Cell Polyploidy: Its Relation to Tissue Growth and Function

Cell Polyploidy: Its Relation to Tissue Growth and Function

w.YA.

BRODSKYAND I.

v. URWAEVA

N. K. Koltsov Institute of Developmental Biology, USSR Academy of Sciences, Moscow, USSR

I. Introduction . . . . . . . . 11. Occurrence and Properties of Polyploid Cell Populations . . . . . , . . 111. Modes of Polyploidy . . . . . . A. Mitotic Polyploidization; Endomitosis . . B. Gz Block; Polyteny . . . . . . C. Special Cases . . . . . . . D. Concluding Remarks , . . . . . IV. Possible Mechanisms of Incomplete Mitotic Cycle A. Interrelation between Cell Differentiation and . . . . . . . Proliferation B. Experimental Reduction in Mitotic Cycle and . . . . . . . Polyploidy C. Competition among Cell Functions . . . D. Concluding Remarks . . . . , V. Functional Consequences of Polyploidy . . VI. Liver Growth and Polyploidy . . . . . A. Normal Growth; Change in Ploidy Class . . B. Mechanisms of Cell Class Transformations in Ontogenesis . . . . . . . C. Transformations of Cell Classes during Liver . . . . . . Regeneration . D. Irreversibility of Polyploidy . . . . E. Proliferative Properties of Polyploid Hepatocytes . . . . . . . and Aging . VII. General Conclusion . . . . . . References . . . . . . . .

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I. Introduction The problem of cell, or somatic, polyploidy was discussed about 20 years ago by Swift (1953)and Vendrely and Vendrely (1956)in the Znternational Review of Cytology. Early cytophotometric investigations of DNA were made in the same period. The number of reported examples of cell polyploidization, which had previously been mentioned in few morphological papers (see Geitler, 1953),immediately began to increase. Cytophotometry revealed cases of doubling of the diploid amount of DNA in mammalian cells. Cell polyploidy ceased to be the “privilege” of invertebrates and plants. 275

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Cytophotometry established a fundamental similarity between polyploidy and polyteny, their common feature being multiple doubling of DNA in the cell. Therefore we combine these morphologically different phenomena under a single term, cell polyploidy, but make a distinction between them when discussing specific cases. More recently, it was found that endomitosis, which had long been considered the only mode of cell polyploidization, is just one of many types of incomplete mitosis. The possibility of an incomplete course of ordinary mitosis was noted in some well-known surveys (Mazia, 1961; Zhinkin, 1966).But the fact that this phenomenon is normal was realized only within the last 10 years, afker cell polyploidy in the histogenesis of many tissues was revealed. Quite recently, the significance of cell polyploidization for tissue growth was substantiated. It was suggested that there exists a relationship between polyploidization and specific features of tissue development. It is our purpose in this article to generalize these and other general questions of cell polyploidy, the most important of which are: (1)What are the possible causes of the formation of a multiplied genome? (2) What are the modes of polyploidization? (3)What is the contribution of cell polyploidy to the functioning of the tissue? 11. Occurrence and Properties of Polyploid Cell Populations

Polyploid nuclei have been found in Protozoa and in the cells of many Metazoa, both animals and plants (see Geitler, 1953; Vendrely and Vendrely, 1956; Leuchtenberger, 1958; D’Amato, 1964; Brodsky, 1966; Raikov, 1967; Tschermak-Woess, 1971; Brodsky and Uryvaeva, 1974). The list is evidently limited only by the scale of application of DNA cytophotometry. Based on the available evidence, cell polyploidy is not due to the morphological complexity of organisms or to their position in the phylogenetic series (Fig. 1). In analyzing the causes and significance of polyploidy, attention must be focused on the biology of the polyploidizing cells themselves. It is cells that synthesize considerable amounts of tissue-specific proteins that usually undergo polyploidization. Such cells are often polyfunctional and can produce various special proteins. Both these properties are more typical of invertebrates than of vertebrates. In worms, molluscs, and insects, tissue functions (e.g., certain nervous or secretory ones) can in some cases be performed by just a few cells. The numerous highly specialized secretory and nervous mammalian cells never attain such degrees of polyploidy as those of invertebrates.

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As a rule, the definitive cells of vertebrates reduplicate their genome only 1to 2 times and, occasionally, 3 to 5 times. Cells of invertebrates often increase their genome by 8 to 10 synchronous reduplications, and examples of 16 to 20 reduplications are also known. In many groups of invertebrates the functional significance of genome reproduction may be due, first, to the much smaller (by a factor of 30 to 50 or more) amount of DNA in their diploid chromosome set as compared with that of mammals (see Britten and Davidson, 1969; Ashmarin, 1974). Second, as already noted, some tissue functions in invertebrates are performed by a few giant cells. In this case polyploidy may cause cell gigantism, a means of increasing protein yield. But in mammals too, it is usually cells that synthesize considerable amounts of tissue proteins (often polyfunctional cells) that undergo polyploidization. Hepatocytes are a good example of such cells. The common property of polyploid cell populations is retention of ability to reproduce DNA by differentiating and often fully differentiated cells. The stem cell phenomenon, which is clearly defined in many diploid cell populations, is in polyploid ones deprived of its main function being the only source of proliferation.

111. Modes of Polyploidy Polyploidization can be regarded as a consequence of blocking of the normal course of mitosis, the omission of certain phases in the cycle of the chromosomes or the achromatic apparatus. Such deviations have been noted repeatedly (Mazia, 1961; Schwarzacher, 1968;

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Nagl, 1970a; Rudkin, 1973). The retention of increased DNA content in the cell may result from different aberrations in the mitotic process (Fig. 2). Accordingly, cells that have not completed their division during a particular phase of mitosis are found to be genetically nonequivalent. In the following discussion we consider types of mitotic polyploidization proper, complete abolition of the mitotic mechanism, and other cases of increases in the DNA content of cells. A. MITOTIC POLYPLOIDIZATION; ENDOMITOSIS In these cases the initial stages of mitosis up to chromosome separation proceed normally, but then mitosis stops. The daughter chromosomes do not drift apart to the poles or, if the chromosomes separate, the cytoplasm does not cleave. It is obvious that mitotic polyploidization results in the formation of true polyploid cells with an increased number of chromosomes. A characteristic feature of the tissue in which such polyploidization typically occurs is mitotic figures, often polyploid ones. These mitoses usually have a normal appearance, because disturbances are generally of a temporary nature and often occur in the late phases. In mammals, polyploid mitotic figures are

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found in the liver, salivary glands, urinary bladder epithelium, and megakaryocytes. 1. Acytokinetic Mitosis The absence of cytotomy after chromosome division results in the formation of a binucleate cell. Binucleate cells are so common in mammals that it would be easier to list the tissues lacking them than to mention the numerous examples of their occurrence. Binucleate cells are especially typical of liver parenchyma, salivary glands, and vegetative ganglia. In the liver of a mouse or a rat, binucleate cells may constitute 50-70% of the total number of hepatocytes. Beams and King (1942) suggested that binucleate cells in the liver are formed via incomplete mitoses. They observed late telophases without signs of cleavage of the cytoplasm. Wilson and Leduc (1948) also found acytokinetic telophases in a growing liver. The mitotic mode of formation of binucleate cells in the liver was confirmed by 1967; Nadal, 1970a; Uryexperiments with t h ~ m i d i n e - ~(Carriere, H vaeva and Lange, 1971).Similar results were obtained when studying the pigment epithelium of the retina (Marschak, 1974).A considerable increase in binucleate cell quantity on completion of DNA synthesis and mitoses was revealed (Fig. 3). A calculation of the increase in labeled binucleate cells per unit time yielded values close to the expected number. The latter had been computed from the observed mitotic activity and the established frequency of acytokinetic mitoses. The new data supported the concept (formulated most clearly by Nadal and Zaidela, 1966)that the problem of polyploidy involving the liver and other similar tissues is largely related to the causes of formation of binucleate cells. Transformations of a cell with two diploid nuclei may yield, in subsequent mitoses, all types of mononucleate and binucleate hepatocytes. It is interesting to note that polyploid clones, which often appear spontaneously in uitro, form along the same lines. At first, binucleate cells appear as a result of diploid acytokinetic mitosis. Then, in the next mitosis, the nuclei fuse together and mononucleate tetraploid cells are formed (Church, 1967; Pera and Schwarzacher, 1968; Yosida et al., 1969).

