Alkaline Phosphatase of the Nucleus

Alkaline Phosphatase of the Nucleus M. CHRVREMONT

AND

H. FIRKET

Institid &Histologic, Universiti de LiPge, Belgium

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I. Introduction ......................................................... 261 11. Discussion of the Localization of Alkaline Phosphatase ............... 263 1. Diffusion of the Enzyme and Nuclear Adsorption ...... 2. Selective Precipitation of Calcium Phosphate on the Nuclei 3. Other Histochemical Methods vs. Gomori Reaction .............. 266 111. Alkaline Phosphatase in Nuclei ...................................... 267 1. Cytological Aspect ............................................... 267 2. Distribution in Various Adult Tissues ............................ 268 3. Alkaline Phosphatase in Chromosomes and during Mitosis. ......... 271 IV. Physiological and Experimental Variations .............. ...... 273 1. Regeneration . . ..................................... 273 2. Carcinogenesis . ..................................... 273 274 3. Embryonic Development .......................................... 4. Growth Variations in Tissue Cultures ............................ 275 5. Phosphatase and Mitotic Poisons ................................ 276 V. Functions of Nuclear Phosphatase ................................... 278 1. Unicity or Plurality of Phosphatases ............................. 280 VI. Addendum ................................................ _ . 282 VII. References .......................................................... 284

I. INTRODUCTION The first observations of mitotic figures by Flemming (18761882) and Van Beneden (1876) and on the morphology of resting nuclei by Heidenhain (1893-1907) were followed by those of numerous authors, and soon a thorough knowledge of the nuclear structure and of its importance for the transmission of hereditary characters was attained. On the other hand. information on the chemical composition of nuclei has been for a long time rather scanty. Until recently the only group of substances known to be present in the nucleus were the nucleic acids. These acids were discovered in isolated nuclei by Miescher (1871-1897), who found that they ivere acids distinct from proteins and who described their high content of organically bound phosphate. They were later studied histochemically by van Herwerden (1913, 1916) and by Feulgen (1924). It is well known that within the last fifteen years a large number of papers have been devoted to the localization and function of desoxyribonucleic acid and ribonucleic acid. Until comparatively recently, the distribution of other substances between nucleus and cytoplasm was studied very little. The important question of the localization of intracellular enzymes, for instance, was hardly

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investigated. To determine the exact function of these catalysts, it is essential to know whether they are dissolved in the cytoplasm, attached to small particles or to mitochondria, or located in parts of the nucleus. Information about this is now being collected rapidly, and it can be roughly said that most enzjmes are present in the cytoplasm, a number of them related to energy transfer are found in the mitochondria, and that perhaps only a few are present in the nuclei, probably with very special functions. One of the very first enzymes to be found in the nucleus was an alkaline phosphatase when Goniori (1939, 194la) and Takaniatsu (1939) introduced simultaneously a method for the histochemical detection of alkaline phosphatase (phosphomonoesterase X or I). More than 300 papers have been devoted to the histochemistry of this enzyme. Many have shown that, hesides specialized localizations studied before by biochemists (intestine, kidney, hone, etc.), nuclei in various tissues have a phosphatase activity and that chroinosonies are highly phosphatase positive. Nuclei isolated by differential centrifugation have also been found to contain this enzyme (Dounce, 1943). I n addition to its part in specialized functions, it is known now that this enzyme must intervene in general cell metabolism and probably in the processes of growth and cell division. A general review of the work published before 1946 on the histochemistry of phosphomonoesterases and their physiological significance has been given by Moog (1946b). In our report we shall entirely leave out acid phosphatases, which have a different optimum pH, probably have different physiological functions, and raise a high number of disputed questions. Neither shall we undertake to cover the interesting question of cytoplasmic phosphatase. W e shall deal only with alkaline phosphatase of nuclei, and we do not even plan to give a final and complete picture of this rapidly changing field. \Ve shall try to give a synthetic survey of what is known today, including not only well-established facts, but also some controversial points. JVe shall end by explaining in some detail the experiments that have produced information about the function of nuclear phosphatase and the hypotheses that can be inferred. The principal questions dealt with are : cytological distribution of the alkaline phosphatase in the nucleus; the amount in the nuclei of various tissues, and variations related to growth and mitosis. The physiological function and the possibility of existence of several phosphatases will be also discussed. B'ut, as some violent criticisms have been put forth about the validity of the localizations obtained and have led some authors to conclude that all pictures of phosphatases in nuclei were artifacts, we shall first enter into a discussion of the technical value of the histochemical methods.

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DI6CUSSION OF THE

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Among the very numerous histochemical studies on alkaline phosphatase, some were not made under technically blameless conditions. Though thesc had been defined by Danielli (1946), several authors used badly fixed material, excessively long incubation periods, or incubating mixtures prepared with too low a p H or unbuffered, etc. In some cases, even if the technique is correct, pictures of false localization appear. These facts have brought the method under adverse criticism, which sometimes has been harsh. We do not intend here to discuss at length technical details, most of which are essential to a correct execution of the methods; this was done recently by Richterich (1952). There is no disagreement concerning the chemical significance of the Gomori reaction, provided that suitable controls are made, and the numerous discussions, written or not, are essentially centered on the presence or absence of alkaline phosphatase in the living nuclei. T o clarify this question, we shall consider separately all the theoretically possible artifacts : (1) diffusion of the enzyme followed by adsorption on the nuclei; (2) selective precipitation of calcium phosphate on the nuclei independently of enzyme distribution and later displacement of this salt or its substitution products (in the Gomori technique), (3) Finally, we shall compare the results of the Gomori reaction with those of other histochemical methods.

1. Difusion of the E m y m e and Nuclear Adsorption Diffusion of the enzyme can theoretically occur in all methods used to localize the enzyme, whether chemical or histochemical. But it is much more dangerous in organs where the Gomori reaction reveals both cytoplasmic and nuclear locations of phosphatase than in tissues where only the nuclei are positive. I n organs rich in phosphatase (small intestine, kidney, etc.), there is always a cytoplasmic structure with a very high activity, the nuclei becoming positive only after a longer incubation. I n these cases, Jacoby and Martin (1949) demonstrated a possibility of artifact. By superimposing an active section on an inactivated one, a positive reaction can be obtained in some nuclei of the section which does not contain an active enzyme. This is probably due to a diffusion of labile enzyme followed by adsorption on nuclei. The possibility of a similar diffusion, either during the preparation of the tissues or during incubation, is also admitted by Lison (1948), Feigin, Wolf, and Kabat (1950), and Yokoyama, Stowell, and Matthews (1951). I t must be stressed that the possible displacement of phosphatase on the nuclei is not due to the adsorbing power of desoxyribonucleic acid (DNA) ; the phosphatase distribution and the possibility of artifact are unchanged if DNA is hydrolyzed

