Antimetabolites in the chemotherapy of leukemia

Experimenfal

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ANTIMETABOLITES

F. M. HUENNEKENS,s Departments and King

9, 441-461

IN THE CHEMOTHERAPY OF LEUKEMIA1

J. R. BERTINO,J

of Biochemistry County

441

(1963)

Central

and Medicine, Blood Bank

R. SILBER4

and B. W. GABRIO

University of Washington, Seattle, Wash., U.S.A.

cells are encountered in a wide variety of histological sizes and shapes, but they have in common one distinguishing feature, namely an abnormal nucleus [ 141. The cancer nuclei are hyperchromatic when stained and there is an increased nuclear to cytoplasmic ratio as compared with normal cells. These visible indications of exaggerated nuclear function are consistent with the elevated rate of cellular proliferation and the occasional but characteristic multiple mitosis seen in cancer cells. On the other hand, the capacity of cancer cells to perform specialized enzymic functions, other than cell division, is somewhat lessened [30], and cancer cells are more akin biochemically to each other than to their tissue of origin. The enzymatic apparatus concerned with cell division is quite complex and is probably housed, in part, within the nucleus. The crucial enzymatic activities include protein synthesis, RNA5 synthesis, and DNA synthesis, all interrelated as illustrated in eq. (1). CANCER

precursors I

i

(1)

ribonucleotides precursors

-

deoxyribonucleotides

-+ DNA I RNA amino

acids

p-

proteins

r Thts work was SUDDOrted bv grants from the American Cancer Society (P-203 and P-301) and from the U.S. Pub& Health S&vice (CY-3310 and CY-6522). a Present address: Division of Biochemistry, -- Scripps Foundation, La Jolla, -_ Clinic and Research California. s Present address: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut. 4 Present address: Department of Medicine, New York University School of Medicine, New York, New York. 6 The following abbreviations are used: RNA, ribonucleic acid; DNA, deoxyribonucleic acid; dCMP, deoxycytidylate; dUMP, deoxyuridylate; dTMP, thymidylate; dATP, dGTP, dCTP and dTTP, the triphosphates of deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine; C,, a one-carbon unit; DPN and TPN, di- and triphosphopyridine nucleotide; DPNH and TPNH, reduced DPN and TPN; and DEAE, diethylaminoethyl. Experimental

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F. M. Huennekens

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The role of DSA in the above sequence [79] has received considerable attention since within this macromolecule is encoded the genetic information [42, 66, 671 which differentiates the cancer cell from its cell of origin. Chromatographic analyses have revealed differences in base composition between the DNA of tumor cells as compared with normal cells [5,14,46]. Enlarging upon (l), the detailed pathway for the biosynthesis of DNA may be outlined in the following manner: dCMP

dU+MP dATP I ~-

‘..

thymidine

--

Cl

dCTP

i dTMP

DNA polymerase

dGTP

p-

dTTP

DNA

(2)

I

The terminal step involves the polymerase-mediated condensation of the four deoxynucleoside triphosphates [42], and in many systems (reviewed by O’Brien [52]), thymidine triphosphate (dTTP) appears to be the limiting component. dTTP is synthesized from its monophosphate (dTMP) via thymidylate kinase [13]. dTMP, in turn, can be produced by two enzymatic pathways: (a) the direct phosphorylation of thymidine via thymidine kinase; and (6) the folic acid coenzyme-dependent methylation of deoxyuridylate via thymidylate synthetase. The multiple reactions involved in DNA synthesis offer a number of potential targets for metabolic inhibitors which, if effective at the enzymatic level, might arrest the growth of tumors. The rationale of this chemotherapeutic approach to cancer stems from the successful employment of such inhibitors, usually called “antimetabolites”, in the destruction of bacteria or protozoa without concomitant harm to the host. In brief, the concept of antimetabolites [88] assumes that for every naturally-occurring vitamin, coenzyme, or substrate, one can substitute a closely-related chemical compound which will possess sufficient affinity for the appropriate enzyme system to interfere with the binding and function of the normal constituent. This simplified definition, of course, does not take into account important pharmacological considerations in the human, such as the possible toxicity of the antimetabolite, its ability to be transported to the appropriate tissue, its ability to penetrate cell membranes, or its resistance to catabolism while in transit to the target site. In two excellent reviews [SO, 811 Welch has discussed a number of antimetabolites currently used in the chemotherapy of cancer. The present Experimentat

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paper will consider only two of these antimetabolites, the folic acid antagonists and the fluoropyrimidines, both of which inhibit DNA synthesis at the site of the thymidylate synthetase. Data for this discussion on the mode of action of these inhibitors will be taken from our recent studies on the biochemical aspects of human leukemic leucocytes. METHODOLOGY

