the bone marrow is very poor in nucleated elements. Further work in this field is certainly desirable. V. Discussion a n d Conclusions

A. AEIKJBICGLYCOLYSIS The problem of aerobic glycolysis, being a component part of the problem of the Pasteur effect, and as it were the opposite side of the coin of this problem, goes beyond the bounds of pathology and acquires general biological significance. Aerobic glycolysis may not only be produced artificially, as the result of some experimental influences (uncoupling agents, damage to the cell, and so forth), but is also a natural attribute of the metabolism of a series of quite different cells. At the present time, we can already list many tissues and cells of the animal organism which have the ability to form lactic acid from sugar in the presence of air. This list would include mammalian erythrocytes, leucocytes, thrombocytes, bone marrow, reticulocytes (Mikhnovich and Seitz, 1959; Seitz, 196l), retina, intestinal mucosa, tonsillar tissue, and apparently the brain, spermatozoa, muscles in a state of normal physiological activity, skin, and possibly still other tissues. Aerobic glycolysis, which is superimposed on respiration under the normal physiological conditions of existence, emerges more and more clearly as a valuable metabolic acquisition, developed during the process of evolution in cells bearing a heavy functional burden and periodically forced to carry on their vital activity under conditions of a limited oxygen supply or oxygen debt. It is true that some unclear aspects remain in the interpretation of the processes connected with aerobic glycolysis. Thus, it is clear from all

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the data presented above that aerobic glycolysis is connected with an inverse Pasteur effect. If the biological significance of the direct Pasteur effect is assumed t o be that the blocking of glycolysis and transition t o a purely or primarily oxidative metabolism represents a more economical utilization of the substrates, then it is difficult to see any possible advantage in the inverse Pasteur effect, in which glycolysis inhibits respiration. Nevertheless, the biological role of aerobic glycolysis itself is obvious. Since in all of the cells that we studied in which the aerobic formation of lactic acid was detected, the respiratory enzyme systems were unsuitable for the broad utilization of glucose as a substrate of oxidation, the possibility of a parallel utilization of this substrate for the resynthesis of ATP by the glycolytic pathway must be looked upon as a favorable adaptation. The more intense the activity, the more intense must be the metabolism, and the more need there is for the involvement of an additional substrate and a related mechanism for the generation of the energy of ATP. It should also not be forgotten that in some of the cells mentioned above (erythrocytes, thrombocytes), aerobic glycolysis is, to some extent, the result of extreme specializationthe evolutionary development of structural and morphological dedifferentiation and structurochemical simplification, as expressed in the partial loss of the mitochondria and even the nucleus, with the corresponding changes in metabolism. It is well known that dedifferentiation also takes place in cancer cells. It might be suggested that aerobic glycolysis is brought about by the functional requirements of the cells, and that dedifferentiation is a secondary phenomenon developing on this new functional basis, since glycolysis does not require those structural elements of the cell with which respiration is connected. The familiar atrophy of the enzymatic and structural apparatus of respiration naturally facilitates the appearance of aerobic glycolysis. It should be clearly understood that the transition from a purely oxidative to a mixed metabolism with active aerobic glycolysis does not give the cell any advantages in the sense of greater economy. On the contrary, the yield of energy per unit of substrate consumed decreases sharply. Consequently, the significance of this rearrangement of cellular metabolism must lie in another direction. It is appropriate to ask whether the significance of this rearrangement may not lie in the transition to another metabolic substrate and the consequent metabolic involvement of compounds which do not play a role (or play a very limited role) inoxidative metabolism, especially of the carbohydrates. As we have seen, the anabolic utilization of the intermediates and end products of aerobic glycolysis in the cells is insignificant. We must therefore think primarily of the energetic significance

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of this metabolic rearrangement, which makes possible the broad utilization of an exogenous nutrient material that enters the cell in excess from the surrounding environment. If we consider what amounts of endogenous substrates a cancer cell would require, for example, in order t o replace the hydrolyzable sugar and still guarantee the characteristic level and intensity of resynthesis of ATP, knowing that a cancer cell hydrolyzes a n amount of sugar equal t o its own weight in 4-5 hours, then it becomes easy to understand the significance of the involvement of an exogenous substrate, glucose, in energy metabolism. This is also true of the leucocytes. If we consider all of the consequences of the fact that respiration and glycolysis basically involve the utilization of different substances and that these two process are separated in the cell not only structurally but also chemically, in the sense of utilizing different substrates, then many of the phenomena of cellular energy metabolism (and particularly both the Pasteur effect and the inverse Pasteur effect) will appear in a new light, and their mechanisms will become more understandable. The concept of oxidative phosphorylation as the basis of the Pasteur effect is in complete agreement with these facts (Seitz, 1955, 1961).

B. AEROBICGLYCOLYSIS AND CANCER Special significance in the biology of neoplastic growth has long been ascribed to the mixed nature of metabolism in cancer cells. The low level of respiration, high glycolysis, and the presence of aerobic glycolysis are looked upon as characteristic features of the metabolism of malignant tumors. One of the most interesting biochemical concepts of the origin and nature of neoplastic growth, the Warburg theory, was built up on this foundation. At the present time, we are significantly more able to evaluate this point of view than we were just a short time ago. The crucial point in the Warburg theory on the origin of malignant tumors is the hypothesis of damaged respiration. I n Warburg’s opinion, the development of an active glycolytic pathway and the appearance of aerobic glycolysis are the result of a developing respiratory insufficiency-quantitative as well as possibly qualitative (uncoupling). According to Warburg, as these phenomena develop, they initiate the process of malignant degeneration. The experimental data collected in this paper show clearly, however, that this basic position of Warburg does not capture the essence of malignant metabolism. This is convincingly demonstrated by the data in Table XXII. The first listings in Table X X I I are human carcinoma and sarcoma, the metabolic indexes of which can be considered classical for human malignant tumors (the initial data of Warburg are presented without correcting for