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(Brachet and Shaver, 1948; Kovikoff, 1951a) (see also p. 271). Leduc and Dempsey, using technical conditions similar to those of Martin and Jacoby ( 1949), suggest that diffusion of natural activators is mainly responsible for the abnormal positive reactions obtained (1951a, b ; ref. 1951a is followed by an interesting discussion). In fact, when a purified enzyme is added to inactivated sections, usually no adsorption on the nuclei takes place, unless the concentration of added phosphatase is very high (Gomori, 195Oa; Novikoff, 1951a) or the p H is below 8.5 (Gomori, 1950a; Leduc and Dempsey, 1951b) . It is to be pointed out that this artifact has been shown to occur only in nuclei situated near cytoplasmic structures especially rich in the enzyme, such as the borders of the intestine and the kidney tubules. The phosphatase is perhaps present in a special physical state or not strongly attached (lyoenzyme?). On the contrary, it does not diffuse to neighboring nuclei from other equally enzyme-rich structures. Such is the case for chorioid plexuses (Wislocki and Dempsey, 1948 ; Gerebtzoff, Ninane, and Firkrt, 1949) and also for the vagina after estrogen administration, where nuclei appear as clear spots in a blackened cytoplasm (Jeener, 1948). By excessive generalization and the erroneous belief that the above artifact occurs in all tissues, a few authors doubt or even deny the presence of alkaline phosphatase in all nuclei (Ruyter and Neumann,* 1949; Novikoff, 1951a). Unfortunately, the method of difereiztial centrifugation and chemical estimation of alkaline phosphatase in the nuclear and the cytoplasmic fractions is subject to similar pitfalls when dealing with the same organs. There is even more danger of diffusion during the destruction of cells and centrifugation than during fixation and embedding. Though it has furnished good evidence in favor of a nuclear location of phosphatase, this method sometimes yields conflicting results. There are other tissues where, under normal conditions, nuclei only give a positive Gomori reaction, no other intra- or extracellular structure being blackened in the neighborhood, even with longer incubation time. A priori, there is then less chance of gross artifact. The validity of the technique has been controlled in only a few such cases, the main example being Falivary glands of Diptera and tissue cultures. The giant nuclei of Diptera salivary glands are Gomori positive in dissected and squashed material ( Danielli and Catcheside, 1945 ; Krugelis, 1945, 1946), and if the nucleus is extracted from the living cell by rapid micromanipulation, the Gomori reaction is still positive (Mulnard, unpublished).

* These authors and, more recently, Goetsch, Reynolds, and Bunting (1952) think that by incuhating nondeparaffinized sections they remedy the enzyme diffusion. This procedure only slows down considerably the enzyme activity and exactly the same pictur+including positive nuclei-is obtained by lengthening the incubation.

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Tissue cultures are a particularly favorable material for testing histochemical techniques ; they are thin and need not be embedded or cut ; cells are rapidly reached by fixatives at optimum concentration, etc. With thls material (fibroblasts and myoblasts) , Firket ( 1952) made a thorough study of possible artifacts. As long as fixation is correct (alcohol 80% or more, ireeze-drying), a normal picture of positive nuclei is obtained, cytoplasm remaining negative. But if the cultures are fixed with 30% alcohol (which does not precipitate all the phosphatase), a large diffusion from the nucleus to the cytoplasm takes place. From this and other experiments, the conclusion seems unavoidable that in the living cells of this type most, if not all, of the phosphatase is located in the nucleus. One of the main causes of the unreliable results obtained with some tissues is the usual embedding procedure. It is well known that after such an embedding, there is a loss which can reach 90% of the total phosphatase activity (Danielli, 1946 ; Capellin, 1947 ; Stafford and Atkinson, 1948). But the enzyme can also be shifted from one place to another. This can be seen by comparing cultures rapidly fixed and directly incubated to cultures fixed and embedded in paraffin (and later deparaffinized in conditions similar to those of the usual histological technique). These last manipulations not only produce a great loss of enzyme activity-nucleoli only appearing positive--but also bring about a tendency to irregular precipitates. The abnormal blackening of nuclei in highly active tissues is considerably reduced when the tissues are embedded by the freeze-drying method. This technique, which has already been used by Deane and Dempsey (1945), Wang and Grossman (1949), Yokoyama and Stowell (1951), Danielli (1953), Firket (1952), etc., prevents to a large extent the possibility of diffusion of the enzyme previous to the incubation and also decreases G r suppresses the loss of enzyme. With a generalized use of this method, perhaps more sites of low phosphatase activity will be found. 2. Selective Precipitation of Calcium Phosphde on th.e Nuclei Another possibility of artifact we shall deal with briefly occurs during incubation. It is the abnormal precipitation of calcium phosphate on nuclei different from, but situated near, structures having an enzymatic activity. This cannot be overlooked, as several authors mention a selective adsorption of Cas (PO4) by nuclei in sections when a nonenzymatic precipitation was produced (Danielli, 1946 ; Gomori, 1950a ; Cleland, 1950; Novikoff, 1951a). However, when the Gomori method is correctly executed (pH, concentration of calcium, relatively short incubation), this probably happens only to a small extent. It can be checked in each case by one of the other histochemical methods for phosphatase in which an organic

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substance is precipitated and visualized. See for instance the demonstrative mirror pictures given by Danielli (1946) ; see also Section 11.3, below. This affinity for calcium phosphate is not a property of all nuclei. I t does not exist, for instance, in tissue cultures. I n this case a nonenzymatic precipitate of Ca3(P04)2 settles irregularly inside and outside the cells, on cytoplasm as well as on the nucleus (Firket, 1952). A displacement of the reaction product after its precipitation during incubation is described by Moe (1952) in intestine. So, in each case it must be verified that there is no shift of the precipitate or its substitution products. Calcium phosphate can be visualized by other techniques : K6ssa’s silver method, B’ourne’s alizarine sulf onate method, or direct visualization in red polarized light (BBanger, 1951a, b) ; these give in tissue cultures the same results as the cobalt sulfide (Firket, 1952). Another aspect of phosphate precipitation should not be forgotten. Very small aiiiounts of phosphatase will produce PO4 ions at a lower rate than the same ions can diffuse in the medium ( Goniori, 194%). Structures appearing positive only after more than about 12 hours’ incubation have such a low activity that this possibility becomes important and it must be feared that the localization of the precipitate is unreliable.