AND

RATIONALE LEUKEMIC

OF

STUDIES

WITH

NORMAL

AND

LEUCOCYTES

Before considering the mode of action of the two antimetabolites mentioned above, it is necessary to describe briefly the methodology and rationale of our studies with leucocytes. Numerous investigations (reviewed in [l, 4, 75, 761) have been undertaken to delineate the biochemical characteristics of normal and leukemic leucocytes. Our group has concentrated largely upon the area of one-carbon metabolism 1381, mediated by folic acid coenzymes, since these reactions are directly involved in the biosynthesis of: (a) the purine ring; (b) the methyl group on the pyrimidine ring of thymidylic acid; and (c) the amino acids-methionine, histidine and serine. Thus, folic acid coenzymes could control the synthesis of DNA, RNA, and proteins. The elevated levels of folic and folinic acids in leukemic cells [53, 71 j, and the routine use of folic acid antagonists in the treatment of acute leukemia further emphasize the relevance of this metabolic area to the leukemic state. Leucocytes are isolated from the blood of normal subjects and leukemic patients by a procedure developed in our laboratory [9], but similar to the method of Fallon et al. [23]. In brief, whole blood is mixed with Dextran (average Mol. Wt. = 180,000) and the suspension is allowed to stand for about 30 min [69]. The supernatant fraction, containing leucocytes and platelets, is removed by decantation, and the leucocytes are separated from platelets by a centrifugation step. At this point, the leucocyte preparation is still contaminated by erythrocytes, but these can be removed by exposing the cells to distilled water for 20 set, a treatment which selectively lyses the residual erythrocytes [78]. A final centrifugation step yields the leucocyte pellet completely free from erythrocytes or hemoglobin and undamaged morphologically, as judged by visual inspection. The entire procedure requires about 2 hr and the yield of leucocytes is about 40 per cent from normal blood and 60-90 per cent from leukemic blood. Lysis of the leucocytes is carried out by subjecting the cells to one of several procedures: (a) repeated freezing and thawing; (b) hi-speed homogenization; or (c) treatment with acetone at - 20”. The last technique is Experimental

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especially useful, since it converts the leucocytes to a stable powder which can be stored at - 20”. After the cell debris is removed by centrifugation, each of these procedures yields a soluble leucocyte preparation, nearly identical with respect to content of the various enzymes of one-carbon metabolism. Using spectrophotometric assays described elsewhere [59], a detailed survey has been made in normal and leukemic leucocytes [9] of these enzymes. The “activity” of an enzyme in a given ireparation refers to the total catalytic capacity of the enzyme, expressed as micromoles of substrate converted per hour. The “level” of the enzyme is then defined as the activity expressed: (a) per mg of protein in the extract; or (b) per 109 cells from which the extract was derived. We have expressed our results routinely in terms of the former parameter, since there is a considerable difference in the amount of soluble protein which can be obtained from the various types of normal and leukemic cells. A statement should also be made about the meaning of the term “leukemic” leucocytes. These cells are obtained, usually prior to therapy, from the peripheral circulation of leukemic patients classified as acute (AL), chronic myelocytic (or granulocytic) (CML), or chronic lymphocytic (CLL). It is probable that the true “leukemic cells” are present mainly in the bone marrow, although the circulating leucocytes may contain a relatively high percentage of immature cells having an increased capacity for DNA synthesis and cellular replication. The circulating immature cells are believed to reflect the proliferative capacity of the malignant tissue. MODE

OF ACTION

OF FOLIC

LEUKEMIC

ACID

ANTAGONISTS

IN

LEUCOCYTES

Soon after the discovery of folic acid in 1943, a number of analogues, or antagonists, of this vitamin were prepared and shown to be extremely toxic to mammals, birds and bacteria. The clinical use of these antagonists in the treatment of leukemia was suggested by the observation that administration of the vitamin actually increased the rate of cell proliferation in ieukemic patients [37]. Farber and his colleagues [24], using aminopterin-one of the first analogues to be synthesized [60], achieved a dramatic series of remissions when children with acute leukemia were treated with this drug. Although the remission is only temporary and the cancer cells inevitably become refractory to further treatment, folic acid antagonists [21, 32, 421 have continued to find wide acceptance in the clinical treatment of leukemia, lymphosarcoma, and more recently, choriocarcinoma [44]. The structures of Experimental

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of leukemia

the most commonly used folic acid antagonists, aminopterin, amethopterin (Methotrexate), and dichloroamethopterin, are shown in Fig. 1. The search for a biochemical explanation for the mode of action of these drugs received its first impetus when it was found that aminopterin blocked the reductive conversion of folic acid to folinic acid (5N-formyl tetrahydrofolic

H2N~JN>cH2eNH+-$eg,”

p$

SF%

(2

(E)

(3

N OH NH2

H J

f

q,

folote (V

Fig.

7,Bdihydrofolace

5.6.7.8.tetrahydrofol.te

Wd

1.

Fig.

VU

2.

Fig. l.-Folic acid antagonists. Folic acid = 2-amino-4-hydroxy pteroylglutamic acid (PGA); Aminopterin =4-amino (PGA); Amethopterin =4-amino, lo-methyl PGA; Dichloroamethopterin = I-amino, lo-methyl, 3’, 5’-dichloro PGA. Fig. 2.-Dihydrofolic (or folic) reductase. Only the pyrazine ring portion of the molecule is shown. F, FH,, and FH, represent folate, dihydrofolate, and tetrahydrofolate.

acid). Later, catalyzes the is inhibited Dihydrofolic many tissues

it was shown that the enzyme, dihydrofolic reductase,l which two-step reduction of folate to tetrahydrofolate shown in Fig. 2, by extremely low levels of the folic acid antagonists [28, 541. reductase, customarily assayed via reaction (3), is present in where it has a two-fold

dihydrofolate

+ TPNH

+ H++ tetrahydrofolate

+ TPN+

(3)

function: (a) in the biosynthesis of the coenzyme (tetrahydrofolate) from the vitamin (folate); and (b) in the synthesis of the methyl group of thymidylate (see p. 453). Most of the early studies on the enzyme, and on its inhibition by folic acid antagonists, were carried out with relatively crude preparations obtained from various mammalian, avian, and microbial sources (reviewed in [38]). More recently, this enzyme has been purified extensively from Streptococcus faecalis [la], calf thymus [50], and chicken liver [45]. The chicken liver enzyme, which has been purified over l,OOO-fold by Mathews, serves as a useful model for the less acessible leucocyte enzyme. Both folate and dihydrofolate are substrates for the chicken liver enzyme although the latter compound is reduced at a faster rate. TPNH, rather than DPNH, is the preferred reductant for both steps. The avian dihydrofolic reductase has two pH optima, one at 4.5 and the other at 7.5; still uncertain is the significance 1 Although both reductive referring to it as “dihydrofolic elsewhere [38].