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I. F. SEITZ

“contamination” by noncancerous material). It is clear that neoplastic cells are indeed characterized by a comparatively low level of respiration, very high anaerobic glycolysis, and high aerobic glycolysis. Warburg emphasizes that the respiration of cancer cells is low not only in an absolute but also in a relative sense, in comparison with glycolysis. For this reason, he ascribes particular significance to the quantitative relationship between glycolysis and respiration, which can best be expressed by the coefficients Qgb2:QO, and QE&:Qo2.According to Warburg, the ratio of aerobic glycolysis to respiration does not exceed 1.0 for normal tissues, while for neoplasms it equals 3 4 . The ratio of anaerobic glycolysis to respiration is even higher in malignant cells. We shall see that this is the case for these indexes in human carcinoma and sarcoma. In 1957, however, an interesting paper appeared by Eschbach in which he showed that both the so-called superficial carcinoma of the uterine cervix and the cornifying carcinoma of the uterine cervix had indexes typical of nonmalignant metabolism (0.9 for the ratio between aerobic glycolysis and respiration). Roetth (1957) was also unable to demonstrate any relationship between the degree of malignancy and the value of aerobic glycolysis for cancer of the uterine cervix. In tissue cultures, according to the data of Phillips and McCarthy (1956), the cells of epidermal carcinoma of the uterine cervix (the so-called HeLa cells) show a paradoxical relationship between respiration and glycolysis: anaerobic glycolysis in these cells is less than one half as active as aerobic glycolysis, so that the QEb, :Qo, ratio is very low (0.9) while the QFo2:Qo, ratio occupies an intermediate position between the corresponding values for normal and malignant tissue (2.0). The metabolism of the leucocytes and some other cells of the hematopoietic system is particularly interesting from this point of view. Here we expected a great surprise. As can be seen from Table XXII, the metabolic indexes of the leucocytes from healthy donors were practically identical to the corresponding values obtained by Warburg for human sarcoma, and comparable to those for carcinoma. As far as the QE&:Qoz and Q&:Qo2 ratios are concerned, they were even more “cancerous” for leucocytes than for human carcinoma. Human thrombocytes and myelokaryocytes were also studied from this point of view, and also revealed ratios between respiration and glycolysis which are typical, according to Warburg, for neoplastic tissues (see Table XXII). Thus, on the basis of the Warburg theory, we would have to include normal leucocytes, the leucocytes of patients with polycythemia or chronic myeloid leukemia, and the thrombocytes and myeloliaryocytes of healthy donors among the neoplastic cells, while excluding the two carcinomas of the uterine cervix studied by Eschbach, as well as the leucocytes and myelokaryocytes of patients with acute leukemia (metabolic type 11).

TABLE X X I I AND THEIRRELATIVEMAGNITUDES I N HUMAN CANCER CELLS, LEUCOCYTES, THROMBOCYTES, AND RESPIRATION, GLTCOLYSIS, BONEMARROW

Q?b2

5.1 4.9 7.6

14.0 15.6 7.1

21.0 27.9 22.2

3.1 3.2 0.9

4.1 5.7 2.9

Warburg (1926) Warburg (1926) Eschbach (1957)

4.5

3.9

13.1

0.9

2.9

Eschbach (1957)

6.0

12.0

5.5

2.0

0.9

Phillips and McCarthy (1956) Luganova et al. (1957a,b,c) Luganova et al. (1957a,b,c)

QOZQ

Carcinoma Sarcoma Carcinoms of uterine cervical epithelium (socalled superficial) Carcinoma of the uterine cervix (cornifying squamous cell epithelium) HeLa (tissue culture of uterine cervical carcinoma) Leucocytes, donor Leucocytes, chronic myeloid leukemia Leucocytes, acute leukemia: Type 1 Type I1 Thrombocytes, donor Bone marrow, donor Bone marrow, cadaver

QcOb,

Q&,

Cells

:Qo,

5.7 5.0

18.8 13.2

31.1 23.3

3.3 2.6

5.5 4.7

10.0 8.3 3.6 4.7 4 4

0 24.0 12.6 13.6 17.4

27.3 29.0 19.3

0 2.9 3.6 2.9 4.0

2.7 3.5 5.4

-

33.5

References

:Qo2

-

7.6

Luganova et al. (1957a,b,c) Luganova et al. (1957a,b,c) Luganova et al. (1958a,b) Luganova et al. (1958a,b) M. N. Blinov (1964)

W 0 0

z

E5

3cc 0 q

3td

F

l?

z

U

zw r M

z z

d H F

r m

~

a

I n the presence of glucose.

w

co co

400

I. F. SEITZ

A comparison of the metabolism of human carcinoma and sarcoma with that of some normal cells thus demonstrates that the values obtained by Warburg for the respiration and glycolysis of neoplasms are not specific for malignant cells. The same type of metabolism is found in normal granulocytes, thrombocytes, bone marrow, and possibly other cells. It can therefore be concluded that the Warburg theory on the inadequacy of respiration in cancer cells and the resultant changes in glycolytic activity are not confirmed experimentally, if the question is considered from a purely quantitative point of view. In a number of cases, the respiration of cancer cells is impaired neither absolutely nor relatively (compared to either aerobic or anaerobic glycolysis), and is in no way inferior to the respiration of some normal cells. It is also impossible to demonstrate any specific qualitative changes in the respiration of cancer cells, a t least on the basis of the date available so far. Our studies on the resynthesis of ATP in ascites carcinoma cells (Seitz, 1961), as well as the work of other authors, have shown that there is also no basis for the second hypothesis of Warburg: that respiration is qualitatively inadequate in malignant tumors. There is no uncoupling of respiration and phosphorylation in ascites carcinoma cells. The respiration of these cells is also effective in the anabolic sense. Thus, C14-labeled fragments arising from the oxidation of uniformly labeled glucose-CI4 and 1 a ~ t a t e - Care ~ ~ assimilated into the proteins and nucleic acids of cancer cells, as well as the leucocytes of patients wibh acute leukemia, and the effectiveness of this assimilation is higher than, for example, in the normal leucocytes of healthy donors. Consequently, the respiration of neoplastic cells is not only quantitatively but also qualitatively adequate, and from the points of view of both catabolism and anabolism. The results of these studies can only be interpreted to mean that neither aerobic glycolysis, the inverse Pasteur effect, nor the ability t o resynthesize important intracellular compounds such as ATP, glycogen, and others effectively under both aerobic and anaerobic conditions, can be considered specific for malignant cells, and that there is no theoretical relationship between these metabolic features and malignancy. Consequently, the question of the relationship between aerobic glycolysis and growth, including malignant growth, should be reexamined. This is especially necessary since a study of the role of aerobic glycolysis in anabolism has demonstrated that this process plays an insignificant role in the general assimilation and growth of cancer cells and leucocytes. The assimilation of C14-fragments arising during the metabolic transformations of g1uc0se-C'~is able to explain only a very Emall proportion of the metabolism and growth rate characteristic of these cells. Apparently, the basic biological significance of aerobic glycolysis is still as a source of energy.