3. Other Histoclietiiical Methods vs. Goiriori Reaction Other histochemical methods, based upoil the precipitation of the organic part of the phosphoric ester molecule, are proposed for the detection of alkaline phosphatase (hienten, lunge, and Green, 1944 ; Danielli, 1946; Loveless and Danielli, 1949 ; Manheimer and Seligman, 1948). In the method of Manheimer and Selignian, which is more readily practicable than the others, a phenol is liberated and is coupled with a diazonium salt, giving a reddish precipitate. These methods can be used to check the xcoiid type of possible artifact mentioned. They are less sensitive to low phosphatase activity than the usual one, but contrary to what is often said (Goniori, 1951), phosphatase activity with them has been detected in the nuclei of several tissues :Danielli (1946) and Loveless and Danielli (1949) in the kidney ; Lorch ( 1947) in kidney and bone ; Firket (1952) in tissue cultures. However, owing to their weak sensitivity, only nucleoli and chromosomes are positive in the favorable case of tissue cultures. To conclude this technical discussion the following points emerge. As usual for histochemistry, good and rapid fixation is essential ; when cells are badly fixed, diffusion of the enzyme can occur. Embedding in the usual histological procedure entails a great loss of enzymatic activity and further enhances the possibility of diffusion. This emphasizes the advantages of very thin preparations (tissue cultures, smears, isolated cells) and,

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for tissues that must be sectioned, of the freezing-drying method. The development of the reaction must be followed from the beginning, and the incubation time must be kept within limits. When submitted to adequate controls, including diffusion controls, the results of the Gomori reaction may be considered as corresponding to the localization of the enzyme in the living cells. In the literature, where the best technical conditions are not always fulfilled, several observations of alkaline phosphatase in nuclei must be considered as due to artifacts (apparently strongly positive nuclei at the top of the intestinal villi, for example). In others, the picture obtained does not correspond quantitatively to the true activity in the living cell: either a diffusion increases the intensity of the reaction or previous treatment results in a loss of enzymatic activity. But other observations undoubtedly correspond to phosphatase activity in the living nuclei. These considerations should be kept in mind when reviewing the literZ-ture, and some results have to be interpreted with caution.

111. ALKALINEPHOSPHATASE IN NUCLEI 1. Cytological Aspect Before comparing the nuclei of various tissues or studying their functional variations, we shall describe the detailed aspect of nuclei stained by the Gomori reaction in a suitable example, i.e., tissue cultures. With these, the technical conditions for the histochemical reactions are favorable ; no artifacts are present, and alkaline phosphatase activity is marked in the nuclei. Here is the picture given by fibroblasts and myoblasts actively growing iiz vitro (Chkvremont and Firket,* 1949a, b). After 6 hours’ incubation, the nucleus is very positive in a completely negative cytoplasm. The single or the two nucleoli look like black spots, which is the sign of a high enzymatic concentration. The chromatin granules, scattered in the nucleus, are dark brown, less positive than nucleoli but clearly defined (Fig. 1). Nuclear membrane and sap are not visible. If the development of the reaction is followed with increasing incubation times, nucleoli appear first (21 hour), then become blacker while the chromatin granules begin to appear ( 2 hours), and are more positive later (6 hours). Even with prolonged incubation, within normal limits, no other structure is positive. These results agree on the whole with those of Willmer (1942), Rodova?

* Gomori reaction

(1949 and 1952).

as standardized by Danielli (1946) and adapted by the authors

t Rodova used cultures containing both fibroblasts and osteoblasts. In these last cells, which have similar morphology but intervene in bone formation, cytoplasm is also positive.

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(1948), Levi and Fajer (1950), Paff, Montagna, and Bloom (1947), and Biesele and Wilson (1951) in cultures of the same or other tissues. A detailed cytological study in ordinary histological sections has been made in rare cases with valid technical conditions. An aspect similar to the

FIG.1. Intermitotic nuclei; cytoplasms are negative ( X 1.350).

nuclei of cultures is found, namely in liver, by Brachet and Jeener (1948), etc. Other investigators mention a positive reaction also in the nuclear membrane (Wachstein, 1945), principally in the nuclear membrane and the nucleoli ( Sulkin and Gardner, 1948) , principally in the nuclear membrane {Baud and Fulleringer, 1948; Baud, 1949) or even in nuclear membrane and caryoplasm (Wang and Grossman, 1949). Most of these results were obtained in liver nuclei. 2. Distribution in Various Adult Tissues

It may be admitted that various kinds of nuclei contain an alkaline phos phatase. This is found in vertebrates and invertebrates. But there are indeed large quantitative variations. Different tissues have a different amount in their nuclei, and in the same tissue the enzymatic activity can vary in relation to spontaneous or experimental metabolic changes. Even in the nuclei richest in phosphatase, the activity is not extremely high. It can probably be estimated at one-twentieth or one-thirtieth of that of the striated border of the intestine. With the Gomori reaction the minimum incubation time to demonstrate calcium phosphate precipitation is a few minutes in the latter and about 1 hour in the former. If variations in the same tissue can be more easily analyzed (see Section IV) , the quantitative

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comparison between nuclei of various origins is rather difficult. Authors sometimes used different technical conditions, and their results are not always comparable. Further, some erroneous interpretations have been given, and, as we already mentioned, in several cases there is a possibility of artifact. Comparative histochemical studies of the distribution of alkaline phosphatase in various organs and tissues were made by Gomori (1941a), Takamatsu (1939), Kabat and Furth (1941), Bourne (1942-1944), Krugelis (1946), Brachet and Jeener (1948), Newman et al. (1950). In sddition to these surveys, many authors have studied one or two organs. In spite of numerous observations quoted in the literature, there is not always agreement for the nuclei of a given cellular type. This can sometimes be explained by differences among the animal species used. Let us take for instance the case of adult hepatic cells which were investigated by Gomori (1941a), Kabat and Furth (1941), Bourne (1942-1944), Deane and Dempsey ( 1945), Wachstein ( 1945), Krugelis ( 1946), Jacoby (1946a), Wachstein and Zak (1946a, b ; 1950), Deane (1947), Zorzoli and Stowell ( 1947), Sulkin and Gardner ( 1948), Wang and Grossman ( 1949), Ebner and Strecker (1950), Hard and Hawkins (1950), Newman ef al. (1950), Leduc and Dernpsey (1951b). In various species (guinea pig, rat, rabbit, mouse, dog, cat, and man) and with long or short incubation times, their results differed considerably. Some showed no phosphatase at all, others found that nuclei (some or all) are positive; bile canalicules sometimes contained the enzyme, sometimes did not. This rather confused situation was not clarified by the results of centrifugations. In ultracentrifuged pieces of frog liver, chromatin and alkaline phosphatase were together displaced at the centrifugal end of the nuclei (Jeener, 1946). In nuclei isolated from centrifuged liver homogenates, Dounce (1943, 195Oa, b) and Mirsky (1947) found most of the phosphatase activity of the tissue. Recently, however, Allfrey, Stern, and Mirsky (1952), employing a modified Behrens technique (Behrens, 1939), questioned the validity of these results. If it is possible to draw a conclusion for liver cells from these observations, we would say that phosphatase activity of liver cells is never high. Tn the guinea pig, the reaction is negative (in cells and in bile canalicules) . I n the rabbit, there is a relatively high activity in the bile canalicules and a lower one in the nuclei (caution for possible diffusion!). In the rat, the reaction is positive only in the nuclei but rather weak" and negativt: elsewhere. Recent evidence suggests variability according to age (Zorzoli, 1951), diet (Ely and Ross, 1951), and other metabolic changes (Annau See the Addendum.