2. Blocking of Other Phases of Mitosis; Endomitosis These types of polyploidizing mitosis result fiom deeper disturbances in the mitotic apparatus. In the liver of humans and other mammals, abnormal forms of polyploid mitoses have been described, namely, c metaphases, monopolar and multipolar mitoses, and patterns

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FIG.3, Mitotic origin of binucleate cells in the liver and in the pigment epithelium of the rat retina. The liver was investigated on the twenty-fourth day, and the pigment epithelium on the fourth day after birth. Labeled binucleate cells (0)appear after completion of the first labeled mitoses (0)and increase in number until these mitoses proceed. (From Marschak, 1974.)

of fusion of anaphase and telophase chromosomes (Wilson and Leduc, 1948; Altmann, 1966; Heine and Stocker, 1970). These forms exceed half of all the mitoses in old rats (Klinge, 1968). They represent the mode of formation of high-polyploidy nuclei and multinuclear cells not only in the liver, but evidently also in the urinary bladder epithelium (Levi et al., 1969) and megakaryocytes of the bone marrow. The latter are characterized by the absence of patterns of the late stages of mitosis (Ode11et al., 1968).The mechanisms of the transition from the diploid state to tetraploidy in megakaryocytes are not yet understood (see Sklarew et al., 1971). There are grounds to believe that the high level of ploidy of megakaryocytes is achieved by blocking the middle phases of mitosis. A block in metaphase-anaphase with reconstruction of the polyploid nucleus is the most likely possibility. Endomitosis is the next step in the disturbance of the division apparatus. We use the term endomitotic cycle, in the narrow sense of its original definition (Geitler, 1953),to refer to the reduplication of chro-

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mosomes with subsequent separation of centromeres inside the nuclear membrane. Endomitosis is common in differentiated tissues of insects and higher plants (D’Amato, 1964; Tschermak-Woess, 1971).A peculiarity of this process is the typical structure of endomitotic nuclei. The terms endomitosis and, especially, endomitotic polyploidy are used in the literature much more frequently than they actually deserve. Geitler drew a sharp distinction between directly observed endomitosis and its assumed effects, that is, patterns of polyploid cells of unknown origin. Later on, such vague cases were referred to as endomitotic polyploidy. Reliable patterns of endomitosis have not been observed in many such cell populations. This is true of all definitive mammalian tissues and many invertebrate tissues. B. G, BLOCK;POLYTENY A complete absence of the mitotic mechanism is the next step in disturbance of the mitotic cycle. Blocking of the cycle immediately before mitosis is a possible means of physiological regulation of proliferation. The cells delayed in the G, period make up, in some tissues, the G, population ready for division (Gelfant, 1963; Terskikh, 1973). A physiological G, block is readily reversed, and therefore G, cells do not accumulate in considerable amounts. An irreversible mitosis block is due to the disturbance of early division mechanisms. Such cells can enter into the new reproduction cycle and synthesize DNA again. This process is called endoreduplication. Repeated reduplication leads to polyteny . 1. E ndoreduplication The term endoreduplication was introduced by Levan and Hauschka (1953), who observed doubling chromosomes (diplochromosomes) in mitotic patterns from mouse tumors. Later it was shown that diplochromosomes form as a result of two consecutive reduplications and may be found in the subsequent mitosis. Thus the first endoreduplication cycle includes two mitotic cycles (Bell, 1964; Mitwoch et al., 1965; Schwarzacher and Schnedl, 1965, 1966; Herreros and Gianelli, 1967). Three consecutive reduplications yield quadruplochromosomes. Besides tumor tissue, diplochromosomes appear spontaneously only in cells in uitro. More frequently, they are found after various treatments that inhibit mitosis in the cell (see Rizzoni and Palitti, 1973).

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2. Polyteny A well-known case of polyteny involves the nuclei of the secretory cells of the salivary glands of Diptera. In these interphase nuclei one can visually discern giant chromosomes which are formed by repeated duplication of DNA. After duplication the cells do not enter into mitosis but again proceed with DNA synthesis. Cytophotometry has also revealed other cases in which, despite the tremendous reproduction of DNA (much greater than in Drosophila), chromosomes are not visually discernible. The greatest amount of DNA found so far, which was of the order of millions of haploid units, was observed in the nucleus of the secretory cell of the silk gland of Bombyx mori (Nakanishi et al., 1969; Rasch, 1974). Very large amounts of DNA, 300,000 haploid units (c) and more, are also found in giant neurons of the molluscs Aplysia and Tritonia (Sakharov, 1965; Cogeshall et al., 1970; Lasek and Dower, 1971). The nuclei of such cells reach a millimeter in diameter. Great amounts of DNA, 100,000 to 300,000 c, have been found in each of the three cells forming the esophageal gland ofdscaris (Anisimov, 1976).Smaller but still considerable amounts of DNA are present in nurse cells in the ovary of certain insects (Jacob and Sirlin, 1959). In mammals, some of the cells of the cytotrophoblast contain up to 1024 c DNA (Zybina, 1963, 1970; Hunt and Avery, 1971; Barlow and Sherman, 1972). In all these giant cells, DNA accumulates without any sign of mitosis. In all the above-mentioned cases of increased DNA content it is the result of complete reduplication. Total replication of the genome is evident from the results of cytophotometry. Thus the data in Fig. 4 show how, with the growth of the ascarid, the total population of all the uterine cells is transferred to the next level of polyploidy. The increase in DNA in neurons of molluscs also occurs synchronously and results in the same geometric progression. Simultaneous reduplication of DNA in neurons was also demonstrated in studies of t h ~ m i d i n e - ~incorporation H (Bezruchko e t al., 1969). As regards the cells of the mouse trophoblast (Sherman et al., 1972) and the silk gland of B. mori (Gage, 1974; Rasch, 1974), not only cytophotometric but also biochemical proof of total replication of the genome has been obtained. In the nuclei of the giant cells of the silk gland and the trophoblast, the appearance of structures, which in the opinion of some investigators (Nakanishi et al., 1969; Snow and Ansell, 1974; Zybina and Chemogryadaskaya, 1976) are similar to polytene chromosomes, has been noted. The presence of one sex chromatin body in trophoblast cells (Zybina and Mosjan, 1967; Nagl, 1972),and also the constancy of

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FIG. 4. Synchronous reduplication of DNA in all the cells of the uterine epithelium during growth ofAscarls suum; DNA-Feulgen cytophotometry of total epithelium preparations. Abscissa: Theoretically expected DNA classes calculated by measured DNA content in AX spermatozoa (c). Horizontal bars left of histograms show changes in worm body length at different stages of growth. (From Anisimov, 1974.)

the number of chromocenters during the development of the cytotrophoblast (Barlow and Sherman, 1972) and association of centromeric heterochromatin (Barlow and Sherman, 1974), are considered cytological proof of total replication of the genome and of its polytene nature. In giant-neuron populations, the first incomplete cycles have not been studied. It is possible that in some cases the cells pass through a stage of mitotic polyploidization prior to polytenization. Then one observes polytenization of the polyploid nucleus, and not the diploid one, as in Drosophila. This assumption is based on the results of Nagl’s experiments (1970a). Giant polytene chromosomes are common in the suspensor cells ofPhaseolus. If, however, repeated endoreduplication cycles are interrupted by polyploid mitoses (4n,8n, and 16n) during early development, in subsequent growth the cells reach the same gigantic size as typical polytene cells, but the nuclear structure remains diffuse. In contrast to the species discussed above, Drosophila exhibited underreduplication, owing to the dissimilar number of replications of DNA in chromocenters and euchromatin during the polytenization of nuclei (see Berendes, 1973). Chironomids, whose nuclei are also 1 Recently, C. Thomas and D. Brown (1976, Deuelop. Biol., 49,89) obtained results that were not in line with the concept of the polytene structure ofB. mod giant nuclei.