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401

C. AEROBICGLYCOLYSIS AND FUNCTIONAL “STRESS” If the aerobic glycolysis in a particular group of cells is looked upon not as the result of pathological changes but as the result of increased functional activity (the synthesis of new cellular substance in cancer cells, phagocytosis, and ameboid movement in leucocytes, etc.) which has become adapted t o a possible deterioration of the oxygen supply, then the general features of the coordination between respiration and glycolysis in cancer cells and certain normal cells can be explained on the basis of normal instead of pathological physiology. It is possible that the true primary regulator of metabolism is the physiological activity itself, even though this rests, in turn, on a chemical basis and is regulated by influences from the body as a whole. The most powerful stimulus for the resynthesis of AT P at the cellular level is an increase in its utilization. The very physiological activity which accompanies a significant expenditure of high-energy phosphate compounds is the most powerful stimulator and regulator of substrate catabolism. In the case of particularly high functional stress, ATP will be degraded more rapidly than it is resynthesized, and a single mechanism for the mobilization of energy may become insufficient for full saturation of the adenylic acceptor, thus creating the possibility of involving (in aerobic cells) or stimulating (in cells with a mixed metabolism) the process of glycolysis. A deterioration in the oxygen supply may thus only facilitate this process of metabolic rearrangement. At the cellular level, the principal factors determining the concentration of phosphate acceptors and, consequently, the ratio between respiration and glycolysis in the presence of a normal supply of nutrient materials are the conditions of aeration and the physiological activity being carried out. Extracellular influeiices, particularly those of humoral and nervous origin, may complicate but do not alter the basic nature of the cellular mechanisms for the regulation of metabolism. The basic effect of such extracellular influences on the cell is to intensify or weaken the genetically determined, elemental, intracellular processes. The more complex and highly developed the organism, the more specialized and less self-sufficient will be its component cells, and the stronger the relationship between intracellular reactions and extracellular factors. Neoplastic cells depart from this principle of the control and regulation of normal tissues mainly in a biological sense: their proliferation and expansion are riot correlated with the functions and vital activity of other systems of the body. From the metabolic point of view, however, this outstanding characteristic of neoplastic tissues is not expressed in any unique or regular way. As we have become convinced by a n examination of all of the material presented above, the basic qualitative characteristics

402

I. F. SEITZ

of metabolism (respiration, glycolysis, their relative magnitudes, etc.) can vary in opposite directions within the limits of a single neoplastic process (leukemia). The metabolic features which often accompany malignant growth can also be demonstrated in normal, functionally complete, cells such as leucocytes, thrombocytes, and myelolcaryocytes. On the other hand, the purely oxidative metabolism which is characteristic of most animal tissues is also found in the undifferentiated leucocytes of approximately half of all patients with acute leukemia, and in the lymphocytes of patients with chronic lymphadenosis. We have obtained the impression that there may be no generally demonstrable qualitative differences between malignant and normal cells in the area of energy metabolism. At best, we may have to be satisfied with constant shifts of a quantitative character. Since the principal function of neoplastic cells is t o reproduce themselves, and since the nucleoproteins are the chemical substrate of this process, it is in this category of substances that one might most justifiably expect to detect changes during leukemia (or cancerous) degeneration, from both the chemical and metabolic points of view. All other processes, including those of catabolism, no matter how significant they are for the vital activity of the cells, are subordinated to the basic function of increasing the cellular mass and cell division. For this reason, the appearance of qualitative changes in those processes which are most common to the functional mechanisms of the whole organism is less probable. Of course, the process of reproduction is also a natural activity in normal cells, although there it is subordinated to the regulatory influences of the body. I n view of the fact that the process of reproduction of malignant cells is not anything basically new, or anything not also found in normal cells, but has merely acquired an uncontrolled character, all of the auxiliary types of metabolism and biochemical mechanisms of the cell may also remain unchanged, a t least qualitatively, to the extent that they support a process which is also characteristic of normal cells. It might therefore be expected that decisive advances in the clarification of the nature of neoplastic growth are most likely to come from the study of the composition, structure, and metabolism of the nucleic acids. This would be the logical culmination of a long series of efiorts, and a triumph for that point of view which considers changes in the genetic material (in the contemporary chemical meaning of that term) to be the primary stimulus toward leukemic or cancerous degeneration. VI. Summary

The characteristics of the chemistry and metabolism of' the cells of the hematopoietic system can be summarized briefly as follows. All of the cells in the blood and bone marrow can be divided into two groups: (a) cells