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and Manginelli, 1950). Apart from those of the guinea pig, the hepatic nuclei may contain alkaline phosphatase but not much. For many other tissues the data are fortunately more in agreement and a quantitative comparison between tissues is valid within limits. I n several organs, however, the phosphatase reaction is not limited to nuclei and the enzyme is usually related to a special metabolism. With these reinarks in mind, the following survey of the distribution of phosphatase in nuclei can be given. Nuclei of stem cells of lymphatic follicles in the spleen and elsewhere (Ivislocki and Dempsey, 1946; Newman et al., 1950) and those of hematopoietic marrow (Bourne, 1944 ; Wachstein, 1946 ; Wislocki ef ul., 1947 ; Rabinovitch and Andreucci, 1949) contain a notable amount of phosphatase. This fact is probably in relation to their mitotic activity. In the skin the most intense reaction is found in the nuclei of the basal cells of the sebaceous glands (Montagna and Noback, 1947 ; Montagna, 1952) that divide actively, and also in hairbuds when they are beginning to grow, decreasing later when the hair root is formed (Johnson and Bevelander, 1946). In the basal area of the epidermis, there is a little phosphatase in nuclei (Kewman et al., 1950). In some lung cells, a variable amount of phosphatase is found in the nuclei only (Policard and Fulleringer, 1949). In striated muscles and myocardium the reaction of nuclei which generally do not divide in normal conditions, is slight even after a prolonged incubation iBourne, 1944; Newman ~t nl., 1950j . Other nuclei have practically no phosphatase activity, for example, bird erythrocytes (Brachet and Jeener, 1948; Allfrey, Stern, and Mirsky, 1952), even with an incubation of 15 hours. Nuclei of adult nervous tissues do not usually show any phosphatase either (Landaw, Kabat. and Newman, 1942 ; Brachet and Jeener, 1948). Several glands (pancreas, salivary glands) produce and excrete a phosphatase and show a strong reaction in the nucleus and cytoplasm of some of their cells (Jacoby, 1946b ; Deane, 1947; Ifrang, Grossman, and Ivy, 1948) . Endocrine glands contain various amounts of phosphatase, the activity appearing essentially nuclear in pituitary (Abolins, 1948) and thyroid (Dempsey and Singer, 1946 ; Grunt and Leathem, 1949; Steger, 1950). But, in general, phosphatase in endocrine organs is essentially related to hormonal factors (Dempsey, Greep, and Deane, 1949 ; Soulairac, Desclaux, and Teysseyre, 1949), and probably plays a part in the gland’s special function. It may be pointed out, however, that Pritchard (1947) mentions that the reaction in placenta is predominantly in nuclei only during the stages of proliferation and differentiation. I n some tissues, nuclei appear to have a rather high enzymatic activity. After half an hour’s in-

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cubation nuclei are positively stained in epithelium of the small intestine or in the kidney tubules; unfortunately, the greater part of this apparent activity is probably due to a diffusion artifact, as stated above. I n most of these cases, it is difficult to appreciate the real phospliatase activity linked with the metabolism of the nuclei themselves. Thus, the nuclei of several adult tissues contain an alkaline phosphatase. but in various amounts. When no other factors interfere (glandular activity, diffusion artifact, etc.), the phosphatase activity in nuclei seems to be roughly parallel to the mitotic activity of the cells. On the other hand, the rate of renewal of the DNA phosphorus measured by radioactive phosphorus (Hevesy, 1948) shows a satisfactory correlation to the intensity of the Gomori reaction in sections of a series of organs (Brachet and J eener, 1948).

3. A I k d h e Phospliatase

ilz

Chromosomes and during Mitosis

All authors agree on the characteristic fact that chromosomes are rich in phosphatase activity. In 1942, Willmer observed that in tissue cultures chromosomes give a very positive Gomori reaction ; this was confirmed briefly by Fell and Danielli (cited by Danielli and Catcheside, 1945). Krugelis found in cells of mouse testes a clearly positive reaction in the chromosomes (1942) ; the reaction was particularly marked in spermatogonia and primary spermatocytes. Chromosomes and nuclei of plant cells are also positive (Ross and Ely, 1951). It is also remarkable that transversal bands of giant chromosomes in Diptera salivary glands are positive for both the Feulgen and the Gomori reactions (Danielli and Catcheside, 1945 ; Krugelis, 1945, 1946). Alkaline phosphatase in these glands was also measured chemicaIly (DoyIe, 1948). Phosphatase is also found in chromatic threads or so-called “chromosomes” isolated by centrifugation from various organs (Mirsky, 1947). The enzyme is firmly bound to the “residual chromosome” after extraction of DNA and shaken off only by autolysis. Other experiments also show that phosphatase is part of a protein fraction readily separable from DNA (Jeener, 1946). W e confirmed in tissue cultures (1949a, b) that chromosomes are inarkedly positive and clearly outlined (Figs. 2 and 3). At very high magnification these somatic chromosomes often show Gomori positive graitis or transversal bands ; these are single at first and are doubled at the longitudinal splitting of the chromosomes. This seems to correspond to the morphological aspect of chromonieres described in other materials (giant chromosomes, some favorable meiotic and vegetal cell chromosomes), During mitosis, the cytoplasm of fibroblasts and myoblasts, which otherwise is negative, become slightly but distinctly Goinori positive, specially

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from the metaphase onwards. This aspect is not due to an artifact and is confirmed by the Mannheimer and Seligman technique (Firket, 1952). I t suggests that the enzyme spreads out into the cytoplasm from the nucleus, perhaps mainly from the nucleolus when it fades out and the nuclear membrane disappears. This, incidentally, is an example of a special process

FIG.2. Metaphase with very positive equatorial plate. The cytoplasni is slightly stained, the spindle is seen only through refringence ( X 1.215).

FIG.3. Chromosomes in a dividing flattened cell (X1.610).

which Seenis t o occur during mitosis. Actiye substances leave their substrate or the structures to which they are attached and are shed in the cytoplasm ; metabolic conditions are very modified. There are more contacts between molecules, and it can be supposed that this is favorable to synthesis. Similar displacements can be inferred from the behavior of mitochondria during mitosis (Chhremont and Frederic, 1952). Does the total amount of alkaline phosphatase increase during mitosis?