steps in Fig. reductase”,

2 are catalyzed rather than

by the same enzyme, “folic reductase”, have

Experimental

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the reasons for been discussed

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of-the fact that the enzymatic pH region. Previous studies from our liver dihydrofolic reductase antagonists. This conclusion data via Michaelis-Menten

activity

is higher

et al. in the non-physiological,

acid

laboratory [54] had indicated that the chicken is inhibited non-competitively by the folic acid was reached from a treatmentof the inhibition kinetics which assumes that both enzyme (E)

Fig. 3.-Hypothetical inhibition.

plot of “stoichiometric”

Amount of Enzyme substrate (S), and enzyme and inhibitor (I), interact reversibly even though the dissociation constants for the E-I and E-S complexes may be extremely low. Werkheiser [83] has proposed an alternate interpretation for the inhibition of rat liver dihydrofolic reductase by aminopterin, namely that the inhibition is “stoichiometric” (i.e., enzyme and inhibitor interact on an equimolal basis with no dissociation of the E-I complex). Stoichiometric inhibition can be detected, as Werkheiser found, by plotting enzyme activity as a function of the amount of enzyme added. -4s shown by the hypotheticai plot in Fig. 3, a straight line (control) is obtained in the absence of inhibitor, but in the presence of a given amount of inhibitor Curve A is obtained. Werkheiser obtained this type of plot for the rat liver enzyme and the point on the x-axis at which enzyme activity emerged represented the “titration” of enzyme by inhibitor. We have carried out the same type of experiment [45] using the highly-purified chicken liver reductase and have found, on the contrary, that the inhibition is not stoichiometric since the data correspond to Curve B in Fig. 3. This latter curve is characteristic of classical Michaelis-Menten inhibition. The disagreement between the two sets of results is probably a reflection of the different enzyme systems used, and perhaps also of the Experimental

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different assay methods for measuring dihydrofolic reductase reaction. Further evidence that the E-I complex is dissociable, at least in the case of the chicken liver enzyme, is afforded by experiments in which the complex is subjected to chromatography. As shown in Fig. 4, passage of the E-I complex through a column of hydroxylapatite regenerates the free enzyme [45]. Similar results were obtained when the E-I complex was chromatographed

Fig. 4.-Dissociation of enzyme-inhibitor complex during chromatography (from Mathews, Ph. D. Thesis, University of Washington, 1962). Tube number

on DEAE-cellulose, except that the free enzyme preceded the inhibitor in the effluent. The aminopterin-inhibited reductase from leucocytes is also labile to chromatography [64]. On the other hand, the chicken liver reductase and the leucocyte enzyme are not entirely equivalent since only the latter E-I complex is reactivated by dialysis against dilute buffers.1 Guided by studies with dihydrofolic reductases from other tissues, which suggested that this enzyme was the probable target site in leukemia for the folic acid antagonists, an investigation was undertaken of the leucocyte dithe enzyme could not be detected hydrofolic reductase. To our surprise, initially in extracts of normal leucocytes by either the direct assay system (eq. (3)), or an indirect assay system, in which the dihydrofolic reductase reaction is coupled with the formate-activating enzyme (eq. (4)). The enzyme could be measured, however, in leukemic leucocytes when the indirect assay tetrahydrofolate

+ ATP + formate

+ WO-formyl

tetrahydrofolate

+ ADP + P (4)

employed. The enzyme was subsequently purified .about 30-fold from leukemic cells and its properties were ascertained. The leucocyte reductase is 1 Werkheiser has reported against high concentrations

that the inhibited of folic acid [83].

rat liver

reductase

can be reactivated

Experimental

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F. M. Huennekens

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similar in most respects to its counterpart from chicken liver, except that the pH optimum for reaction (3) occurs at 8.3, rather than at 7.5. The enzyme is almost completely specific for both TPNH and dihydrofolate. The sensitivity of the leucocyte dihydrofolic reductase to the folic acid antagonists1 is documented in Table I [8]. More extensive data, showing inhibition of this enzyme by various concentrations of the drugs between I. Inhibition

TABLE

of leucocyte dihydrofolic antagonists [9]. Inhibitor (10-S M)

reductase

Inhibition 70

Amethopterin Aminopterin Dichloroamethopterin

TABLE

II. Activity

57 64 85

of dihydrofolic

Diagnosis

No.

reductase

in leucocytes

of patients

a Mean

leukemia leukemia

+ standard

15 8 10 22

[8].

Activity @f/hr/mg

Normal Chronic lymphocytic Chronic myelogenous Acute leukemia

by folic acid

ca 0.002 0.042 0.034

of protein 0.001 +0.001= + 0.008 + 0.004

error.