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

403

having aerobic glycolysis, and ( b ) cells not having aerobic glycolysis. The first group includes the granulocytes of normal blood as well as the blood of patients with chronic myeloid leukemia and polycythemia, the undifferentiated leucocytes of patients with acute leukemia of metabolic type I, the thrombocytes, and the myelokaryocytes of normal subjects and of patients with acute leukemia of metabolic type I. The second group includes the lymphocytes and the undifferentiated cells of the blood and bone marrow in patients with acute leukemia of metabolic type 11. With the exception of the young, uiidifferetitiatd cells of the white series, all of these formed elements are distinguished by a relatively low level of respiration, carried on principally at the expense of a substrate other than glucose. All of the leucocytes of the peripheral blood, the blood platelets, and the nucleated cells of the bone marrow show very high anaerobic glycolysis. The cells of the blood and bone marrow which have aerobic glycolysis are also characterized by the presence of a pronounced inverse Pasteur effect. Another quite characteristic property of the peripheral leucocytes, thrombocytes, and myelokaryocytes is their ability t o maintain normal levels of the most important intracellular compounds (ATP, glycogen, nucleic acids, etc.) under anaerobic conditions. This metabolic property of the cells of the hematopoietic system guarantees their adequate functional activity under both aerobic and anaerobic conditions. The glycogen content in terms of dry cell weight is about 3.5y0for the peripheral leucocytes of healthy donors, 1.5% for the thrombocytes, and 1.7% for the nucleated cells of the bone marrow. In the young, undifferentiated cells of the peripheral blood and bone marrow during acute leukemia, there is a sharp drop in the glycogen content. This decrease in the polysaccharide content of the leucocytes is less pronounced in chronic myeloid leukemia than in acute leukemia, but is still quite distinct and statistically significant. There is also some decrease in the glycogen contentj of the thrombocytes during chronic myeloid leukemia, and the lymphocytes of patients with chronic lymphadenosis are poor in glycogen. As a rule, the turnover rate of glycogen is increased during leukemia in both the peripheral leucocytes and the nucleated cells of the bone marrow. The only exceptions are the lymphocytes in patients with chronic lymphadenosis and the young, undifferentiated cells of the bone marrow in patients with acute leukemia of metabolic type 11. The turnover rate of glycogen is also increased in the thrombocytes during chronic myeloid leukemia. A direct relationship, although not a strict proportionality, is observed between the glycogen content in the formed elements of the blood and bone marrow aiid the activity of the enzymes of the uridiiie diphosphoglucose pathway for glycogen synthesis. The activity of phosphoglucomutase,

404

I. F. SEITZ

UDPG-pyrophosphorylase and UDPG-glycogen glycosyl transferase is highest of all in the leucocytes of patients with polycythemia, which also contain the largest amounts of glycogen; in agreement with the decrease in the glycogen content of the cells, the enzymatic activity decreases in the following order: donor leucocytes, leucocytes of patients with chronic myeloid leukemia, lymphocytes of patients with chronic lymphadenosis, and the young, undifferentiated leucocytes in patients with acute leukemia. Abnormally low activity of the enzymes involved in glycogen synthesis is also observed in the young, undifferentiated cells of the bone marrow during acute leukemia.

REFERENCES Anderson, B., and Odell, T. 1958. Proc. SOC.Exptl. Biol. Med. 99, 765-768. Arroba, C. V., and Lagunilla, M. I. A. 1961. Rev. Clin. Espan. 80, 34S347. Astaldi, G., and Verga, L. 1957. Acta Haematol. 17, 129-135. Barker, S., and Summerson, W. 1941. J. Biol. Chem. 138, 535-554. Baron, E. J., and Harrop, G. 1929. J. Biol. Chem. 84, 89-100. Bazin, S., and Avice, C. 1953. Compt. Rend. SOC.Biol. 147, 1025. Beck, W. S. 1955. J. Biol. Chem. 216, 333-350. Beck, W. S. 1958a. J. Biol. Chem. 232, 251-270. Beck, W. S. 1958b. J. Biol. Chem. 232, 271-283. Beck, W. S. 195%. Ann. N . Y . Acad. Sci. 75, 4 1 5 . Beck, W. S., and Valentine, W. N. 1951. J. Lab. Clin. Med. 38, 245-253. Beck, W. S., and Valentine, W. N. 1952. Cancer Res. 21, 818-821. Beck, W. S., and Valentine, W. N. 1953. Cancer Res. 13, 309-317. Berni, C. M., Di Francesco, L., Rea, F., and Schettini, F. 1960. Boll. SOC.Ztal. Biol. Sper. 36, 1482-1483. Berni, C. M., Di Francesco, L., Rea, F., and Schettini, F. 1961a. Boll. SOC.Ztal. Biol. Sper. 37, 793-795. Berni, C. M., Di Francesco, L., Rea, F., and Schettini, F. 1961b. Boll. SOC.Ztal. Biol. Sper. 37, 1204-1206. Berni, C. M., Rea, F. D., Francesco, L., and Schettini, F. 1961~.Boll. SOC.Ztal. B i d . Sper. 37, 101S1015. Bird, R. M., Clements, J. A., and Backer, L. M. 1950. Federation Proc. 9, 11. Blinov, M. V. 1964. Vopr. Med. Khim. 10, 163-170. Bloom, M. L., and Wislocki, G. B. 1950. Blood 5, 79-88. Boll, I. 1957. Acta Haematol. 18, 390-403. Born, G. V. R. 1956a. Biochem. J. 62, 33P. Born, G. V. R. 1956b. J. Physiol. (London) 133, 61P. Born, G. V. R. 1962. Nature 194, 927-929. Born, G. V. R., and Esnouf, M. 1959. Proc, 4th Intern. Congr. Biochem., Vienna, 1958, Vol. X, pp. 97-104. Macmillan (Pergamon), New York. Boyd, E. M. 1936. Arch. Pathol. 21, 739. Boyd, E. M., and Murray, R. B. 1937. Can. Med. Assoc. J . 37, 229. Breckenridge, B. M., and Crawford, E. J. 1960. J. Biol. Chem. 235, 3054-3057. Bridge, E. W., and Holt, L. E. 1945. J. Pediat. 27, 299. Briickel, K. W., and Remmele, W. 1954. Z. Vitamin-, Hormon- Fermentforsch. 6, 50-63.