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As there is no precise basis for quantitative estimation, it is difficult to conclude with certainty, but we believe that this enzymatic activity of the cell is raised.

IV. PHYSIOLOGICAL AND EXPERIMENTAL VARIATIONS W e shall now see that, for a given type of cell, the phosphatase activity

of the nuclei can vary considerably in a number of physiological states and experimental conditions. W e shall consider mainly regeneration, carcinogenesis, embryonic development, induced variations of growth in tissue cultures, and action of some antimitotic substances.

1. Regenm~tion The Gomori reaction becomes more intense in the process of regeneration of many tissues. It is well known that within a few days after partial hepatectomy liver is regenerating actively. When growth and mitotic activity are most intense, the cobalt precipitate in the nuclei is notably increased (Brachet and Jeener, 1948; Sulkin and Gardner, 1948). The same increase of phosphatase is also measured in nuclei isolated by centrifugation (Novikoff, 1951b). At the same time, the turnover rate of DNA-phosphorus is considerably higher (Brues, Tracy, and Cohn, 1944), and there is also a rise of R N A in the cytoplasm (Drochmans, 1947, 1950; Stowell, 1948). It was mentioned above that normal chick erythrocytes contain practically no phosphatase. But when an anemia is provoked by injection of phenylhydrazine, the regenerating erythrocytes become numerous in the blood and show a notably positive reaction only in their nuclei (Brachet and Jeener, 1948). Chemical estimations on isolated nuclei give the same results (Jeener, 1946). Regeneration of planaria and amphibian tail is also accompanied by an increased enzymatic activity of the nuclei, a lesser increase being found in cytoplasm (Moyson, 1946 ; Junqueira, 1950). I n the regeneration of mammal skin the picture is more complex, as the main phosphatase activity is linked to neoformation of collqgen fibers (Fell and Danielli, 1943). When the maximum mitotic activity occurs at a different time, an increased reaction can be observed in the nuclei of the epidermis (Firket, 1950, 1951). 2. Caycinogewsis

In the course of various cases of spontaneous or experimental carcinogenesis, alkaline phosphatase in the nuclei of tumor cells is increased in comparison to that of the original tissue. Except for the special case of osteogenic sarcoma (Kabat and Furth, 1941), with a high phosphatase content in the cytoplasm (related to bone

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formation) and nuclei, this rise is generally confined to nuclear phosphatase. I t is found in fibroadenoma of the breast (Kabat and Furth, 1941 ; Rondoni, 1947), in several hepatonias (White, Dalton, and Edwards, 1932), in embryonal carcinoma of the testis and niyogenic carcinoma (Woodward, 1942), in tumorous mastcells (Paff, Montagna, and Bloom, 1947) and in others (Hard, et d.,1948; King and Nigrelli, 1949). Cultures of cancer tissues also contain a notable amount of enzyme in the nuclei (Biesele and Wilson, 1951). It is remarkable that in several cases of experimental carcinogenesis the increase in alkaline phosphatase of the iluclei is one of the first changes to be observed with proliferation. It was found in chemical cancers of epidermis (Biesele and Biesele, 1944) and of liver ( Woodward, 1943 ; Mellors and Subiura, 1948 ; Pearson, Novikoff, and Morrione, 1950). Other data on phosphatase in cancer tissues are found in chemical estimations that, of course, do not give information on cytological distribution of the enzyme. Greenstein (1942 and 1943) found an increase of alkaline phosphatase in some mouse and most rat cancers. Before him, Edlbacher and Koller (1934) already mentioned a similar rise in Jensen sarcoma, and Kohler (1940) made the interesting observation that phosphatase was increased in tumor-bearing animals during the early stages of growth and decreased later. X few other data do not show a notable difference between the normal tissue and the cancer derived from it. These results are sometimes misleading, as the pieces of tissue used for tests can contain both active tumor tissue and necrotic parts. The rate of yroliferation of the tumor is also a factor which must be taken into account.

3. Etiibi-yoitic Llevelopnzent The results of histochemical and chemical studies show that embryonic tissues usually have a higher phosphatase content than adult tissues. An important synthesis of phosphatase in nuclei takes place, mainly when the embryo i s entering an active stage of growth and differentiation (Moog, 1946a; Brachet, 1946; Brachet and Jeener, 1948; etc.). The Goniori reaction is usually not strong in germinal vesicles and in fertilized oiicytes (B'rachet, 1945 ; Krugelis, 1947a, h ; Wicklund, 1948 ; Bradfield, 1949) ; the increase is very small during early cleavage (Brachet, 1946 ; Krugelis, 1947a, h ) . From gastrulation onward, a marked rise in enzyme actkity takes place. In the gastrula and pluteus of Arbaciu the phosphatase content of nuclei increases together with their basophilia and the intensity of Feulgen reaction (Krugelis, 1947a). I n amphibians the reaction is intense in cells of the animal pole during cleavage, and in the gastrula a cytoplasmic and nuclear gradient is found from ectodermis to

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mesoderm and entoderm (Brachet, 1946 ; Krugelis, 194713). Later, nuclei remain more positive than in adults. Usually their reaction decreases progressively while specialized cytoplasmic localizations appear (Moog, 1944 ; Elftman and Copenhaver, 1947), but we will not enter here into a detailed description of these changes. By means of chemical estimations, several authors confirmed the steep rise of phosphatase, beginning at gastrulatioil in various zoological orders : in chick (Moog, 1946a) ; in sea urchin (Mazia, Blumenthal, and Bknson, 1948 ; Gustafson and Hasselberg, 1950), and in amphibian (Krugelis,

1950).

Some embryologists interpreted these results as indicating that the main function of alkaline phosphatase during embryonic development must be correlated to processes of differentiation and organogenesis (Moog ; Brachet and Jeener; Krugelis). There is a little phosphatase in nuclei during cleavage though divisions are very active, contrary to what is found in the other cases of high mitotic activity. To explain this exception it may be pointed out that cell divisions during segmentation are not identical to somatic mitoses. They tend to distribute cytoplasmic and nuclear material in a number of smaller territories; the total volume remains the same, and there is no increase of total protein and of RNA (Steinert, 1951) or of the amount of desoxyribosides until late cleavage (Hoff-Jorgensen and Zeuthen 1952). On the whole, during embryonic development phosphatase is not abundant in the nuclei at first, later it increases notably, and finally it decreases, more o r less rapidly according to the tissue, toward values obtained for adult nuclei.