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FIG.5. Complete replication of DNA in cells ofthe Malpighian tubes (M.t.) and salivary gland of Chlronomus thummf at the beginning (IV Beg) and the end (IV End) of the fourth instar of a larva and a prepupa (Pre). Each line represents the intervals between.the DNA values (mean 23 standard deviations) for every DNA class of nuclei. Abscissa: Mean values expected theoretically from the results of DNA-Feulgen cyto fluorometry in metaphase I of spermatogenesis. (Courtesy of Vlasova et al., 1972.)

characterized by clearly defined banding but have little heterochromatin, showed total replication of the genome (Vlasova et al., 1972; Fig. 5). Thus underreplication of DNA characterizes chromatin, but not the process of reduction in the mitotic cycle. It seems obvious that the considerable accumulation of DNA in nuclei with visually undiscernible chromosomes, and cases of typical polyteny and endoreduplication, are in fact the same phenomena as DNA reduplication with complete blocking of mitosis (Brodsky and Uryvaeva, 1970). The same opinion is voiced by Rudkin (1973) and Pearson (1974),who propose to extend the narrow morphological definition of the term polyteny, restoring its initial use in reference to a multistrand structure. The polytene cell cycle is characterized by the omission of mitosis (Fig. S),and in this sense it does not differ from the endoreduplication cycle. In the early stages of development of polyteny, the cells retain the ability to divide mitotically. Then the mul-

FIG.6. The cell cycle in an endopolyploid cell (A) and a polytene cell (B). In (A) GI, S, Ge,and M follow as in a normal mitotic cell; M differs in that there is no spindle for-

mation and the sequence of chromosomal events takes place within an intact nuclear membrane. In (B) there is no M phase, so the cell is in permanent interphase. (Courtesy of Pearson, 1974.)

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tistrand structure is visually discernible as diplochromosomes. So far, only the puffing phenomenon can be considered an obvious distinction between “true” and “concealed” polytene nuclei. It is not clear, however, whether or not puffing occurs in a hidden form in the nuclei or silkworms and molluscs. As to the visual polynemic pattern, its significance is not at all clear. It is known that visual expression of polyteny depends on life condition and even on nutrition. Megaselia (Phoridae) larvae have typical polytene cells in the salivary glands when their food is rich in amino acids and fats. When animals receive a poor diet, the same nuclei have diffuse chromatin (Barigozzi and Semenza, 1952). Large nurse cells of Calliphora gonad may have diffuse chromatin or polytene structures for the same amount of DNA (2048-4096 c). Both types of nuclei develop from small (16 c) typical polytene nuclei (Bier, 1959). C. SPECIALCASES Besides polyploidization and polytenization, partial replication of the genome also increases the DNA content. Two types have been described. In one case, part of the genome regularly drops out of reproduction (Sauaia and Alves, 1969; Fox, 1970a,b).In the other case of partial replication, amplification of a few genes takes place (see Adrian, 1971; Sherman et al., 1972). In both cases, nuclei with a DNA content inconsistent with the 2” series are obtained. Recently, a similar (intermediate between 2c and 4c)amount of DNA was found in the Purkinje cells of the rat cerebellum (Brodsky et al., 1974).This is evidently a rare phenomenon, characteristic only of certain cells in a few animal specimens. Possibly, this and other (Fontaine and Swartz, 1972; Lohmann, 1972,1975) observations of a nonmultiple DNA content are due to partial replication of the genome at some stage of development or functioning of the cells. Fusion results in the formation of bi- and multinucleate cells. Subsequent synchronization of nuclei during the cycle and their union during mitosis may produce a clone of polyploid cells. This phenomenon is assumed to occur in the fat body of Diptera, where typical endomitosis has also been described (Wigglesworth, 1966, 1967). I n mammalian development, cell fusion is rare. It evidently promotes the formation of multinucleate giant cells of the urinary bladder epithelium (Martin, 1972). Recently, Le Bouton (1976) proposed that cell fusion takes place in the liver of young rats. Modem methods have failed to confirm the possibility of the fusion of nuclei and cells in the trophoblast and in the uterine decidua, where it was once considered quite common (Chapman et al., 1972; Gearhart and Mintz, 1972; Ansell et al., 1974).

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Some reports draw attention to the possibility of a special kind of fusion-the union of daughter cells immediately after division or even during cytokinesis (Uryvaeva and Lange, 1971)-what Wheatley (1972) called “refusion.” This phenomenon has been observed in living megakaryocytes of bone marrow in uitro (Kinosita et aZ., 1959)and in cultures of other cells (Sisken and Kinosita, 1961; Oftebro and Wolf, 1967).The decisive factor for refusion, as well as for acytokinetic mitosis, may be a change in the properties of the cell surface. Such changes result, for instance, in the fusion of myoblasts in normal differentiation of skeletal muscles.

D. CONCLUDINGREMARKS Cell polyploidy seems to be the consequence of an incomplete mitotic cycle. The extreme degree of cycle reduction is omission of the entire mitotic mechanism. Such a (polytene) cycle is typical of cells with a particularly high rate of growth. The real occurrence of endomitosis is not clear at this time. The chromatin may sometimes imitate mitosis in the nuclear membranes. An example is known in which the endomitotic patterns probably represent the interphase state of the nuclei (Kiknadze et al., 1975).The endomitotic cycle usually results in the formation of a polyploid nucleus. Other cases of polyploidizing mitosis also lead to duplication of the number of chromosome sets. Mitotic polyploidization has proved to be common in definitive tissues of mammals and other vertebrates. In the cells of vertebrates, the disturbances in mitosis are usually moderate. In many cases, only the final act of cell division is omitted, and the cell goes on living with two nuclei. Tetraploid binuclear cells are common among fibroblasts, peripheral neurons of some mammals, and pigment cells of the retina. Binuclearity is probably a prerequisite for polyploidy. The following sequence of events is possible. Acytokinetic mitosis of a diploid cell leads to the formation of a binucleate cell. In the next mitosis of the binucleate cell, two mononucleate tetraploid cells are formed, which undergo acytokinetic mitosis again, and so on. This pattern of polyploidization is typical of the liver and perhaps of other large glands in mammals. In certain situations, particularly in aged animals, mitoses may stop at earlier stages. The blocking of intermediate stages of mitosis is typical of the normal development of megakaryocytes, whereas binucleation is not at all characteristic of such cells. The morphological basis for polyploidizing mitosis is still not clear. The mitotic machinery is very likely disturbed but not the centrioles. Sets of centrioles are retained in polyploid cells of a mitotic origin. The

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number of centrioles determined on ultrathin sections of hepatocytes and megakaryocytes is exactly the same as the degree of ploidy. Reduction of the mitotic machinery in polytene cells of Chironomid salivary glands involves a reduction of centrioles as well (Onishchenko, personal communication). Incomplete mitosis as a means of cell polyploidization is characteristic not only of vertebrates, but also of many invertebrate tissues (Aisenstadt and Marschak, 1969; Anisimov et al., 1974; Vorobjev and Leibson, 1974) and plants (Brunori, 1971).

IV. Possible Mechanisms of Incomplete Mitotic Cycle Why do we observe regular reduction in the mitotic cycle and polyploidization in many cell populations? Genome growth is not dependent on the number of chromosomes at the beginning of development; cells of both diploid and polyploid form undergo polyploidization (Klimenko and Spiridonova, 1974). A plausible answer lies in the specific features of the development of such cells. The most remarkable trait of polyploidizing cells is their retention of the ability to reproduce during specialization. A. INTERRELATION BETWEEN CELL DIFFERENTIATION AND PROLIFERATION Mutual relations between cell differentiation and proliferation in developing tissues can be reduced to two principal types. In one, proliferative syntheses do not coincide with differentiation. Thus, in the histogenesis of skeletal muscles, the synthesis of large amounts of contractile proteins begins only after completion of the growth of myoblasts and their irreversible withdrawal from the mitotic cycle (Stockdale and Holtzer, 1961). The neuroblasts of the central nervous system do not accumulate neurofibrils and do not form axons until mitoses terminate and the cells migrate out of the ventricular germinal epithelium (Fujita, 1964).The Paneth cells of the duodenum (Cairnie, 1970) and the cells of the lens fibers (Modak et al., 1968) also belong to this type of population. During its history each such cell experiences an abrupt change in program, hence in metabolism; proliferative syntheses are replaced by tissue-specific ones (Fig. 7). Having completed their division, such cells remain diploid and function until they die. It is precisely the development of such, and only such, cells that corresponds to the old embryological tenet: “A cell either divides or differentiates.” Their growth depends exclusively on the number of divisions of immature cells.

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FIG.7. Change in intracellular syntheses during cell life. Left: Tissues in which tissue-specific syntheses replace sharply proliferative ones and the cells drop out from the mitotic cycle with the beginning of differentiation. Right: Tissues in which these syntheses concur and the cells can undergo polyploidization during differentiation.