BIOCHEMISTRY OF NORMAL AND LEUKEMIC CELLS

405

Burk, D., Laszlo, J., Stengle, J., and Wight, K. 1957. Federation Proc. 16, 692. Burk, D., Laszlo, J., and White, K. 1959. Federation Proc. 18, 785. Burk, D., Laszlo, J., Seitz, J., Stambuk, B., and Woods, M. 1961. Proc. 6th Intent. Congr. Biochem. Moscow, 1962. Abstracts Sect. 14-28, pp. 325-326. Macmillan (Pergamon), New York and Izdat. Akad. Nauk SSSR. Burk, D., Laszlo, J., Seitz, J., Stambuk, B., and Woods, M. 1962. Acta Biol. Med. Ger. 8, 128-133. Byrom, F. B., and Kay, H. D. 1928. Brit. J . Exptl. Pathol. 9, 72. Campbell, E. W., Small, W. J., and Dameshek, W. 1956. J . Lab. Clin. Med. 47, 835843.

Campbell, E. W., Small, W. J., Lo Pilato, E., and Dameshek, W. 1957. Proc. SOC. Exptl. Biol. Med. 94, 506510. Chernyak, N. B. 1957. Vopr. Med. Khim. 3, 21EL227. Chernyak, N. B. 1958. Dokl. Akad. Nauk SSSR 118, 1004-1006. Chernyak, N. B. 1960. Vopr. Med. Khim. 6, 459-463. Chernyak, N. B. 1961. Vopr. Med. Khim. 7, 339-353. Chernyak, N. B., and Guscinov, Ch. S.1960. Dokl. Akad. Nauk SSSR 133, 476479. Chernyak, N. B., and Totskaya, A. A . 1963. Vopr. Med. Khim. 9, 146154. Chernyak, N. B., Sventsitskaya, M. B., and Guseinov, Ch. S. 1960a. Byul. Eksperim. Biol. i ilfed. 3, 51-54. Chernyak, N. B., Sventsitskaya, M. B., and Guseinov, Ch. S. 1960b. Probl. Gematol. i Pereliv. Krovi 5, 39-45. Claude, A. 1944. J . Exptl. Med. 80, 19-29. Cohn, Z. A., and Morse, S. I. 1960. J . Exptl. Med. 111, 667. Coxon, P. V., and Robinson, R. J. 1956. Proc. R o y . SOC.B145, 232-248. Dale, R. A. 1960. Clin. Chim. Acta 5, 652-663. Dameshek, W.,and Gunz, F. 1958. “Leukemia.” Grune & Stratton, New York. Davidson, J. N., Leslie, I., and Whitr, J. C. 1948. J . Pathol. Buctel-iol. 60, 1-20. Davidson, J. N., Leslie, I., and White, J. C. 1951. Blood 6, 9&915. Di Fmncesco, L., Berni, C. M., and Schettini, F. 1960a. Boll. SOC.Ztal. Biol. Sper. 36, 1484-1486. Di Francesco, L., Berni, C. M., and Schettini, F. 1960b. Boll. SOC.Ital. Biol. Sper. 36, 1477-1479. Di Francesco, L., Berni, C. M., Rea, F., and Schettini, F. 1961. Boll. SOC.Ztal. Biol. Sper. 37, 796797. Dubos, R. 1957. “Biological Factors in Microbial Diseases.” Publishing House for Foreign Literature, Moscow. (In Russian.) Endres, G., and Kubowitz, F. 1927. Biochem. 2 191, 395-397. Erickson, B., Williams, H., Avrin, J., and Lee, P. 1939. J . Clin. Invest. 18, 81-85. Eschbach, W. 1957. 2. Gebursthilfe Gynaekol. Beilageh. 148, 1 4 . Fantl, P., and Ward, H. 1956. Biochem. J . 64, 747-754. Fieschi, A,, Bianchini, E., and Cambiaggi, G. 1956. Acta Haematol. 16, 126-136. Firkin, B. G., and Williams, W. J. 1961. J . Clin. Invest. 40, 423-432. Fisher, I. N., and Ginsburg, H. S. 1956. Virology 2, 656-664. Fleischmann, W. 1939. Cold Spring Harbor Symp. Quant. Biol. 7, 29CL300. Furth, J., and Baldini, M. 1959. I n “Physiopathology of Cancer” pp. 364-468. Harper (Hoeber), New York. Gavosto, F., Maraini, G., Perelli, G., and Pileri, A. 1960. Boll. SOC.Ital. Biol. Sper. 36, 237-239.

Genkin, A. M. 1939. Byul. Eksperim. Biol. i Med. 7, 213-218.