4 . Growth Variations in Tissue Cultures Most of the observed changes in alkaline phosphatase of the nuclei are perhaps more easily understood when the variations found in tissue cultures are considered. These constitute a very favorable material, for controlled inodifications of growth can be produced in the same type of cells and in relatively simple conditions. ChPvremont and Firket (1949a, b) have demonstrated that the phosphatase activity is then closely related to the mitotic activity. Under normal growth conditions (fibroblasts and myoblasts in hanging drop) a correlation exists between the intensity of the Gomori reaction and the growth of the cultures estimated by mitotic indexes and area measurements. In a given culture the zone where mitosis frequency is the highest is also the one where the reaction is the strongest. The same relation is constantly observed if the growth of the cultures is modified

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experimentally. When growth slows down or stops after a few days of cultivation, the reaction becomes weaker or negative. If growth is maintained by washing and partial renewal of the medium, the reaction remains positive. Tissues cultivated in several media, variously favorable to proliferation, give very demonstrative results. The frequency of mitoses and the alkaline phosphatase of the nuclei present remarkably parallel changes. For instance, when, after 3 days, cultures with only a small increase of area (1.25 times) are compared to others proliferating actively (surface increased more than 15 times), the nuclei of the former are weakly or not stained at all, whereas in the latter they are very dark. When the surface increase is moderate, the reaction in the nuclei is also medium. The phosphatase activity of the nucleus is modified together with the proliferation intensity. This phenomenon is clearly demonstrated in tissue cultures but is not limited to them. The nature of this relation and the function of nuclear phosphatase in the cell will be more precisely analyzed below.

5. Phosphatase and Mitotic Poisons As alkaline phosphatase of the nucleus is in some way related to the processes of cell division, it would be interesting to determine whether known antimitotic substances have an action on this enzyme and whether phosphatase inhibitors are able to disturb mitosis. Only a few data are now available on the action of antimitotic substances on phosphatase. In oocytes of Asellus oquutkus, a high mustard gas concentration, which produces chromosomic abnormalities and rapidly kiUs the cell, inhibits alkaline phosphatase in chromosomes, but not in nucleoli ; mercury salts have a similar action (Montalenti and de Nicola, 1948a). On the contrary, under the influence of colchicine injected into the rat, alkaline phosphatase is increased in nuclei of liver cells, especially in nucleoli (Ebner and Strecker, 1950). As Lang, Siebert, and Oswald (1949) conclude from chemical investigations, the action of colchicine on the spindle at very low concentrations cannot be related to the inhibition cf phosphate-splitting enzymes obtained only at very high concentrations. Similarly, cyanide prevents cytodieresis in tissue culture at much lower concentration than it inhibits phosphatase (Chhremont and Firket, 1950). Lithium inhibits growth of the same material but has no action on the Gomori reaction in nuclei (Chkvremont-Comhaire, 1952). In chemical tests Schoetensack (1948, 1950) found an activation with colchicine, a decrease with hydroquinone, and no effect with urethane on the alkaline phosphatase activity of kidney extracts. It seems thus that the antimitotic substances studied do not exert their

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action through phosphatase inhibition. The only positive evidence of a link between the two (Montalenti and de Nicola, 1948a) seems unconvincing. If alkaline phosphatase of the nucleus has a function during mitosis, specific inhibitors of this enzyme added to living cells must produce disturbances of mitosis and decrease growth. Unfortunately, most chemical agents which can modify phosphatase activity are also toxic for other enzymes and other cell mechanisms, often at lower doses. They cannot, therefore, be used to study the effects of selective inhibition of phosphatase in living tissues. One substance is of special interest for this researchberyllium. Recent biochemical investigations showed that it inhibits alkaline phosphatase at low concentration (Grier, Hood, and Hoagland, 1949 ; Klemperer, Miller, and Hill, 1949; Dubois, Cochran, and Mazur, 1949) but it has no similar action on numerous other enzymes (Klemperer, 1950). Added to living cells, beryllium ions produce mitotic abnormalities and a marked inhibition of growth; in the same cells the Gomori reaction is negative (Chevremont and Firket, 195la, b, c, 1952a, b) . I n living cultures observed by phase contrast and cinemicrography, half of the mitoses present peculiar abnormalities : after a prolonged metaphase, synthesis of chromatin does not take place, chromosomes do not split, and one resting nucleus is reconstructed in the cell which does not divide ; there is neither anaphase nor telophase. This effect is different from that obtained with other mitotic poisons. The histochemical analysis of these cultures shows a decrease of DNA in nuclei, marked by a Feulgen reaction weaker than in controls, and probably an increased cytoplasmic basophilia in the round metaphases and the reconstruction stages of the mononucleated single cells. Cytoplasmic granules, the exact chemical nature of which has not yet been established but which are believed to be phosphates, are stained by the cobalt sulfide technique used in the Gomori method (Chkremont and Firket, 19.51~). Beryllium enters the nuclei and by Denz’s histochemical method is visualized there, in higher amounts in the chromosomes than in the cytoplasm (Chbremont and Firket, 1952b). Such mitotic abnormalities are also produced when beryllium is injected into animals. Besides stress effects and direct alteration of the liver, Ninane and Pepinster (1951) observed a decrease in the total number of mitoses and a relative increase of the number of metaphases in orgati sections, similar to that found in fixed cultures. In treated animals, the Gomori reaction is decreased in nuclei of several organs (intestine, adrenal cortex, etc.) but not in brush borders of kidney and intestine or in adrenal cortex cytoplasm. This would suggest a more specific action of beryllium on nuclear alkaline phosphatase. If the main effect of beryllium in the living cell is to inhibit alkaline

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phosphatase, the observed mitotic alterations would be consequences of this inhibition. To prove this without the shadow of a doubt, we should demonstrate that beryllium has no direct effect on any chemical substance, known or unknown, within the cell but on alkaline phosphatase. However, a number of arguments strongly support the view that the morphological effects of beryllium are due to inhibition of phosphatase and, more generally, to interferences in the phosphate metabolism. The lowest concentration of beryllium able to inhibit phosphatase in the living cell is the same at which mitotic abnormalities begin to appear. The action of this metal on living cultures can be partially prevented by the addition of high amounts of magnesium, and the alkaline phosphatase activity of these cultures is also protected (Firket and Chevremont, 1952). This antagonism can be explained only by a competition of the two ions for an enzyme. The action of beryllium has been tested by biochemists on a large number of enzymatic processes, and practically all of them are unaffected,* except alkaline phosplionionoesterase, which is already sensitive to very low concentration of this poison. Its action can be counteracted by magnesium in large excess (Klemperer, Miller, and Hill, 1949; Grier, Hood, and IIoagland, 1949 ; Aldridge, 1950). Two other enzymes of phosphorus metabolism also have been mentioned to be inhibited by beryllium, namely, phosphoglucomutase and adenosinetriphosphatase. But for the former the data are rather contradictory (Cochran, Zerwic, and DuB'ois, 1951 ; Stickland, 1949) ; for the latter they are also contradictory and much higher concentrations of beryllium are required to produce an effect (Cochran, Zerwic, and DuBois, 1951 ; Klemperer, 1950). All the chemical and histochemical evidence indicates that the action of beryllium on mitosis is due to an inhibition of alkaline phosphatase causing an interference in the phosphate metabolism. I t is possible that phosphoglucomutase, for which we have no histochemical reaction, is also involved, but this is not at present demonstrated. Other hypotheses put forward to explain the action c,f beryllium (precipitation of phosphate or unspecific adsorption on proteins) (Aldridge, Barnes, and Denz, 1949) can be easily ruled out (cf., Ch6vreinont and Firket, 1952b).