The growth of other tissues depends not only on immature cells but also on the division of maturing ones. For instance, erythroblasts of the chicken embryo synthesize and accumulate hemoglobin, undergoing six division cycles prior to their terminal differentiation (Campbell et al., 1971). In contrast to the neurons of the central nervous system, peripheral adrenergic neuroblasts synthesize DNA and divide simultaneously with the accumulation of neurotransmitter and the formation of specific structures (Cohen, 1974). Proliferation ceases after a considerable accumulation of catecholamine granules. The populations of mast cells (Combs, 1966), p cells of the islets of Langerhans (von Denffer, 1970), and white adipose tissue cells (Pilgrim, 1971) also increase, all as a result of the division of immature cells and cells that have attained a considerable degree of maturity. Megakaryocytes increase their ploidy at the same time at which they undergo primary differentiation (Odell and Jackson, 1968; Odell et al., 1969). Cardial myocytes differentiate early in embryogenesis, ensuring spontaneous rhythmical heart pulsations. Differentiation does not decrease the proliferative pool, which contains almost all the myocardial cells (Jeter and Cameron, 1971). New muscle cells form during embryogenesis and early postnatal development from proliferating differentiated cells containing myofibrils (Rumjantsev, 1963; Rumjantsev and Snigirevskaya, 1968; Manasek, 1968; Weinstein and Hay, 1970; Polinger, 1973). In myocardium cultures some investigators have also observed the division of contracting cells (Mark and Strasser, 1966; De Haan, 1969; Chacko, 1973). During development of the pigment epithelium of the gray rat retina, pigmentation begins during embryogenesis. It occurs in all cells

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and does not affect the increase in their number. Electron microscopy has shown that dividing cells contain premelanosomes and mature granules and do not differ from nondividing cells as regards their degree of differentiation (Marschak et al., 1972; Stroeva and Gorbunova, 1972). Figure 7 shows the relationship between proliferation and differentiation in such cell populations. The scheme refers mainly to the situation in which not only differentiating but also completely differentiated cells are capable of proliferation. These include mature hepatocytes (Goss, 1967), giant cells of the urinary bladder (Walker, 1959; Levi et al., 1969), secretory cells of the salivary glands (Redman and Sreebny, 1970) and the exocrine region of the pancreas (Pictet et al., 1972),goblet cells of the duodenum (Troughton and Trier, 1969), and smooth muscle cells (Cobb and Bennett, 1970). The coexistence of proliferative and tissue-specific syntheses in the cell is an important feature of these populations. Their simultaneous occurrence may be the cause of the reduction in the mitotic cycle, and the latter may result in polyploidy (Brodsky and Uryvaeva, 1970). It may be assumed that the increase in differentiation is accompanied by suppression of syntheses, ensuring proliferation. Here, however, the mitotic cycle may not be blocked completely, but may proceed according to one of the abbreviated versions. B. EXPERIMENTAL REDUCTION IN MITOTIC CYCLE AND POLYPLOIDY It is common knowledge that the initiation of DNA synthesis and the entry of the cell into mitosis, as well as the realization of each of its separate phases, require the synthesis of special proteins (see Epiphanova, 1973).These proteins are synthesized stage by stage. The sequence of their formation determines the progress of the cell through the cycle. The initiation of reduplication does not mean automatic entry of the cell into mitosis, and prophase is not necessarily followed by the final stages of division. If the mitosis program depends on adequate provision of interphase syntheses, the effects of transcription and translation inhibitors must stop mitosis and lead to polyploidy. The concept of “division proteins” was based on the arrest of the cell in some stage of division after the inhibition of protein synthesis during interphase. Tetraploid mitoses and mitoses with diplochromosomes arise in transformed lymphocytes in uftro after the blocking of protein synthesis by chloramphenicol or streptonigrin (Nasjleti and Spenser, 1967, 1968).The effect of 8-azaguanine is to suppress mitoses in the cells of pea root meristem, which switch over to the endoreduplication cycle

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2o Hours FIG.8. Effect of actinomycin D on the mitotic (-) and endomitotic (--) indexes in the meristem and differentiating region, respectively, of root tips ofA. carinaturn (each determination is based on the examination of lo00 cells). Note that the mitotic index is more strongly affected than the endomitotic one. (Courtesy of Nagl, 1970b.)

(Nuti Ronchi et aZ., 1965).Of particular interest are the results of Nagl (1970b), according to which (Figs. 8 and 9) mitoses in the meristem of AZZium carinatum are more readily suppressed by actinomycin D than endomitoses in differentiated tissue. A definite dose of the antibiotic caused the mitotic cycle in the meristem to be replaced by an endomi-

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._.M ( P - T )

/"

I

.-. .-.

/ O.lmg/ml

2c 4c D 8 C

Act.D

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Rc. 9. Frequency distribution (in percent) of nuclear stages in the root tip meristem ofA. carinaturn after treatment with actinomycin D and recovery (each determination is based on the examination of 300 to 500 cells). M, Mitosis; P-T, prophase to telophase; Z, dispersion stage (early prophase); S, period of DNA replication, c, unit for the DNA content of the haploid chromosome set. Note that the treatment suppresses mitosis to endomitosis (blocking mitosis at the dispersion stage) and the tetraploid nuclei originate within the meristem. (Courtesy of Nagl, 1970b.)

CELL POLYPLOIDY

29 1

totic one. It may be assumed that the resistance of endomitosis to inhibition is due to its reduced program, that is, the lack of spindle protein synthesis, reproduction of centrioles, and cytokinesis. The same may be true of the effect of high doses of colchicine on mammalian cells. Low doses, as is well known, block mitosis in metaphase. This is due to the formation of complexes between colchicine and the protein subunits of the microtubules (Borisy and Taylor, 1967a,b).High doses of colchicine, which affect a greater number of sensitive sites, completely exclude mitosis, causing endoreduplication (Rizzoni and Palitti, 1973). Neurotransmitter antagonists evidently produce the same effect. The use of these substances, which inhibit protein synthesis, results in the appearance of binucleate blastomeres after division of sea urchin eggs (Buznikov, 1973). These experiments possibly reproduce the situation occurring in normal development. The mechanism of reduction in the mitotic cycle may consist of metabolic competition among cell functions.

c.

COMPETITION AMONG CELL FUNCTIONS It may be assumed that the cell possesses limited metabolic resources. For example, one can estimate the limit of possible formation of proteins based on a definite amount of ribosomes, precursors, or ATP. Three categories of cell syntheses can be distinguished. Those in the first group are essential for the very life of the cell. The second, proliferative syntheses, are necessary for cell division and growth. The third, which determine cell differentiation, are called “luxury” syntheses in the terminology of Holtzer et al. (1972).It is assumed that the total extent of these three types of synthesis cannot exceed a certain level, which is inherent to the cell and may depend on the amount of ribosomes, ATP, enzyme activity, and so on. Those and other factors limit the translational machinery yield of a cell to 105-106 molecules per minute (see Palmiter, 1975 and Leitin, 1975). Metabolites common for several reactions (UTP, for example, see further data of Marzullo and Lash, 1970) may be a limiting factor. Therefore the expression of one cell function may depend on the intensity of the others. Below, we consider examples of balancing proliferative and tissue-specific functions. Their competition may cause a weakening of one of the functions in the course of intensifying the other.