1O-s and 10es M, are presented elsewhere [64]. It will be noted that all three antagonists do not produce the same percentage inhibition at 1O-8 M and this is further evidence that these agents do not react “stoichiometrically” with the enzyme. Similar findings on the different degrees of inhibition caused by the antagonists have been reported [48] for the dihydrofolic reductase in the spleen and liver of L-1210 leukemic mice. The level of diliydrofolic reductase [8, 91 was measured in normal and leukemic leucocytes and the results are summarized in Table II. The enzyme is present at approximately the same level2 in acute leukemic cells and chronic 1 0~01s [55] has shown that dihydroaminopterin is about as effective as aminopterin in the inhibition of the chicken liver reductase, while tetrahydroaminopterin is a much poorer inhibitor. a From the data in Table II and from unpublished studies on the binding of aminopterin, it can be calculated [7] that each leukemic cell contains approximately 10” to lo6 molecules of dihydrofolic reductase and that the enzyme has a turnover number of between 10 and 100. Experimenfal

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myelogenous leukemic cells, but it is barely detectable in chronic lymphocytic leukemic cells or in normal cells. Similar findings have been reported by Wilmanns [85]. It is difficult to decide whether the enzyme is really absent from normal cells since there may be present an endogenous inhibitor which prevents the enzyme activity from being fully expressed. Perhaps relevant to this point, Craddock [17] has presented evidence for the occurrence in normal human granulocytes of a non-dialyzable factor that suppresses DNA synthesis in dog thoracic duct lymphocytes. Although the leukemic leucocyte contains an enzyme,, dihydrofolic reductase, which is sensitive to minute amounts of the folic acid antagonists, it may be questioned whether this enzyme is the unique target site for these drugs. To partially answer this question, several other enzyme systems, in which tetrahydrofolate serves as a coenzyme, have been examined in leuoccytes [9]. These include formate-activating enzyme, serine hydroxymethylase, 6N,10Nmethylene tetrahydrofolic dehydrogenase, and cyclohydrolase. None of these enzymes is inhibited by the folic acid antagonists unless the drug is present at high levels (ca lo--% M). Thus, it is probable that, at the concentrations present in the cell during therapy, the drugs inhibit only dihydrofolic reductase and do not affect the other enzymes concerned with C, metabolism. We cannot exclude the possibility, however unlikely it seems, that other enzymes or proteins in the leucocyte might also have an appreciable avidity for the drugs and thereby act as secondary “target sites” without any metabolic consequences. The availability of 3H-labeled aminopterin should facilitate an investigation of this point. At this point, brief mention should be made of certain other enzymes whose levels are consistently higher in leukemic cells, when compared with normal cells: DNA polymerase [ll]; formate-activating enzyme [9, 841; serine hydroxymethylasel [9, 841; 5N10N-methylene tetrahydrofolic dehydrogenase [9, 841; TD transhydrogenasez [63]; and several enzymes involved in pyrimidine metabolism [70]. On the other hand, leukemic cells have a lower level of glucose-6-phosphate dehydrogenase [3] and alkaline phosphatase [75, 761, 1 The level of serine hydroxymethylase is about lo-fold higher in chronic lymphocytic leukemic cells and about a-fold higher in acute leukemic cells as compared with normal or chronic mvelonenous leukemic cells [9]. The greatly elevated level of this enzyme in the lymphocytes p;ovides a possible explanation for Winzler’s earlier finding that the incorporation of r*C-labeled formate into the protein fraction (presumably into the se&e residues) is the highest in this type of leucocyte [87]. 8 TD transhydrogenase catalyzes the reaction: TPNH + DPN%TPN + DPNH. It is of interest that the level of DPN is about 4-fold higher in acute leukemic cells and a-fold higher in chronic myelogenous leukemic cells, while the levels of TPN, DPNH, and TPNH are essentially the same in all types of leucocytes [Ml. 29 - 631807

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as well as a depressed rate of glycolysis [4]. It is difficult to know, however, whether these alterations in metabolic pathways are the basis for the primary leukemic lesion or whether they are a consequence of the lesion. At present, it is our belief that dihydrofolic reductase and thymidylate synthetase, being found in leukemic cells and not in normal cells, are the most likely systems involved in the primary neoplastic process. The fact that acute and chronic myelogenous leukemic leucocytes contain an amethopterin-sensitive dihydrofolic reductase led to a study of the effect of amethopterin therapy upon the reductase level in the cells of these patients undergoing treatment. Typical data illustrating this point have been presented elsewhere (cf. Fig. 1 in ref. [6]). This acute myeloblastic leukemic patient, who had an initial elevated white count, responded satisfactorily to a massive treatment with amethopterin, as indicated by the decline in total white count and in the percentage of immature forms. Subsequently, the patient was maintained in this state by additional dosages of amethopterin and after about six weeks, when the white count began to rise again, there was one other transient response to the drug. The leukemia eventually became refractory to amethopterin, and other regimens were employed. During the early course of therapy, the level of dihydrofolic reductase increased more or less consistently, even during periods in which the total white count was decreasing. By comparison, the level of formate-activating enzyme remained almost constant during the entire course of therapy. The rise in reductase level as a result of amethopterin therapy has been observed in a number of othkr patients [6] (cf. Part A, Table III). In this Table the pre-treatment level of reductase is compared with the highest level obtained during the course of amethopterin therapy. In the cases studied, there was a 5- to 20-fold increase in reductase level over the pretreatment baseline. The rise in level could not be attributed to any alteration in the properties of the enzyme since dihydrofolic reductase, isolated from the same patient prior to, and during, the course of therapy, showed no changes in the following parameters: K,,, for substrates, KI for inhibitors, and pH optimum for reaction (3). When patients are treated, alternatively, with drugs other than the folic acid antagonists (e.g. 6-mercaptopurine, Myleran, or prednisone), no rise in dihydrofolic reductase activity is noted during the course of therapy (cf. Part B, Table III). Similar findings regarding the rise in reductase level upon treatment with folic acid antagonists have been reported for L-1210 leukemia in mice [27, 481, and cells grown in tissue culture [25, 311. On the other hand, an initial increase in dichloroamethopterin resistance, unaccompanied by an increase in reductase, has been reported [27] for a Experimental

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Chemotherapy of leukemia subline of L-1210 cells; intensive treatment with the drug, however, ultimately results in the selection of cells with an elevated enzyme content. The above phenomenon, in which the level of dihydrofolic reductase in leukemic cells is increased from a finite value to a higher value, is paralleled by the observation that the enzyme can also be “induced”,1 i.e. increased TABLE

III.