406

I. F. SEITZ

Gibb, R . P., and Stowell, R. E. 1949. Blood 4, 569L576. Green, R. 1954. Proc. Soc. Exptl. Biol. M e d . 87, 412-414. Greenstein, J. P. 1954. “Biochemistry of Cancer,” 2nd ed. Academic Press, New York. Grignani, F., and Lohr, G. W. 1960. Klin. Wochschr. 38, 796-799. Gross, R., Lohr, G. W., and Waller, H. D. 1958. Proc. 4th Intern. Congr. Biochem., Vienna, 1958, Vol. X, pp. 92-96. Macmillan (Pergamon), New York. Gude, W., Upton, A., and Odell, T. 1956. Lab. Invest. 5, 348-358. Gurbanov, V. P. 1961. Probl. Gematol. i Pereliv. Krovi 6, 3-11. Haight, W. F., and Rossiter, J. 1950. Blood 5, 267-277. Hartman, J. D. 1952. Proc. Soc. Ezptl. Biol. M e d . 79, 3-7. Heckner, F. 1956. Actu Haematol. 16, 1-10. Holzer, H., Haan, J., and Pette, D. 1955s. Biochem. Z. 327, 19&201. Holzer, H., Holzer, E., and Schultz, M. 1955b. Biochem. Z. 326, 385-404. Hiilsmann, W. C., Oli, T . L., and Criveld, S. 1961. 2, N7202, 581-583. Illingworth, B., Cori, G., and Cori, C. F., 1956. J . Biol. Chem. 218, 123-129. Karnovslry, M. L. 1962. Physiol. Rev. 42, 143-168. Kassirskii, I. A., and Alekseyev, G. A. 1962. “Clinical Hematology.” Medgiz, Moscow. (In Russian.) Icnzanova, N. I., and Kozinet,s, G. I . 1963. Probl. Gemutol. i Pereliv. Krovi 8, 1922. Kedrovskii, B. V. 1951a. Usp. Sovrem. Biol. 31, 3&56. Kedrovskii, B. V. 1951b. Usp. Sovrem. Biol. 32, 309-329. Kempner, W. 1939. J . Clin. Invest. 18, 291-300. Kerby, G., and Langley, N. 1959. J. Lab. Clin. Med. 53, 708-713. Kozinets, G. I. 1962. Probl. Gematol. i Pereliv. Krovi 7, 41-48. Kugelmas, N. 1959. “Biochemistry of Blood in Health and Disease.” Thomas, Springfield, Illinois. Lajtha, L. G. 1957. Physiol. Rev. 37, 50-65. Lajtha, L. G., Oliver, R., and Ellis, F. 1954. Brit. J. Cancer 8, 367-379. Lawkowicz, W., Porembinska, H., and Czrrski, P. 1956. Folia iMorphol. 7, 101-107. Lawrence, J. S. 1955. J. Am. Med. Assoc. 157, 1212-1218. Leloir, L. F., and Cardini, C. E. 1957. J . Am. Chem. SOC.79, 6340-6341. Leloir, 1,. F., and Goldembcrg, S. H. 1960. J. Biol. Chem. 235, 91S923. Leloir, L. F., Olavarria, J. H., Goldemberg, S. H., and Carmin:ttt,i, H. 1959. Arch. Biochem. Biophys. 81, 508-520. Leslie, I. 1955. I n “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), pp. 7-50. Academic Press, New York. Russian translation Publishing House for Foreign Literature, Moscow, 1957. LettrB, H. 1954. Scientia (Milan) 89, 19-22. Libinzon, R. Ye. 1961. Vopr. Med. Khim. 7, 39s.396. Lohr, G. W., Waller, H. D., and Gross, R. 1961. Deut. M e d . Wochschr. 86, 807-904. Luck, D. I. Z. 1961. J. Biophys. Biochem. Cytol. 10, 195-209. Luganova, I. S. 1958. Respiration, Glycolysis and the Metabolism of Phosphoruscontaining Compounds in the Leukocytes of Normal and Leukemic Subjects. Thesis, Leningrad. (In Russian.) Luganova, I. S., and Seitz, I. F. 1958a. Byzkl. Eksperim. Biol. i M e d . 8, 58-61. Luganova, I. S., and Seitz, I . F. 1958b. Byul. Eksperim. Biol. i M e d . 12, 57-60. Luganova, I . S., and Seitz, I. F. 1959a. Aktual‘nyye Vopr. Pereliv. Krovi 7, 109-114. Luganova, I. S., and Seitz, I. F. 1959b. Aktual‘nyye Vopr. Pereliv. Krovi 7, 115-118. Luganova, I. S., and Seitz, I. F. 1959~.Aktuul’nyye Vopr. Pereliv. Krovi 7, 11&121. Luganova, I . S., and Seitz, I. F. 1959d. Aktual‘nyye Vopr. Pereliv. Krovi 7, 122-128.

BIOCHEMISTRY O F NORMAL AND LEUKEMIC CELLS

407

Luganova, I. S., and Seitz, I. F. 1959,. Probl. Gematol. i Pereliv. Krovi 11, 33-38. Luganova, I. S., and Seitz, I. F. 1959f. Vopr. Med. Khim. 5, 285-292. Luganova, I. S., and Seitz, I. F. 1960a. Vopr. Med. Khim. 6, 309-315. Luganova, I. S., and Seitz, I. F. 1960b. Probl. Leikozov i Immunogematol. 9, 64-68. Luganova, I. S., and Scitz, I. F. 1961a. Vopr. Gematol. Kanservirovaniya Krovi i l'kanei 12, 15-20. Luganova, I. S.,and Seitz, I. F. 1961b. Vopr. Gematol. Konscrvirovniiiya Krovi i Tknnei 12, 21-25. Luganova, I. S., and Seitz, I. F. 1963a. Vopr. M e d . Khim. 9, No. 4. Luganova, I. S., and Seitz, I. F. 196311. Aktual'nylje Vopr. Pereliv. Krovi 14, 231. Luganova, I. S., Seitz, I. F., and Teoclorovich, V. I. 1957a. Dolcl. Alcad. Nazkk SSSR 112, 108%1085. Luganova, I. S., Seitz, I. F., and Teodorovich, V. I. 1957b. Vopr. Med. Khim. 3, 428439. Luganova, I. S., Seitz, I. F., and Teodorovich, V. I. 1957~.Aktual'nyye Vopr. Pereliv. Krovi 5, 99-109. Luganova, I. S.,Scitz, I. F., and Teodorovich, V. I. 1957d. Aktztal'nyye Vopr. Pereliv. Krovi 5, 109-119. Luganova, I. S., Seitz, I. F., and Teodorovich, V. I. 1957~.Proc. All-Union Conf. Peaceful CJses A t . Energy pp. 96-103. Luganova, I. S.,Seitz, I. F., and Tcodorovich, V. I. 1957f. Dokl. Akatl. N u u k S S S R 113, 149-151. Luganova, I. S.,Scitz, I. F., and Teodorvirh, V. I. 1958a. Dokl. Akatl. N a u k SSSR 118, 537-539. Luganova, I. S.,Seitz, I. F., and Teodorovich, V. I. 1958b. Biokhimiya 23, 405-411. Luganova, I. S., Seitz, I. F., and Teodorovich, V. I. 1959. Alctual'nyye Vopr. Pereliv. Krovi 7, 105-109. Luganova, I. S., Rozanova, L. M., and Scitz, I. F. 1962. Vopr. Pereliv. Krovi i Klin. Med. Kirov pp. 247-256. Luganova, I. S., Egorol-a, V. A,, St:it,z, I. F. 1963a. A c f d ' r i y y e Vopr. Pereliv. Kroui 14, 215-220. Luganova, I. S.,Vladirnirova, .4.D., Sriiz, I. F. 1963b. Aclual'rr2/rle Vopr. Pereliv. Krovi 14, 231-239. MacKinney, G. H., and Rundless, R. W. 1956. Cancer Res. 16, 67-69. MacICinney, G. H., Martin, S. P., Rundless, R. W., and Green, R. 1953. J. AppE. Physiol. 5, 335. Mandel, P., and Metais, P. 1952. Semaine Hop., Paris 28, 12S132. Martin, S. P., McKynney, G. R., and Green, R. 1955. Ann. N. Y. Acad. Sci. 59, 996-1003. Menten, M. L. 1952. Cancer Res. 12, 281-282. Menten, M. L., and Willms, M. 1953. Cancer Res. 13, 733-736. Mikhnovich, Ye. P., and Seitz, I. F. 1959. Aktzial'nyye Vopr. Pereliv. Krovi 7, 128135. Moloney, W. C., and Lange, R. D. 1954. Texas Rept. Biol. Med. 12, 887-897. Morita, H., and Asada, T. 1956. Acta Haematol. Japon. 19, 426449. Morita, H., and Asada, T. 1957. Sang 28, 827. Maupin, B. 1954. Compt. Rend. Soc. Biol. 148, 439-441. Maupin, B., Schnabcli, J., and Ardry, R. 1954. Compt. R e n d . Soc. Biol. 148, 441-443. Manpin, B., Saint-Blaucerd, J., and Storck, J. 1962. Rev. Franr. Etntles Clin. Biol. 7, 16S173. Nigani, V. N., MacDonald, H. L., and Cantero, A. 1962. Cancer Res. 22, 131-138.