V. FUNCTIONS OF NUCLEAR PHOSPHATASE Several authors have speculated on the special function of the alkaline phosphatase in the nucleus. Among others, Danielli (1946), struck by the identical localization of D N A and phosphatase in giant chromosomes,

* To list some of them: respiration, glycolysis (and thus the enzymes playing a part in these processes), several other phosphatases, and a number of magnesium activated enzymes (Klemperer, 1950 ; Cochran, Zerwic, and DuBois, 1951).

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admitted the possibility of the enzyme’s acting in nucleic acid metabolism. Using nucleic acids as substrates, Krugelis (1946) concluded that nuclear phosphatase is specific for nucleic acid chains (see p. 281). Brachet and Jeener (1948) pointed out the relations between phosphatase activity and DNA-phosphorus turnover. Many of the data we mentioned above, showing phosphatase activity to be correlated to the mitotic frequency, strongly suggest that the enzyme is taking a part in mitotic phenomena: variations in adult tissues, regeneration, carcinogenesis, tissue cultures, etc. This function during mitosis is, we think, demonstrated by the experiments with beryllium. From these we can also infer some details about this function. When phosphatase is selectively inhibited, chromosomes do not split at metaphase, and the mitosis, unable to continue normally, ends up in a reconstruction of one nucleus without division of the cytoplasm. I t is thus likely that phosphatase is one of the enzymes necessary for the synthesis of chromatin or, more specifically, of desoxyribonucleoproteins. The histochemical evidence of tissue cultures treated by beryllium also supports this view. Phosphatase plays an essential part during metaphase ; this suggests that some step of the synthesis of desoxyribonucleoproteins takes place at this stage. This would involve fixation or perhaps transfer of phosphate ; that alkaline phosphatase can act as a transphosphorylating enzyme was shown by the chemical investigations of Meyerhof and Green (1950). But we do not know what is the natural substrate of phosphatase at metaphase. Measurements of DNA within the cells indicate that the last steps in the synthesis of DNA take place at the telophase or after (Pasteels and Lison,* 1950). On the other hand, phosphorus is apparently introduced in the nucleus when the cell is “preparing for division” (Howard and Pelc, 1951). Phosphatase is likely to be acting somewhere on the line of chemical reactions linking these two processes. These views are still hypothetical. But it is certain that alkaline phosphatase plays an essential part during mitosis. Thus we begin to know one of the numerous enzymatic processes which must occur in cell division. Even when the cell is not in mitosis, phosphatase probably intervenes in DNA synthesis. This is strongly suggested by their nearly identical localization and the relation found in several instances between phosphatase activity and DNA phosphorus turnover. It was stated above that during mitosis some phosphatase is found in the cytoplasm, probably coming from the nucleus. There is no indication of its exact function there and its relations to RNA or some other metabolic process. I t may well be asked if nuclear phosphatase itself is linked

* These results, or at least the conclusion drawn from them, are contradicted by those of Swift (1950).

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only to the synthesis of nuclear substances or if it has some influence on phosphorus metabolism in the cytoplasm. Some facts suggest a control of phosphate uptake in the cytoplasm by the nucleus. If an amoeba is cut in two, the assimilation of radioactive phosphorus can continue only in the nucleated part ( Mazia and Hirshfield, 1950). Oxidative phosphorylations are possible in isolated mitochondria, but they are considerably accelerated if nuclei are added to the system (Potter, Lyle, and Schneider, 1951). From these and other experiments Brachet ( 1952) assumes the existence of a nuclear control of the coupling between oxidations and phosphorylations and thus of cytoplasmic synthesis. In our opinion, the possibility should not be excluded that nuclear enzymes, namely phosphatase, take a part in these mechanisms, but something other than a simple diffusion of the enzyme molecule into the cytoplasm would have to be involved, Phosphatase is active in mitotic processes, but it is possible to produce disturbances in mitosis without affecting this enzyme. Mitotic poisons, such as colchicine and lithium and probably many others, do not decrease phosphatase activity, but inhibit growth and modify mitosis (see above). They affect other chemical mechanisms, but these are generally not known. Tndeed, most of the chemical reactions taking place when the cell divides are unknown. Rare attempts along similar lines of reasoning have been made in recent years. Marshak and Fager (1950) tentatively explained the mitotic alteration they obtained on sea urchin oocytes with usnic acid (an antibiotic substance) as being caused by inhibition of a desoxyribonucleodepolyriierase. LettrC ( 1950) found that A T P reverses colchicin action on mitosis. H e concluded that A T P is necessary for the function of the spindle and that colchicine prevents the synthesis of this essential metabolite. It is to be hoped that in future years information will be gathered about the chemical action of other mitotic poisons and a fuller knowledge of the chemical phenomena of mitosis will be reached.

1. Unicity or Plurality of Phosplzutases The special functions that may be attributed to phosphatase in the nuclei raise the question of the identity of this enzyme with other alkaline phosphomonoesterases. The problem of unicity or plurality of these phosphatases has already been discussed by many authors, and it is one that probably can not be solved by histochemistry alone. The action of enzymes can be modified by small changes of various factors : coenzymes, activators, and physical conditions. T o be sure of differences between enzyme molecules of various origins, it is necessary to purify them to the utmost before they are compared (Roche and Nguyen-Van Thoai, 1948; Roche, 1950). Histochemistry can help in this problem, however, if, by varying substrates and other conditions, strikingly different localizations appear. Several