Changes in dihydrofolic reductase level in patients treated with amethopterin and other chemotherapeutic agents [7]. Dihydrofolic Reductase Activity (yM/ml/hr/mg protein) Chemotherapy

before

treatment

after

x IO-2 Part

x 10-a

A

Acute Acute Acute Acute Acute Acute Part

treatment

lymphocytic’ lymphocytic’ myelogeno& myelogenous myelogenous monocytic

Amethopterin Amethopterin Amethopterin Amethopterin Amethopterin Amethopterin

3.0 3.4 3.1 3.3 6.7

23 23 25 25 60 37

4.8 4.7 3.7

Ob 4.6 2.5

8.1

1.9

R

Chronic myelogenous Acute lymphocytica Acute myelogenous Acute monocytic

a Denotes child. b Hematological

values

Myleran Prednisone Mercaptopurine Mercaptopurine and Prednisone

returned

to normal

following

treatment.

from a barely detectable level to a finite level. Thus, in a recent study [7] all seven patients with nonhematologic neoplasms developed a measurable level of dihydrofolic reductase following amethopterin therapy. Furthermore, when both leukemic and non-leukemic patients were given a single 20 mg. infusion of the drug, dihydrofolic reductase appeared in both the ieucocytes and erythrocytes within one week. After reaching a peak value, however, the reductase activity thereafter declined in both types of cells, with the rate of decrease being more rapid in the leucocytes. Typical data [7] for the induc1 The term “induced” the stimulus of folic acid

is used here antagonists.

to indicate

only

the

appearance

Experimental

of enzyme

in response

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452

F. M. Huennekens et al. CM. leukocyte

,5 0.10 ',k 0.00 -

data

F 0.06 5 0.04 z a 0.02 5 o*----

0-e

-'

/Q

a’

age52

Apenocarc~noma

r '

I'

\

/'

9

olrtomach

\ '\

Dihydrofolic

o-------~~~~~~

reductase

---------O-

-“*

WBC ,o t ---______

4 ----

o- _____

- ----

0 ---------o-w--

---e----o--,

-0

5

B

9

10

11

12

13

14

15

16

17

18

Fig. 5.-Effect of a single dose of amethopterin submitted to J. Clin. Inuest.).

19 20 June

21 22

23

24 25

upon dihydrofolic

26

27

28 29

30

reductase (from Bertino et al.,

tion and decline of reductase in leucocytes, following a single dose of amethopterin, are shown in Fig. 5. A discussion of this effect and of the general problem of resistance to amethopterin is presented in a later section.

MODE

OF ACTION LEUKEMIC

OF FLUOROPYRIMIDINES

IN

LEUCOCYTES

Of comparable importance to the folic acid antagonists has been the introduction of fluoropyrimidines [22, 341 into cancer chemotherapy. These compounds, especially 5-fluoro-2’-deoxyuridine-5’-monophosphate (FUDRP) (cf. Fig. 6) selectively block the synthesis of thymidylate from deoxyuridylate [15, 331. In recent years, the mechanism of this reaction has been elucidated by studies from the laboratories of Friedkin [77], Greenberg [40], Blakley [47], Heidelberger [33] and Cohen [15]. The enzyme, thymidylate synthetase, catalyzes reaction [5] (shown across the top of Fig. 7) in which “active formaldehyde” 5N,10N-methylene tetrahydrofolate

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+ deoxyuridylate-+dihydrofolate + thymidylate

(5)

Chemotherapy

of leukemia

453

(SN1oN-methylene tetrahydrofolate) reacts with deoxyuridylate. In this complex reaction, which is apparently carried out by a single enzyme, the C, group is simultaneously transferred to the pyrimidine and reduced to the methyl level.1 Dihydrofolate, the other product of the reaction, is then recycled to tetrahydrofolate via the dihydrofolic reductase reaction, followed by the addition of a new C, unit at the oxidation level of formaldehyde (perhaps arising from serine). Since the fluoropyrimidines inhibit thymidylate synthetase and the folic acid antagonists inhibit dihydrofolic reductase, it is evident that the cycle in Fig. 7, in which the two enzymes are linked together functionally, represents one of the most important metabolic areas in cancer chemotherapy. The close relationship of these two reactions was anticipated, in fact, by the results of Totter and Best [73] who found that in bone marrow cells incorporation of 14C-formate into thymine is inhibited by aminopterin, and of Winzler et al. [86] who reported that in leukemic leucocytes amethopterin inhibits the incorporation of 14C-labeled formate or formaldehyde into DNA thymidylate. Even though folic acid coenzymes are required in rapidly dividing cells for the transfer of one-carbon units in several important metabolic reactions, OH I

iI O’H Fig.

sN, ‘ON-Methylcne

6.-5-Fluoro-2’-deoxyuridine-5’-phosphate.

Tetrahydrofolate t

+ dUMP

“C,“+ Tetrahydrofolate

+..

Fig.