408

I. F. SEITZ

Notario, A., and Nespoli, M. 1961. Haematol. Latina (Milan) 46, 505-550. Notario, A., Casiorola, G., and Nespoli, M. 1961. Haematol. Latina (Milan) 46, 609-6 17. Odell, T., and Anderson, B. 1957. Proc. SOC.Exptl. Biol. Med. 94, 151-156. Ondarza, R. N. 1960. Acta Physiol. Latinoam. 10, 129-134. Paladini, A. C., and Leloir, L. F. 1952. Biochem. J. 51, 426-430. Pavlov, B. A. 1960. Terapevt. Ark. 32, 44-48. Peschel, E. 1930. Klin. Wochschr. 9, 1061-1062. Petrakis, M. L. 1953. Blood 8, 905-915. Phillips, H. J., and McCarthy, H. L. 1956. Proc. SOC.Exptl. Biol. Med. 93, 573-576. Polli, E. 1959. Contemporary Problems of Oncology. (In Russian.) (Collected Translations and Abstracts), Publishing House for Foreign Literature, Moscow, ( 9 0 , 21. Polli, E., and Semenza, G. 1955. Rend. Zst. Lombard0 Sci. Lettere B89, 16CL181. Ponder, E., and MacLeod, J. 1936. J. Gen. Physiol. 20, 267. Puchkov, N. V. 1955. Biokhimiya 20, 709-713. Ramonova-Tskhovrebova, 0. D. 1948. Sovrem. Probl. Gematol. i Pereliv. Krovi 24/ 25, 234239. Remmele, W. 1955. Acta Haematol. 13, 103-123. Rigas, D. A., and Osgood, E. E. 1955. Federation Proc. 14, 269. Rigas, D. A., Duerst, M. L., Jump, M. E., and Osgood, E. E. 1956. J. Lab. Clin. M e d . 48, 356378. Robbins, P. W., Traut, R. R., and Lipmann, F. 1959. Proc. Natl. Acad. Sci. U . S. 45, 612. Roetth, H. 1957. Cancer Res. 17, 833. Rohdewald, M. 1946. Naturwissenschaften 33, 314. Rohdewald, M. 1952. Biochem. Z . 323, 146156. Rossi, F., and Zatti, M. 1961. Exptl. Cell Res. 25, 182-183. Salvidio, E. 1958. Proc. 6th Congr. European Sac. Haematol., 1957, Vol. 2, pp. 319323. Karger, Basel. Sauer, H., and Rauschstrooman, J. G. 1955. Deut. Arch. Klin. Med. 202, 282-290. Schettini, F., Di Francesco, L., and Berni, C. M. 1960. Boll. Sac. Ztal. Biol. Sper. 36, 1484-1486. Schettini, F., Di Francesco, L., Berni, C. M., and Rea, F. 1961. Boll. Sac. Ztal. Biol. Sper. 37, 797-801. Schmid, R., and Mahler, R. 1959. J. Clin. Invest. 38, 1040. Schmid, R., Robbins, Ph. W., and Traut, R. R. 1959. Proc. Natl. Acad. Sci. U . S. 45, 1236-1240. Seelich, F. 1957. Wien. Klin. Wochschr. 69, 448-449. Seelich, F., Letnansky, K., Frisch, R., and Schneck, 0. 1957. Z . Krebsforsch. 62, 1-8. Seitz, I. F. 1955. Coordination of Aerobic and Anaerobic Metabolism in Cellular Respiratory Phosphorylation. Thesis, Institute of Experimental Medicine, USSR Academy of Medical Sciences, Leningrad. (In Russian.) Seitz, I. F. 1957. Byul. Eksperim. Biol. i Med. 43, 119-123. Seitz, I. F. 1959. Vopr. Med. Khim. 5, 114-123. Seitz, I. F. 1958. Dokl. Akad. Nauk SSSR 118, 344-347. Seitz, I. F. 1961. %teraction of Respiration and Glycolysis in the Cell.” Medgiz, Leningrad. (In Russian.) Seitz, I. F., and Luganova, I. S. 1961a. Usp. Sovrem. Biol. 51, 317-336.