1. Variations of the Cell Phenotype In Vitro It is well known that differentiated cells, after being explanted in uitro, reversibly lose certain features of differentiation. One of the alleged causes of this phenomenon is stimulation of cell division in the explant. For instance, in a neuroblastoma culture, cells did not

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possess the tissue phenotype as long as division was stimulated by the serum. When the latter was removed, the growth of the culture was repressed, and the cells began to differentiate, form axons, and produce specific enzymes (see Prasad, 1975). Experiments on the effect of culture conditions on the synthesis of polysaccharides by chondroblasts are well known (Nameroff and Holtzer, 1967; Holtzer and Abbot, 1968).When cultured in pellets the cells retained the same properties as those that had been in intact cartilage. In the monolayer, growth was induced and special syntheses weakened. In the growth phase of the primary culture of a kidney, the synthesis of proteins essential for cell proliferation intensified, whereas the synthesis of lactate dehydrogenase slowed down (Ruddle and Rapola, 1970). In the stationary phase, the synthesis of lactate dehydrogenase increased, while the synthesis of “division” proteins dropped. The total protein production during different phases of the cell cycle was constant. It is obvious that intensification of one synthesis did not completely repress the other. Similar results were obtained in a study of cultured embryonic chicken liver cells made by Grieninger and Granick (1975). The translational machinery of these cells functioned at a stable level, whereas a change in the types of synthesized plasma proteins took place. These investigators suggest that translational competition occurs in the stationary monolayer. An interesting example of the balancing of functions is given by Whittaker (1968a,b). His experiments demonstrated a decrease in intensity of the synthesis of tyrosinase, an enzyme involved in melanin formation, after explantation of pigment epithelium of a chicken retina. Visually, this effect manifests itself in cell depigmentation. The decrease in tyrosinase synthesis is not due to termination of the synthesis of tyrosinase mRNA but is caused by the weakening of its translation. After explantation the pigment cells begin to prepare for mitosis. Such cells display considerable intensification of protein synthesis, and during this period new mRNA appears in the cell. According to Whittaker’s hypothesis, the reduced expression of differentiation is due to the competition for ribosomes between the tyrosinase mRNA and the newly formed mRNA. This hypothesis is well supported by the observed intensification of tyrosinase synthesis after the blocking of transcription by actinomycin D (Whittaker, 1970). Redifferentiation of the pigment cells also occurs after contact inhibition of cell division. The most probable cause of both effects is the removal of mitotic messengers from the protein-synthesizing system. Marzullo and Lash (1970) concluded that the loss of differentiation traits in chicken chondroblasts in uitro is due to the competition for

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UTP. The limiting factor is the concentration of UTP in the medium. Under optimum cultivation conditions no competition is observed; the UTP is sufficient both for the synthesis of chondroitin sulfate and for mitoses. A metabolite deficit in the medium leads to a preference for,proliferation, and this results in loss of the mature phenotype. Competition between the two types of syntheses in uitro usually leads to supplanting of the tissue-specific “luxury” processes which are not essential for the cell life. The reproduction processes are intensified at their expense. This mechanism possibly operates during normal development of cells in vivo as well, when the reproduction function is supplanted by the specialized functions of the cells. There are no direct data on this so far, but some observations may be interpreted in this light. 2. I n Vivo Analogies Among the examples of cell development in vivo we mention, first, the pigment epithelium of the retina. In gray rats, the first polyploid (binucleate) cells appear in the pigment epithelium on the fifth day after birth. At this time, proliferation is retarded, while melanin synthesis continues (Fig. 10). These data may be interpreted in different ways. First, the two processes, that is, the increase in differentiation and the retardation of proliferation, may proceed independently, coinciding only in time. Another possibility is that one aspect of cell life, for instance, differentiation, is rigidly fixed during development, while the other, proliferation, depends on the successful accomplishment of the first. A third possibility is the regulation of one function by the other; derepression of one gene represses the other. The choice from among these possibilities is determined by the fact that, during the formation of the first polyploid cells in the pigment epithelium, proliferation and melanin synthesis are inhibited simultaneously. The last mitotic cycle terminates in acytokinetic mitosis. The resulting binucleate cells at first show a decreased (in proportion to the diploid cell) melanin content. The repression of the two processes points to the competitive nature of their relationship, rather than to the regulation of one function by the other. Tissue and reproductive functions evidently develop in parallel, but manifestation of each depends on expression of the other function. A similar example was described earlier by Prokofjeva-Belgovskaya (1959,1960).It was shown that, as starch accumulates in potato tubers, mitoses in the starch forming cells were retarded. As in the pigment epithelium and other polyploidizing cell populations, the inhibition of proliferation was not of the all-or-none type. The mitoses did not cease,

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90 %

50

0 I

3

5

7

9

II days

FIG.10. The development of pigment retinal cells of gray SK rats. Changes in the melanin content (cytophotometry of unstained sections; A = 590 nm), the number of DNA-synthesizing cells and binucleate cells from birth to the eleventh day of life. Each point represents one animal. For melanin at least 30 cells make up a point (mean -C standard error) and, for thymidine -SH and binucleates, 2000 to 4000 cells. Note the decrease (relative to didoid cells) in melanin content in newlv formed binucleate cells. (rrom MarscnaK et ah., IY ro.)

but they did not proceed to completion, and binucleate cells were formed. The competition between starch formation and proliferation is indicated by the nonselectivity of inhibition; stimulation of mitoses retarded starch accumulation, while intensified starch formation repressed mitoses. Cardial myocytes are also characterized by concurrent processes of reproduction and differentiation within a single cell. This may possibly explain the polyploidization of myocytes which, although it does not develop completely in ontogenesis, progresses vigorously under conditions of reactive proliferation. The growth of the heart muscle is due to the division of differentiated cells. The dividing cells undergo a chain of modifications consisting of disintegration of the Z disks and disorientation of the myofibrils (Hay and Law, 1972; Rumjantsev, 1972). Signs of “partial dedifferentiation” have also been noted in the DNA synthesis phase (Rumjantsev, 1973). As already mentioned, in cultures one can observe the division of contracting cells. But during

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metaphase-anaphase myocardial (Kasten, 1972) and also smooth muscle (Chamley and Campbell, 1974)cells the contractions cease. In Kasten’s opinion, the temporary loss of the contractile function is due to the energy requirement for mitosis. In competition for energy, preference is given to mitosis rather than to contractions. Here, however, the mitotic myocytes do not divide and form binucleate cells. Chacko (1973)detected acytokinetic mitosis in 10-15% of myocytes in oitro. Other examples of polyploidizing cell populations also confirm the hypothesis suggesting that proliferative syntheses are supplanted by tissue-specific ones. In the parotid and other salivary glands of the rat, differentiated cells proliferate during the final stages of histogenesis (Redman and Sreebny, 1970).Isoproterenol induces mitoses in the glands of adult animals (Novi and Baserga, 1971).The retardation of secretion observed in both cases is interpreted as being the result of competition between the secretory and mitotic processes. In these situations, the course of mitosis is affected too. In normal growth of the salivary glands, cells with a low degree of polyploidy appear. Stimulation of additional growth by isoproterenol results in the formation of high-ploidy cells which are unusual in normal development (Radley, 1967;Schneyer et al., 1967). Compensatory regeneration of the liver, as well as its postnatal growth, are ensured by the proliferation of parenchymal cells. Here a certain antagonism between mitotic activity and certain specific functions is observed. I n a regenerating liver, the activity of microsomal drug-metabolizing enzymes is reduced (Fouts et al., 1961;Henderson and Kersten, 1971), and the formation of inducible enzymes is repressed (Seidman et al., 1966).A temporary decline in the activity of drug-metabolizing enzymes results in a resistance of the regenerating liver to the hepatotoxins carbon tetrachloride and paracetamol (Uryvaeva and Faktor, 1976a,b).The toxic action of these compounds manifests itself only after their enzymic conversion. Examples of a reverse effect are also known, for example, retardation of the regeneratory response on intensification of the tissue-specific function, which takes place, for instance, after the animals are saturated with glucose (Takata, 1974). Antagonism between tissue functions and mitosis does not necessarily imply a competitive relationship. Thus the activity of one of the microsomal enzymes-cholesterol-7a-hydroxylase-and the mitotic reproduction of hepatocytes do not coincide in time either in regenerating or in growing livers. The circadian rhythms of mitoses and enzymic activity are in counterphase (Van Cantfort and Barbason, 1972; Barbason et al., 1974).In the opinion of the authors cited, this type of function interaction suggests regulation at the gene level.

296

W. YA. BRODSKY AND I. V. URYVAEVA Localization

Localization

of CCI,-

of

DNA

i n d u c e d necroses in liver lobule

synthesis in liver

lobule

FIG.11. Reciprocal relation of drug-metabolizing and mitotic functions in the mouse regenerating liver. Carbon tetrachloride poisoning induces centrolobular necrosis in the normal mouse, because of toxic conversion of carbon tetrachloride by drugmetabolizing enzymes in centrolobular hepatocytes. Thus necrogenic action served as an indicator of the activity of the drug-metabolizing enzymes. Adult CBAIC57BL micr were subjected to partial (two-thirds) hepatectomy and then poisoned with carbon tet, rachloride at different times after the operation. During the period of active proliferation the necroses do not appear. (From Uryvaeva and Faktor, 1976b.)