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authors used this method, but their results are not always easy to interpret. Some of these research workers tried to differentiate between phosphatases in various tissues without attempting particularly to work on the cytological level. The histochemical methods for phosphatase with an acid optimum pH (Gomori, 1941b) and for 5-nucleotidase (Gomori, 1949b) doubtlessly demonstrate enzymes different from alkaline phosphatase with an altogether different distribution in tissues. The problem of histochemical demonstration of adenosinetriphosphatase has been studied by Glick and Fischer (1945), Moog and Steinbach (1946), and Glick (1946) ; and that of hexosediphosphatase by Allen and B’ourne ( 1943) , Gomori ( 1943), and Zorzoli and Stowell (1947). When purified, these enzymes do not act on glycerophosphate, but ordinary alkaline phosphatase hydrolyzes their specific substrates. Thus a positive reaction with glycerophosphate (ordinary Gomori reaction) cannot be attributed to them, and a precipitate produced with their specific substrates demonstrates their presence only where the ordinary Gomori reaction is negative. By varying substrates and eventually activators, different patterns can be obtained. This is mentioned in several papers of the school of Dempsey and Wislocki (Dempsey and Deane, 1946; Dempsey and Singer, 1946; Dempsey and Wislocki, 1947), but Gomori (1949a) thinks it is doubtful that these differences are essential. He also believes that his own results do not warrant “the presence in paraffin embedded mammalia tissues of phosphatases other than the common nonspecific alkaline and acid variety.” On the contrary, Maengwyn-Davies and Friedenwald ( 1950) believe that in fresh-f rozen tissues phosphatases are several and substrate specific. Baradi and Bourne (1951) express a similar view for olfactory mucosa. Other differences based upon variable resistance to inhibitors are described by Emmel (1946, 1950). Dealing more particularly with nuclear phosphatase, several authors have obtained different histochemical pictures by using different substrates, namely, nucleic acids or derivatives. Purified phosphatase can split phosphate off native RNA (Zittle, 1946) but acts on DNA only proportionately to its depolymerization (Ross and Ely, 1949). Krugelis (1946) said that nuclear phosphatase appears to be able to attack depolymerized DNA but not RNA, whereas the reverse was observed in cytoplasm of intestine. Her results are largely confirmed on fresh frozen cornea by Friedenwald and Crowell (1949), who find further that a very small amount of glucose activates this reaction. With a very artificial organic substrate Loveless and Danielli (1949) obtained in the kidney two different pictures (either brush borders or nuclei) according to the reaction product (phosphate or an organic molecule) that must be added in trace amounts to make the reaction start. They say that the “simplest explanation at present available,

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is that there are many alkaline phosphatases” acting with various rates on different substrates. Newman et ul. (1950) compare the variable pictures obtained by changing the substrates, adding activators and inhibitors, and evaluating the rate of hydrolysis. Using sometimes dangerously long incubation times, they conclude that three groups of alkaline phosphatases may be characterized. Group I can be demonstrated with almost all substrates. Group I1 hydrolyzes A T P or muscle adenylic acid (compare to the 5-nucleotidase of Gomori), and group I11 is found in nuclei whether enzymes of group I and I1 are present or not. These nuclear enzymes show a “comparatively slower rate of hydrolysis and greater resistance to inhibitors than the cytoplasmic phosphatases.” The question is still open, and it is difficult to appreciate how much of the detailed conclusions of all these authors will remain valid, or to give an unifying picture of them, but the merit of their experiments is to direct attention to an interesting point : What are the natural substrates and working conditions of nuclear phosphatase ? I n view of what is said above about its function, these are important problems to be dealt with in the future.

VI.

,4DDENDUM

A number of relevant papers have been published or have come to our knowledge since this review was written. The technical conditions and the artifacts have been reviewed by Pearse ( 1953) and Danielli (1953) and further investigated by Taft (1952) and Fredricsson ( 1952a, b) . The advantages of the freeze-drying technique have further been stressed by Novikoff, Korson, and Spater (1952) and also by Yokoyama, Berenbom, and Stowell (1952), who find the losses in enzyme content to be small by this method. Firket and Michel (in preparation) compare blocks, f rozen-dried or embedded after alcohol fixation, for diffusion artifacts (Jacoby and Martin type) and find that both diffusion and adsorption on nuclei become almost negligible in frozendried preparation. Artificial staining of nuclei is more pronounced in paraffin sections than in tissues sectioned after freezing only and fixed (Herman and Deane, 1952), and by the cobalt sulfide technique than by the silver technique for revelation of calcium phosphate (Feigin and Wolf, 1952). Ruyter (1952) investigating the phenomenon described by Moe (1952) points also to the advantages of the silver technique. Johansen and Linderstr#m-Lang ( 1951, 1952), in a theoretical analysis of the calcium phosphate precipitation, conclude that it occurs only on crystal “nuclei” or on cell sites having an affinity for this salt. Although, as already pointed out by Gomori (discussion of the paper of Feigin and Wolf, 1952), this study is based on the false assumption that the enzyme

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is distributed evenly within the cell, it makes it even more imperative to test the affinity of various cell structures for calcium phosphate in each material. For the other methods involving precipitation of the organic part of the ester molecule, Grogg and Pearse (1952) have proposed n number of new favorable azo-dyes. Recent attempts at photometric measurements of the Gomori reaction (Neumann, 1950; Abolins, 1952 ; Taft and Scott, 1952) lead us to mention other quantitative estimations (Follis, 1949; Cleland, 1950; Danielli, 1950; Gomori, 1950b) often involving chemical (Doyle, 1950; Doyle, Omoto, and Doyle, 1951) or physical measurements (Dalgaard, 1948; Barka et al. 1952). I n spite of the considerable interest that quantitative histochemistry evokes, the numerous pitfalls inherent to these techniques calls for great caution. Alkaline phosphatase of normal and malignant tissues growing in vitro has again been studied by Sacerdote de Lustig and Sacerdote (1951) and of bone marrow by Takeuchi (1952). The enzyme is found in the nucleus of lymphocytes in lymph nodes, or in tissue cultures (Ackermann, Knouff, and Hoster, 195210). Cancerization brings about an increase in enzyme content (Ackermann et al., 1952a). Contrary to previous reports, isolated normal liver cell nuclei do not seem to contain the enzyme (Zajdela and Morin, 1952 ; Tsuboi, 1952). Also, Michel (unpublished) finds, in frozendried rat liver, that the Gomori reaction is positive only in bile canalicules, nuclei and cytoplasm of hepatic cells remaining negative. This is not surprising, as normal liver cells hardly ever divide. On the contrary, during active regeneration, there is an increase of enzyme content in nuclei isolated by centrifugation as confirmed by Tsuboi ( 1952). Raven and Spronk (1952) studied the action of beryllium on the embryonic development and confirmed that localization and time of appearance of the Be effects are closely related to the alkaline phosphatase activity. Speculating about the function of the alkaline phosphatase in nuclei, Danielli (1953) thinks there are at least three possibilities : protective function of the genes against phosphorylating agents such as ATP ; dephosphorylation as a final stage in synthesis of the genes, or a part in the nucleic acid synthesis. W e must also mention recent work on 5-nucleotidase (Pearse and Reis, 1952), an enzyme that has been found in nuclei ( Wachstein and Meisel, 1952). Maengwyn-Davies, Friedenwald, and White (1952) have continued their work on substrate specificity. Aneurinpyrophosphatase was studied histochemically by Naidoo and Pratt (1951; 1952). Green and Meyerhoff (1952) have further tested the possibilities of transphosphorylation by phosphatases, an investigation that may lead to the finding of the chemical reactions actually performed by phosphatases in the living cell.

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