7.-Pathway

Thymidylate Synthetase

-

dTMP

Dihydrofolic Reductace G of thymidylate

+ Dihydrofolate

(;TPpzH

biosynthesis.

r This reaction appears to be generally utilized by mammalian, avian .and bacterial systems. In phage-infected Escherichia coli, however, a variation on this reaction, catalyzed by deoxycytidylate hydroxymethylase, has been described [26] in which sN, IoN-methylene tetrahydrofolate interacts with deoxycytidylate to yield 5-hydroxymethyl deoxycytidylate and tetrahydrofolate. If this reaction followed the pathway of reaction (5), B-methyl deoxycytidylate and dihydrofolate would result, whereupon deamination of the former product would again yield thymidylate. Experimental

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F. M. Huennekens

et al.

the synthesis of the methyl group of thymidylate is evidently the reaction most susceptible to deprivation of folate coenzymes, or to inhibition by the folic acid antagonists. This conclusion is supported by numerous studies (reviewed by O’Brien [52]) in which treatment of viruses, tissue culture cells, bacteria, yeast, and mammalian tissues with folic antagonists causes greater reduction in DNA synthesis than in RNA (or protein) synthesis. Such inhibited cells develop an asynchronism between growth and cell duplicative processes. During the so-called “thymine-less death” of cells in culture, caused by withholding thymine or by treatment with folic acid antagonists, RNA synthesis continues but DNA synthesis is arrested [49]. Reisner [57] has described the morphological effects on cells in which a block occurs in the conversion of uracil to thymine: Since only an occasional cell synthesizes enough DNA to divide, there is an accumulation of immature “resting” cells. Thymidylate synthetase has been detected in leukemic leucocytes [62], but owing to the extremely low level of the enzyme,1 it is necessary to use a sensitive tracer technique to follow reaction (5). 5N,10N-methylene tetrahydrofolate, prepared in situ by the chemical interaction of 14C-formaldehyde of high specific activity (10 pcuries/,umole) with tetrahydrofolate, is allowed to react with deoxyuridylate and the resulting thymidylate is isolated (after degradation to thymine) with the aid of carrier and counted. Despite its scarcity and lability, the enzyme has been purified about 1O-fold from chronic myelogenous leukemic leucocytes and some of its properties have been measured. A component study for the synthetase reaction (pH optimum at 6.5) reveals that there is an absolute requirement for deoxyuridylate and 5N,10N-methylene tetrahydrofolate. The functions of Mg2+ and EDTA in this process remain to be elucidated. The enzyme is inhibited by the reaction product, dTMP, which provides a mechanism to prevent accumulation of this material when not needed for DNA synthesis. Leucocytes also contain an active deaminase [62] which converts deoxycytidylate to deoxyuridylate (eq. (6)) thereby enabling the former deoxycytidylate

+ H,O+deoxyuridylate

nucleotide to serve as a potential substrate The pH ogtimum for the deaminase occurs

+ NH,

for the thymidylate synthetase.2 at 8.0. In addition to the deoxyri-

r By comparison, in chronic myelogenous leukemia cells the formate-activating enzyme 5000-fold higher, and the lactic dehydrogenase is about 7.5 x 106 higher, than the level dylate synthetase. Thymidylate synthetase, therefore, is probably the rate-limiting step synthesis in the leukemic leucocyte (cf. also Bianchi 1111). * The dCMP deaminase in leukemic leucocytes offers a possible pathway for the in version of halogenated derivatives of cytosine [81] (e.g., 5-iodo-2’-deoxycytidine (ICDR)) corresponding uracil derivatives which might inhibit the thymidylate synthetase or incorporated into nucleic acids. Experimental

Cell

Research,

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9

is about of thymiin DNA uiuo conto the even be

Chemotherapy

of leukemia

455

botide, deamination also occurs with the deoxyriboside (deoxycytidine), the 5-methyl deoxyriboside, and the riboside (@dine). Neither the free base (cytosine), nor the various ribotides (q&dine mono-, di-, or triphosphates) are deaminated. Like its counterpart in other tissues, the leucocyte thymidylate synthetase is insensitive to the folic acid antagonists but it is inhibited very specifically TABLE

IV. Levels of thymidylate synthetase leucocytes [62].

Cell

Number subjects

type

of

in normal

and leukemic

Thymidylate synthetase m/lmoles/hr/mg

Normal Chronic lymphocytic Chronic myelocytic Acute leukemia

leukemia leukemiab

17 8 12 4 3

protein 0 0 0.19 + 0.02= 0.07 $0.03 not detectable

a Mean f standard error. b Thymidylate synthetase was found in all 12 patients who were in relapse, and the level could be correlated, in general, with the percentage of immature forms in the blood. In two additional chronic myelocytic leukemic patients who were in acute crises, the enzyme levels were 0.56 and 1.1 m,uM/hr/mg protein, respectively. Aminopterin, 4-amino PGA; Amethopterin, 4-amino, lo-methyl PGA; Dichloroamethopterin, 4-amino, lo-methyl, 3’, 5’-dichloro PGA.