The example given in Fig. 11also demonstrates the fall in the activity of specific enzymes during liver regeneration. But here the phenomena may be interpreted differently. In these experiments, the necrogenic effect of carbon tetrachloride was used as an indicator of the drug-metabolizing enzyme’s activity (for the reason behind this analysis, see Gerhard et aZ., 1970, 1972). The loss of specific function was compared with the rate of mitotic events as related to their localization in the liver lobule. It is doubtful that the interaction of these two functions occurred at the gene level. The synthesis of new types of mRNA, which is regarded as genome reprogramming (Markov et aZ., 1975), takes place after the stimulation of divisions simultaneously in all the zones of the liver lobule (Rabes and Brandle, 1968). The loss of the drug-metabolizing function, however, occurs asynchronously in the cells of a competent zone of the lobule. It begins at the periphery and progresses toward the center, in accord with the wavelike propagation of DNA synthesis and mitoses through the lobule (Fabrikant, 1968). After partial hepatectomy, not only proliferative but also many other syntheses are stimulated. Thus, in addition to the processes essential to mitosis itself, syntheses ensuring cell growth are intensified. For instance, the activity of the enzymes involved in the metabolism of ga-

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lactose (which is a component of the cell membranes) is enhanced (Bauer et al., 1976). It is possible that the temporary drop in the activity of drug-metabolizing enzymes results from competition not only with the proliferative function, but also with other functions stimulated during regeneration. These explanations are in agreement with the hypothesis proposed by Wilson and co-workers (Wilson and Frohman, 1974; Wilson and Spelsberg, 1976),wherein tissue-specific functions of the liver not associated with growth decrease after stimulation of growth, probably as a result of the diversion of cellular resources to growth requirements. In view of the function interactions some examples of accelerated differentiation are of interest. Isoproterenol causes proliferation and hypertrophy of the cells in the parotid gland when injected into adult rats. However, if isoproterenol is injected during the first postnatal weeks, differentiation of the gland cells speeds up, whereas proliferation is suppressed (Schneyer, 1973): The adrenomimetic drug isoproterenol (isopropylnorepinephrine), acting through a system of adenylcyclase-CAMP, stimulates nonspecifically various cell syntheses (Robison et al., 1968). It may be assumed that its original effect on a differentiating tissue is due to the stimulation of the process predominant at this stage and the consequent displacement of the competing one. Another example refers to erythropoietic cells. Inducement of anemia in pigeons with the use of phenylhydrazine rapidly results in the appearance of atypical hemoglobin-containing cells in the blood, which bypass the usual stages of maturation. In this case too, quick differentiation is attained by the omission of cell divisions (Gazaryan and Kul’minskaya, 1975).*

D. CONCLUDING REMARKS Cell polyploidization evidently depends on tissue development conditions. Tissues in which polyploid cells form are characterized by the retention of proliferative ability in maturing and mature cells. In contrast to other tissues, whose development is attended by an abrupt change in programs and an overall repression of proliferation, in polyploidizing tissues proliferation and tissue-specific programs coexist in the same cell. Transition from a complete mitotic cycle to an incomplete one seems to represent not qualitative replacement, but rather quantitative changes. The cause of incomplete mitotic cycles, which lead to polyploidy, may lie in a deficiency of premitotic syntheses. To

* Recently, in ajoint study (Kul’minskaya,A. S., Gazaryan, K. G.,and Brodsky, W. Ya. (1977).Dokl. Acad. Nauk S S S R , in press), it has been shown that acceleration of differentiation resulted in the appearance of DNA-tetraploid erythroblasts and reticulocytes.

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confirm this hypothesis, experiments were carried out with polyploidy induction by partial inhibition of premitotic syntheses. The mechanism of displacement of proliferative syntheses during differentiation may involve competitive relationships with other cell activities. The loss of phenotypic traits in differentiating cells explantedin vitro may, in the opinion ofthe investigators who performed these experiments, result from translational competition or competition for energy. This mechanism is most suitable for explaining cases of balance between cell growth and differentiation. From this point of view, we regard examples of tissues differentiating in an organism (retinal pigment epithelium, myocardium, salivary glands, liver) and cases of variations in cell phenotype in a culture as similar. It is possible that the reciprocal relationships between division and the specific functions are a particular case of competition among different functions, and not just between reproduction and differentiation. Examples have already been cited. This mechanism of coordination of tissue functions may be of special importance of polyfunctional cells such as hepatocytes.

V. Functional Consequences of Polyploidy The mechanism of supplanting mitotic syntheses by tissue-specific ones as the cause of reduction in the stages of mitosis may be common for different cell populations. But polyploidy in different tissues may have a different meaning associated with the specific function of the tissue. Polyploidization, as a rule, accompanies cell differentiation. The first question that arises is: How are these two processes related and does polyploidization determine the specific features of the cell type? One can cite examples of cell lines whose definitive function is performed both in the diploid and polyploid state. Thus the livers of young and aged mice differ considerably in the ploidy of the parenchymal cells, they still perform fundamentally the same functions. In some mammals, such as the mouse, rat, dog, and human, the liver is formed of polyploid hepatocytes. In others, for example, guinea pig and cat, the same functions are performed by diploid cells. The mature pigment epithelium of the rat or mouse consists of polyploid cells and, in the ram and bull, of diploid cells (Berman et al., 1974). For animals whose livers function normally with polyploid cells, it is possible to obtain experimentally a diploid analog of the organ. Thus the liver parenchyma of dwarf mice is formed of diploid cells (Geschwind et al., 1960). Liver regeneration in such animals is achieved by the proliferation of diploid cells in adult species as well

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(Swartz, 1967). The stimulation of growth of dwarf mice by injecting growth hormone (especially coupled with thyroxine) caused considerable growth of the liver. Many polyploid cells appear in the parenchyma. These observations also point to the importance of cell polyploidization in tissue growth rather than in differentiation. The hormone effect in this case is clearly indirect: The liver remains diploid in undersized animals poorly fed in early life (Naora, 1957). Examples of polyploidy with stimulated additional growth of the organ are of some interest. Partial removal of the liver from a mouse and, even more so, repeated resections, cause polyploidization of the hepatocytes to degrees not observed in normal development. The myocardium of the rat auricle represents a diploid, nonrenewing cell population. Normally, this tissue contains no more than 5% tetraploid (in DNA content) cells. After an infarction of the ventricle the auricula atrii is hypertrophied. Induction of DNA synthesis and mitoses leads to almost complete (up to 90%)polyploidization of the auricular myocardium. Here not only tetraploid but also octaploid nuclei are found (Rumjantsev and Mirakjan, 1968). In hypertrophy of the human myocardium, stemming from cardiac insufficiency and other heart diseases, it also exhibits a great number of polyploid cells (Sandritter and Scomazzoni, 1964; Eisenstein and Wied, 1970). Isoproterenol causes growth of the salivary glands, which contributes nothing to the requirements of the organism and is accompanied b y considerable polyploidization of the acinary cells. In all the above-discussed cases, polyploidy seems to be a forced consequence of stimulation of tissue growth, rather than a necessity. The cells, which are forced to prepare for division and are at the same time performing their tissue functions, are incapable of adequate preparative action. As a result, the proliferation of equivalent cells is replaced by mere replication of the genome. Both during cell proliferation and polyploidization, the number of templates for cell syntheses increases, and thus the corresponding cell functions are compensated for. The actual means of reproduction, that is, cell proliferation or polyploidization, depends on the conditions of tissue renewal and growth. The same is suggested by consideration of the development of cell lines in which polyploidization precedes differentiation. It is well known that high-polyploidy and polytene cells of plants and invertebrates, as well as the giant cells of the trophoblast and the megakaryocytes of mammals, represent the final stage of development of these types of cells. The reduction in the mitotic cycle occurs here in parallel with an increase in differentiation. It is obvious that the diploid cells of each specialization series are not entirely differentiated.