BIOCHEMISTRY O F NORMAL AND L EU K EM I C CELLS

409

Seitz, I. F., and Luganova, I. S. l96lb. Conference on the Utilization of Radioisotopes in Animal Biology and the Medical Sciences, Mexico. Seitz, I. F., and Yel’tsina, N. V. 1951. Biokhimiya 16, 62-68. Seitz, I. F., Luganova, I. S., and Vladimirova, A. D. 1963. Biokhimiya 28, 295-302. Shacter, B. 1957. J . Natl. Cancer Inst. 18, 455-462. Sherstneva, 0. S. 1958. Byul. Eksperim. Biol. i Med. 45, 67-69. Sieracki, J. C. 1955. Ann. N . Y . Acad. Sci. 59, 690. Siyanitskaya, M . F., and Scitz, I. F. 1958. Aktual‘nyye Vopr. Pereliv. Krovi 6, 189193. Siyanikkaya, M. F., and Seitz, I. F. 1962. Vopr. Med. Khim. 9, 19-27. Soffer, L. J. and Wintrobe, M. M. 1932. J . Clin. Invest. 11, 661-676. Stetten, De Witt, Jr., and Stetten, M. R. 1960. Physiol. Rev. 40, 506537. Storti, E. 1953. Acta Haematol. 10, 144. Strominger, J. L. 1960. Physiol. R e v . 40, 55-111. Teodorovich, V. I. 1958. Aktual’nyye Vopr. Pereliv. Krovi 6, 309-312. Terent’yeva, E. I., Zosimovskaya, A. I., and Kazanova, L. I. 1957. Probl. Gematol. i Pereliv. Krovi 5, 2431. Terent’yeva, E. I., Zosimovskaya, A. I., Kazanova, L. I., and Fainshtein, F. E. 1959. Probl. Gematol. i Pereliv. Krovi 11, 39-49. Terent’yeva, E. I., Kazanova, L. I., and Fainshtein, F. E. 1960. Probl. Gematol. i Pereliv. Krovi 5, %8. Thorell, B. 1947. Acta filed. Scand., Suppl. 200. Trubowitz, S., Feldman, D., Benante, D., and Kirman, D. 1957. Proc. SOC.Exptl. Biol. Med. 95, 35-38. Tullis, J. L., ed. 1953. “Blood Cells and Plasma Proteins: Their State in Nature,” pp. 257-280. Academic Press, Kew York. Valentine, W. N. 1951. Blood 6, 845854. Valentine, W. N. 1955a. Ann. R e v . Med. 6, 77-89. Valentine, W. N. 1955b. Ann. N . Y . Acad. Sci. 59, 1003-1011. Valentine, W. N. 1960. A m . J . Med. 28, 699-710. Valentine, W. N., and Beck, W. S. 1951. J . Lab. Clin. Med. 38, 39-55. Valentine, W. N., Beck, W. S., Follette. J. H., Mills, H., and Lawrence, J. S. 1952. Blood 7, 959-977. Valentine, W. N., Follette, J. H., and Lawrence, J. S. 1953. J . Clin. Invest. 32, 251-257. Valentine, W. N., Follette, J. H., Hardin, E. B., Beck, W. S., and Lawrence, J. S. 1954. J . Lab. Clin. Med. 44, 219-228. Valentine, W. N., Lawrence, J. S., Pearce, M . L., and Beck, W. S. 1955. Blood 10, 154-159. Villar-Palasi, C., and Larner, J. 1958. Biochim. Biophys. Acta 30, 449. Wachstein, M. 1946. J . Lab. Clin. Med. 31, 1. Wagner, R. 1946. Arch. Biochem. 11, 249-258. Wagner, R. 1947. Blood 2, 235-243. Wagner, R., and Yourke, A. 1952. Arch. Biochem. 39, 174-187. Wagner, R., Meyericks, N., and Sparaco, R. 1956. Arch. Biochem. Biophys. 61, 278290. Warburg, 0. 1926. “tlrber den Stoffwechsel der Tumoren.” Springer, Berlin. Warburg, 0. 1956. Science 123, 309-314. Warburg, O., Gawehn, K., and Geissler, A. 1958. 2. Naturforsch. 138b, 515-516.

410

I. F. SEITZ

Weber, G. 1961. Cancer Res. 6, 403487. Weinhousc, S. 1958. Theses reports and presentations a t the International Congress against Cancer in London. I n “Contemporary Problems of Oncology” (Collected Translations and Abstracts), Vol. 4(91), p. 14. Publishing House for Foreign Literature, Moscow, 1959. (In Russian.) Weinhouse, S. 1962. Cancer Res. 22, 1372-1380. Wells, W., and Wineler, R. J. 1959. Cancer Res. 19, 1086-1090. White, J. C., Leslie, E., and Davidson, J. N. 1953. J . Pathol. Bacteriol. 66, 291-306. Will, J. J., Glazer, H. S., and Vilter, R. W. 1957. I n “The Leukemias” (J. W. Rebuck, ed.), pp. 417422. Academic Press, New York. Williams, H. E., and Field, J. B. 1961. J. Clin. Invest. 40, 1841-1845. Willstattcr, R., and Rohdcwald, M. 1937. 2. Physiol. Chem. 247, 11S126. Wintrobe, M. M. 1947. “Clinical Hematology,” 2nd ed. Lea & Febiger, Philadelphia, Pennsylvania. Wineler, R. 1958. Ann. N . Y . Acad. Sci. 75, 3744. Winder, R., Williams, A. D., and Best, W. R. 1957. Cancer Res. 17, 108-116. Wislocki, G., Rheingold, J. J., and Dempsey, E. 1949. Blood 4, 56%568. Woodside, E. E., and Kocholaty, W. 1960. Blood 16, 1173-1183. Wureel, H., McCreary, Th., Baker, L., and Gumermann, L. 1961. Blood 17, 314-318. Yel‘tsina, N. V., and Seite, I. F. 1950. Dokl. Akad. N a u k SSSR 70, 457-460. Yel’tsina, N. V., and Seitz, I. F. 1951. Dokl. Akad. N a u k SSSR 77, 653-656. Zatti, M., and Rossi, E. 1961. Ital. J . Biochem. 10, 19-41. Zilversmit, R. D., Marcus, A. J., and Ullman, H. L. 1961. J . Biol. Chem. 236, 47-49.