by fluoropyrimidines. Neither fluorouracil nor the deoxyriboside (FUDR) are effective as inhibitors even at 1O-3 M, but the deoxyribotide (FUDRP) is a most potent inhibitor (40 per cent inhibition at lo-’ M). By comparison, Hartmann and Heidelberger [33] have reported a value of K, = 2.2 x 1O-8 M for the competitive inhibition of thymidylate synthetase from Ehrlich ascites cells by FUDRP. Of interest is the fact that the leucocyte contains an ATPdependent kinase which is capable of converting the otherwise non-inhibitory FUDR to an excellent inhibitor of the enzyme, presumably FUDRP. Thus, the leukemic cell may assist in its own destruction by virtue of this kinase which carries out a “lethal synthesis” of FUDRP.l The level of thymidylate synthetase has been measured in normal and leukemic leucocytes (cf. Table IV). Like the dihydrofolic reductase, this 1 FUDR would appear to be potentially useful in the treatment leukemia since it is a more readily available drug than FUDRP and, it passes through cell membranes more readily. Experimental

of chronic myelogenous being non-phosphorylated,

Cell

Research,

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9

F. M. Huennekens enzyme is present only extent) and in chronic found for the presence lymphocytic leukemic in normal rat liver, but TABLE

in acute leukemic cells (but in this case only to a small myelogenous leukemic cells.1 No evidence could be of the enzyme in the normal leucocyte or in chronic cells.* Thymidylate synthetase is barely detectable is lo-fold higher in regenerating rat liver, Morris 5123

V. Criteria

of proliferative

Mitotic Cell type

activity

Incorporation into thymidylate

activity

Marrow

Normal Chronic lymphocytic leukemia Chronic myelocytic leukemia Acute leukemia

ef al.

=HCOOH or 14HCH0

Blood

in leucocytes

[62].

DNA

%H Thymidine

Thymidylate synthetase

Dihydrofolic rednctase

+

0

0

0

0

i

0

0

0

+

0

+

+

+ + -t -1 +

+++-k

++ ++

++++

-t+++

++++ ++

+

++++

hepatoma, or the lymph node of a patient with Hodgkin’s disease [33]. Preliminary studies from our laboratory have shown that the level of thymidylate synthetase does not rise in leucocytes following therapy with FUDR. In Ehrlich ascites tumor cells made resistant to fluoropyrimidines, the purified synthetase is inhibited to a lesser degree by FUDRP [35]. The problem of tumor resistance to fluoropyrimidines has been discussed elsewhere [56]. It is instructive to compare, as in Table V, the various criteria of cellular proliferative activity of the leucocyte. The previously used indices, namely the incorporation of 14C-formate and l*C-formaldehyde, or 3H-thymidine [18, 58, 821, into DNA thymidylate, as well as the frequency of mitotic figures [2, 19, 291, are all maximal in the chronic myelogenous leukemic cells and are present to a smaller degree in the acute leukemic cells.3 These findings 1 By comparison, the mean level of deoxycytidylate deaminase in the leucocytes normal subjects and leukemic patients is 0.5 pM/hr/mg protein. a Thymidylate synthetase has also been detected in two patients with monocytic and in two non-leukemic patients with marked leucocytosis, i.e. a leukemoid reaction to carcinoma and an infectious mononucleosis. s Paradoxically, the acute leukemia cell may actually require a longer time for DNA than the proliferating cells of chronic myelogenous leukemia. As Craddock and Nakai pointed out, the intermitotic interval of acute leukemic cells could be longer than that precursors and still retain a growth advantage because of: (a) the large numbers of divisible cells; (b) the failure of maturation of leukemic blasts and retention of “stem ferative capacity; and (c) the longevity of the leukemic blast as compared with granulocyte. Experimental

Cell Research,

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of both leukemia secondary replication [18] have of normal potentially cell” prolithe normal

Chemotherapy

of leukemia

are paralleled by the present enzymatic studies reductase and thymidylate synthetase in normal

TREATMENT

OF LEUKEMIA

WITH

on the level of dihydrofolic and leukemic cells.

REFERENCE

TO DRUG

RESISTANCE

Studies on the biochemistry of leukemia reported in this paper, and most previous investigations as well, have utilized peripheral human leucocytes as the experimental system. In chronic myelogenous leukemia and, to a lesser extent, in acute leukemia there is present in the circulation a number of cells undergoing mitosis. These cells take up 3H-thymidine and fix it in the form of newly-synthesized DNA. From the pathways outlined in eq. (2) it is not surprising to find that these cells also contain an active thymidylate synthetase -dihydrofolic reductase system. This dual system has been detected in other tumors and ‘in growing bacteria, phage-infected cells, regenerating liver, embryonic tissue, and intestinal epithelial cells. Thymidylate synthetase and dihydrofolic reductase are reasonably well-documented enzymes from a biochemical standpoint and each is sensitive to a specific agent whose inhibitor constant (K,) is less than lo-’ M. Thus, although some improvement might still be desirable in the rate of uptake of these drugs by leukemic cells, it is evident that they are specific and effective in attacking their target sites. Even though leukemia is a disseminated neoplasm, with malignant cells being found in the, marrow, the peripheral circulation, the central nervous system [SO, 811, and even infiltrated into the soft tissues, massive amounts of these particular drugs should be able to destroy the leukemic cells by preventing cell division. Pursuant to this point, it has been shown that amethopterin is effective against mouse leukemia provided that the number of cells in the innoculum is small and the drug is administered promptly [68]. Unfortunately, other tissues of the body, notably the normal proliferating hemopoietic cells and the intestinal epithelial cells, cannot tolerate high doses of the drugs,1 so that the therapeutic regimen must be restricted to a level which is inadequate to destroy all of the leukemic leucocytes. Even if all of the leukemic cells could be prevented from multiplying, it is not yet certain whether the “leukemic” DNA might not persist and be re-utilized by cells in later generations. Finally, there is no evidence that leukemic leucocytes have any greater avidity, or faster rate of uptake, for the folic acid antagonists and this, too, limits the size and frequency of the therapeutic regimen. Similarly, 1 The observation of Welch and his colleagues [81] that azauridine, an agent which tively toxic for some types of human neoplasms, is essentially non-toxic to any normal cell, offers some hope that more selective folic acid antagonists might also be prepared. Experimental

Cell

Research,

is selechuman

Suppl.