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It seems that the differentiation of such cells is the result of their polyploidization. Thus thrombopoiesis has been observed only beginning with the octaploid state of the megakaryocytes (Paulus, 1968; Ode11 et aZ., 1970). Among the diploid cells of the bone marrow, the megakaryocyte series has not yet been identified. In chronic myeloid leukemia in humans, however, diploid megakaryocytes have been observed which are capable of producing blood platelets (Undritz and Nusselt-Bohaumilitzky, 1968). The evolution of the thrombopoietic cells can be represented as follows. In lower vertebrates, the nucleus-containing spindlelike cells, and not the platelets, take part in blood coagulation. In intermediate species (as regards the development of the blood-forming system) these cells participate in blood coagulation, both as a whole and by separating blood platelets from their cytoplasm. In maturing mammalian megakaryocytes some of the final cycles of DNA replication end in polyploidizing mitosis. Terminal differentiation is achieved by cells with 8,16, and 32 chromosome sets. Hence it is not polyploidy that determines the specific features of such cells as megakaryocytes, hepatocytes , and melanocytes. Nevertheless, the development of the thrombopoietic function in phylogenesis (transition from diploid to polyploid cells-producers of blood platelets) draws one’s attention to the progressive role of polyploidy and the causes of the fixation of this property during evolution. This conclusion applies to many cases of cell gigantism. Thus endomitotic polyploidy is thought to cause the larger size of the tuber in cultivated species of potato plants as compared with the wild species of the genus SoZanurn, in which growth of the tuber is limited by rapidly inhibited cell divisions (Goroshchenko and Chuksanova, 1965). With similar specialization of the starch-forming cells in agricultural and wild species, the former accumulate much more starch than the latter. Polyploidization of the macronuclei of Znfusoria and the nuclei of other Protozoa is considered an important factor in their evolution (Poljansky, 1972). Polyploidization repeatedly increases the genome, and therefore a single cell combines within itself the possibilities of hundreds of equivalent cells. The concealed multicellularity (polygenomy) can also be traced in the development of certain cells of Metazoa, particularly invertebrates. The giant neurons of molluscs can be regarded as a “single-cell” ganglion, and some secretory cells of worms and insects as “single-cell” glands. In these cases polyploidy is a necessary element of cell development. Still greater and

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more rapid growth of oocytes occurs on the basis of polygenomy, external in this case. The growth of the oocytes in most animals is due to the influx of ribosomes, precursors, yolk proteins, and many other substances from special nurse cells and nongonadal tissues. The nurse cells may be few in number (insects), in which case they have high-polyploidy nuclei (Jacob and Sirlin, 1959); or they may amount to several thousand (worms), in which case they are diploid (Aisenstadt and Marschak, 1969). Can one speak of advantages of polyploidy? Two concrete questions can be posed: What does the cell gain from genome multiplication? And how does the reduction in the mitotic cycle influence cell functions? One of the obvious consequences of polyploidization is enlargement of the cells. The volume of the nucleus and cytoplasm usually increases proportionally to the increase in the number of chromosome sets (Jacoby, 1925). But the surfaces increase only 235, that is, 1.59 times, with each doubling of the volume. Thus polyploidy reduces the surfaceholume ratio (Brodsky, 1966; Epstein, 1967; Harris, 1971). Some investigators regard this as the cause of the reduction in functions whose initiation and performance depend on the surface membranes (Alfert and Das, 1969; De Leeuw-Israel et al., 1972).It is probably not accidentally that the nuclei with the highest ploidy have extremely branched nuclear membranes. These include, for instance, the nuclei of secretory cells of the silk gland of the silkworm and the esophageal gland of the ascarid. In the giant neurons of the mollusc Tritonia, numerous submicroscopic invaginations of the cytoplasm arrive at the nucleolus, and the nuclear evaginations perceptibly penetrate into the cytoplasm (Sakharov et al., 1965). Polyploidization is always attended by the intensification of transcription and translation, hence b y an increase in the strength of various cell functions. In any case, after the first DNA reduplications cytoplasmic syntheses may increase proportionally to genome reproduction (Fig. 12). Thus, after two replications, incorporation of ~ r i d i n e - ~into H the wall cells of the testis of the locust Chrysochraon dispar increases proportionally (Kiknadze and Tuturova, 1970). Transcription in 2n, 4n, and 8n cells of the onion also increases proportionally to ploidy (Nagl, 1973) (Fig. 13). In the liver cells of the rat the amino acid label and the activity of many enzymes increase proportionally to their ploidy (Brodsky, 1966). The DNA synthesis rate in a tetraploid nucleus of a hepatocyte is twice as high as in a diploid one. This conclusion follows from the similar direction of t h ~ m i d i n e - ~incorporation H curves for 2n and 4n

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m V

a

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nuclei in the S period (Fig. 14) and the equal duration of the cycle (Fig. 15).The growth dynamics of 2n and 4n nuclei are also similar (Fig. 16). If one takes into account the self-absorption of tritium, the label intensity doubles with the doubling of the number of chromosomes in the hepatocyte nucleus (Uryvaeva and Faktor, 1971). Hence individual chromosomes of a tetraploid genome of a hepatocyte reduplicate in the same sequence as in a diploid one. In this case, the properties of the chromosomes evidently remain unchanged, and polyploidy only means doubling of the indexes of the diploid genome. This conclusion can probably be extended to other cells in which the

i16001

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FIG.13. Diagrams illustrating the amount of RNA synthesized ( ~ r i d i n e - ~incorpoH rated) during different stages of mitotic (A) and endomitotic (B) chromosome cycles in terms of mean grain numbers per onion cell nucleus GI and Go,Interphase stages before and after DNA replication, respectively; D, mitotic dispersion stage and endomitotic dispersion stage; M, mitotic metaphase. Each symbol represents the mean of 15 to 25 nuclei; the bars indicate the standard error. (Courtesy of Nagl, 1973.)

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FIG. 14. Incorporation of th~midine-~H into diploid, tetraploid, and octaploid hepatocyte nuclei during the S period. Two adult CBNC57BL mice were subjected to partial hepatectomy and after 32 hours injected with th~midine-~H 1 hour before being killed. The liver was separated by a method similar to that of Jacob and Bhargava (1962). The cell suspension was smeared on glass slides and stained by Feulgen, and autoradiograms were prepared. After a grain count the label was removed from the emulsion and DNA-Feulgen was measured cytophotometrically in the same nuclei. The arrows indicate the 95% confidence interval for the DNA values in unlabeled nuclei. (From Uryvaeva and Faktor, 1971.)

-z In

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FIG. 15. Graph of labeled mitoses for mouse hepatocytes of different ploidy. (0)Diploid; ( 0 )tetraploid;).( octaploid. Chromosome preparations were made with colchicine hypotonic citrate and the air-drying technique. All the experimental data refer to 50 hours after partial hepatectomy and 4 hours after colchicine injection. Th~rnidine-~H was injected at various times after the operation, as indicated on the lower abscissa. (From Faktor and Uryvaeva, 1972.)

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2 200 150

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FIG.16. The occurrence of liver nuclei throughout the mitotic cycle periods, represented as the plot of nuclear area versus DNA content. Each dot or circle represents one nucleus on a smear of isolated cells of a regenerating liver from one mouse 1 hour after injection of the precursor. Solid circles, Unlabeled nuclei; open circles, th~midine-~H-labeled nuclei. DNA-Feulgen cytophotometry and autoradiography were performed on the same nuclei as described in the legend to Fig. 14.

duration of the S period is equal in the diploid and polyploid nuclei: HeLa cells (Firket and Hoppes, 1970), urinary bladder cells (Levi et al., 1969; Fig. 17), plant meristem cells (Van't Hof, 1966; Friedberg and Davidson, 1970), and diploids and autotetraploids of various plants (Troy and Wimber, 1968; Yang and Dodson, 1970; Seithodschajev, 1973). In the cells of the tetraploid strain of an ascites Ehrlich carcinoma, however, the duration of DN'A synthesis is longer than in the diploid cells of this cancer (Defendi and Manson, 1963). The same results were obtained in studying the 2n and 4n forms of some plants (Titu and Popovici, 1970; Karpovskaja and Beljaeva, 1973). It may be.assumed that in such cases polyploidization is attended by a change in the sequence of replication, and possibly in the chromosome structure. As for the consequences of the incompleteness of the mitotic cycle, they are most obvious in the extreme forms ofreduction, that is, in polyteny and endomitosis. In these cases the specific activity of the cells is not interrupted during growth (Fig. 13). The chromosomes of the polytene cells remain permanently in the interphase state. Continuous transcription is sometimes pointed out as being a feature demonstrating a radical difference between polytene and polyploid cells,

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whose genome activity is periodically inhibited during mitosis. The actual significance of this property of the polytene nucleus, however, is not clear. The average lifetime of mRNA in many eukaryotes exceeds by far the duration of mitosis. In polytene cells too, incorporation of amino acids into proteins is not inhibited for a long time after repression of transcription by actinomycin D (Pearson, 1974).The main difference between polyteny and polyploidy evidently lies in the reduction in the mitotic cycle and its frequent repetition, hence the rapid growth of the cell, rather than in continuous transcription. In mitotic polyploidization, in particular, that of hepatocytes, the reduction in the mitotic cycle is minimal, and therefore its effect on the cell growth rate is practically undetectable. Other functional effects of polyploidy on the liver are not clear either. (See Section VI,B,2.) Thus, so far there are no data indicating that an increase in the cell genome, that is, polyploidy proper, determines the specific features of differentiation. In some cases, however, this gives the tissue certain advantages over the performance of its functions by the more numerous diploid cells. The mode of polyploidization-the reduction in mitosis-could have been used in evolution as an adaptation for speeding up the growth of certain cells. The development of giant polyploid, and especially polytene, cells shows that an increase in the

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W. YA. BRODSKY AND I. V. URWAEVA

cell genome, which is not in itself a factor in the differentiation of these cells, becomes a necessary condition for adequate performance of their functions.