9

F. M. Huennekens

et al.

the rate of uptake of fluorouracil is not sufficiently more rapid by tumors to make this agent selectively toxic even though most tumors incorporate 14Curacil, a normal metabolite, more rapidly than normal tissues [Se]. Because of the host-imposed upper limit for chemotherapy with the folic acid antagonists and the fluoropyrimidines, the effect of these drugs is only temporary. The number of malignant cells may be decreased and the patient may show hematologic improvement, but there is an inevitable recurrence to the previous clinical state as the patient becomes resistant to the drugs [43, 511. What is the basis of this acquired resistance whereby neoplastic cells exhibit this remarkable ability to adapt to the presence of drugs which are initially potent inhibitors of their metabolism? A variety of mechanisms [20, 801 may be responsible for the development of drug-induced “resistance”, inter alia decreased penetration of the drug into the cell, increased destruction of the drug, increased formation of metabolic antagonists to the drug, increased concentration of the enzyme affected by the drug, decreased requirement for the reaction inhibited by the drug, development of an alternate metabolic pathway by-passing the inhibited enzyme, and decreased affinity of the target enzyme for the drug. Our studies [B, 71 and those from other groups [25; 27, 31, 481 have shown clearly that treatment of malignant cells with amethopterin results in a rise in level of dihydrofolic reductase. But this is by no means a general mechanims since developing chick embryos [65] and the intestinal mucosal cells of the guinea pig [lo] do not develop an increased level of dihydrofolic reductase following amethopterin therapy, even though both tissues are rapidly proliferating and contain amethopterin-sensitive reductases. We had originally thought that the drug-induced rise in dihydrofolic reductase level was due to a process of cell selection, since evidence in support of this type of mechanism has been given for amethopterin-treated bacteria [51], cells in tissue culture [25, 311, and transplantable L-1210 leukemia cells [27]. A process of cell selection, in which cells containing a lower level of the target enzyme, are destroyed by the sub-optimal dose of drug, should be characterized by the persistance of an elevated level of enzyme in subsequent generations even after the drug is removed. On the other hand, human leukemic cells (or even normal leucocytes and erythrocytes) do not conform to this mechanism since a single dose of amethopterin produces a rise in the reductase level, but this is followed by a decline in the enzyme [7]. Interestingly enough, Hakala ef al. [31] have also observed a minor decrease in the reductase level in cultured S-180 cells when amethopterin was removed from the medium. Thus, there appears to be another, and as yet unexplained, Experimental

Cell

Research,

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9

Chemotherapy

of leukemia

459

mechanism by which the drug inhibits the enzyme and thereby induces the formation of higher amounts of enzyme in subsequent cell populations. In these single-dose experiments [ 71, the elevation and decline of the enzyme was roughly consistent with the life-span of the particular cells.1 Despite the fact that an elevated level of dihydrofolic reductase is the most clearly demonstrable biochemical event accompanying drug resistance, other factors cannot be overlooked. For example, isolated peripheral leucocytes (even from an untreated acute leukemia patient) do not take up 3Hlabeled aminopterin rapidly [74] and the resistant cells may be even less permeable to the drug. Inactivation of the drug must also be considered since Jacobsen and Cathie [41] have presented evidence that aminopterin is converted to an inactive form by leukemic bone marrow, leukemic cells of an acute lymphoblastic mouse leukemia, normal human red cells, chick fibroblasts, and mouse liver; normal bone marrow, normal lymphoblasts, or normal lymphocytes are unable to inactivate aminopterin. It is also possible that the level of thymidine kinase, ordinarily quite low in both normal and luekemic leucocytes [ 111. may rise when the dUMP+dTMP system is blocked and thereby provide an alternate metabolic pathway for the synthesis of DNA. Thus, ultimate success in the chemotherapy of leukemia would appear to depend upon three favorable circumstances: (a) improved drugs that are taken up more rapidly or more selectively by the leucocyte; (b) the ability to treat the leucocyte mass separately from the other body tissues (or conversely, protection of other tissues while the leukemic cells are being treated); and (c) prevention of the rise in level of dihydrofolic reductase and thymidylate synthetase in subsequent generations of drug-treated cells. The authors are deeply indebted to Dr. J. Richard Czajkowski, Director of the King County Central Blood Bank, whose interest and support made this work possible. We also wish to thank our colleagues, Dr. M. I. Freeman, Mrs. Aline Alenty, Mrs. Margaret Albrecht, and Mr. Paul Nieman for their assistance on these problems, and Drs. C. A. Finch, D. H. Coleman, Q. B. de Marsh, D. M. Donohue and A. Stevens for providing the leukemic blood samples and for many valuable discussions. REFERENCES 1. AISENBERG, A. C., The Glycolysis and Respiration of Tumors, pp. 20-23. Academic Press, New York, 1961. 2. ASTALDI, G., in CIBA Foundation Symposium on Hemopoiesis, p. 99. G. E. W. WOLSTENHOLME and M. O'CONNOR (eds.) Little, Brown and Co., Boston, 1960. 1 Condit [16] has shown that single, massive, intravenous doses of amethopterin result in a decrease in number of peripheral reticulocytes, leucocytes, and platelets with minimum values being reached in 4.6, 6.2, and 9.3 days, respectively. These time intervals are essentially the same as the life spans of the individual cells and indicate that amethopterin arrests the production or release of cells from the bone marrow. Experimental

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p. New

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