VI. Liver Growth and Polyploidy A. NORMALGROWTH;CHANGEIN PLOIDYCLASS The liver parenchyma of many adult mammals is a mixed population of cells differing in the number of nuclei and DNA content. The transformation of the diploid population of hepatocytes in newborn animals into a practically exclusively polyploid one occurs during the postnatal growth of the organ. The relative number of polyploid and binucleate cells in animals of different ages as determined by many investigators has been reviewed by Carriere (1969). We have compared these results with more recent data. The first polyploid cell class-a binucleate cell with diploid nuclei-appears in young animals. It is only after a considerable number of binucleate cells (up to 30-50%) has been accumulated that the frequency of cells with a single tetraploid nucleus begins to increase. This is followed, in adult animals, by an accumulation of binucleate cells with tetraploid nuclei, and then with a single octaploid nucleus. In mice, more high-ploidy and multinucleate cells appear. The number of the latter is insignificant, therefore these cell classes are excluded from further discussion. Only in some lines of mice characterized by particularly high polyploidization do they reach about 1% (Gerhard et al., 1971). Each of the ontogenesis periods shows the predominance of some cell classes characteristic of this period. These classes can be represented as a series: 2n, 2n x 2,4n, 4n x 2, 8n, 8n x 2, . , . ,which reflects the sequence of their appearance and the changes during postnatal growth. The polyploidization process is illustrated by the results of experiments in which determination of the DNA content of the class of hepatocyte in mice was carried out in a selected population with a double thymidine label (Brodsky et al., 1973). It can be seen from Fig. 18 that in young mice it is cells with diploid nuclei that proliferate. But among the newly formed cells the percentage of those with tetraploid nuclei is high. For the next age group (Table I), the results of DNA photometry refer both to labeled and unlabeled cells. Most of these 1-month-old mice already have a polyploid parenchyma, but binucleate cells with diploid nuclei predominate. On completion of the mitotic cycle, the label is found mainly in tetra- and octaploid nuclei of cells of the 4n, 92 x 2, and 8n types. In the newly formed popula-

307

CELL POLYPLOIDY 2

mouse I

3

4

20

I

a 9 1

20

tion a reduction in the number of 2n and 2n x 2 cells is observed. The behavior of the diploid cells, which are the precursors of the whole hepatocyte series, is noteworthy. Being the most active in reproduction (Busanny-Caspari, 1962; Post and Hoffman, 1965), they cease to self-maintain themselves and transfer to the next ploidy class. This phenomenon is also observed in older animals and in regenerating livers. This fact is important, since it may point to the existence of a source that supplements the hepatocyte population. In adult animals, liver growth is retarded to such an extent that DNA synthesis is practically undetectable. In three of nine 4month-old mice, 14 injections of t h ~ m i d i n e - ~labeled H less than 1%of hepatocytes within days. The others showed practically no label. At this age, the parenchyma contains only about 1%diploid cells. No diploid cells were found among the labeled, newly formed cells, and binucleate cells with diploid nuclei were also few in number (Table 11).

Mouse number

Number ofcells

1 2 3 4 5 6

li)4

124 98 84

110 144

Percent in each cell class, unlabeled

2n

2n x 2

4n

4n x 2

8n

12.5 12.1 14.3 21.4 10.0 11.8

51.9 46.0 45.9 34.5 34.5 41.6

22.1 19.4 28.6 32.1 39.1 25.7

13.5 20.2 9.2 9.5 14.5 18.8

-

1.6 1.0 2.4 1.9 1.4

8n x 2

-

0.8 1.0 -

-

0.7

Mouse number 1 2 3 4 5 6

Number ofcells

6 0 81 87 88 77 35

Percent in each cell class, labeled

2n

-

-

2.3 8.0 3.9

-

2n x 2

4n

11.7 4.9 18.4 18.2 2.6 25.7

21.7 40.8 10.3 51.1 42.8 37.2

91.

X

56.7 44.4 59.8 20.5 46.8 37.1

2

8n

8n x 2

10.0 7.4 6.9 1.1 3.9

2.5 2.3 1.1

-

-

-

a Mice weighing 15-17 g m were given six injections of thymidineSH (each 0.7~Cilgm body weight) at &hour intervals. Killing took place 40 hours after the last injection. Liver cells were isolated and smeared on glass slides. The preparations were Feulgen-stained, covered with emulsion, exposed, and developed. Then mononucleate and binucleate hepatocytes were estimated by means of phase-contrast, and 3Hlabeled and unlabeled cells were marked. The frequency of accumulated labeled hepatocytes varied from 0.7 to 4.8% in different mice. DNA cytophotometry was performed on the marked hepatocytes after grain removal. The labeled cells were newly formed ones. (From Brodsky et aZ., 1973.)

0

8

Percent in each class, unlabeled NumMouse ber number of cells 2n 2n x 2 4n 4n x 2 8n 8n x 2 16n 1 2 3

104 121 136

1.5 0.8 1.5

14.4 24.8 10.3

32.0 27.6 40.4

44.4 36.8 41.2

4.8 5.8 3.7

2.9 3.3 2.2

0.9 0.7

NumMouse ber of number cells 1 2 3

Percent in each class, labeled

2n 2n x 2

6 0 207 258 -

1.7 1.5 2.3

4n

4n x 2

26.7 5.8 10.1

58.0

62.3 62.4

8n 8n x 2 16n 16n x 2 10.1 26.6 15.9

1.7 3.4 6.2

1.8 0.4

2.7

-

-

0.4

3 10

W. YA. BRODSKY AND I. V. URYVAEVA -

- 100 -

I

-

50

-

-

2 I

W P

-

-0

-

BODY

12 WEEKS

I

2 3 4 MONTHS

1.5 2 YEARS

W&grT

FIG.19. Relative changes in liver weight (A), mean hepatocyte ploidy ( E ) , and percentage of diploid cells (C) in postnatal development of CBNC57BL mice and calculated values of cell quantity (AIE)and of changes in absolute quantity of diploid hepatocytes (AIE x C). (From the data of Brodsky et al., 1973.)

Some experimental results are summarized in Fig. 19, which show that the intensive proliferation of diploid hepatocytes occurs only in baby mice during the first 2 weeks of life. Then, toward 1month, the diploid cells cease to maintain themselves and transform into polyploid cells. Ultimately, in aged animals the parenchyma retains only 0.02 of the diploid cells of the newborn animal. While the weight of the liver increases almost 30 times within 2 years, the number of cells increases much less than the weight or mean ploidy. Hence the postnatal growth of the liver parenchyma is due to cell polyploidization. B. MECHANISMSO F CELL CLASS TRANSFORMATIONS IN ONTOGENESIS

1. Polyploidizing Mitoses In the course of growth of a organ each diploid cell goes through a regular chain of transformations. The first stage of polyploidization is

CELL POLYPLOIDY

311

the formation of binucleate cells from diploid ones by way of acytokinetic mitosis (see Section 111).In the next mitotic cycle the nuclei ofthe binucleate cell replicate the DNA and simultaneously enter into prophase. In metaphase, one bipolar spindle is formed, and consequently the chromosomes of both nuclei are united into a single metaphase nuclear plate. Anaphase and telophase proceed normally, resulting in two daughter mononucleate tetraploid cells (Nadal and Zaidela, 1966). Mitosis in binucleate cells of another origin proceeds in a different way. Caffeine induces the formation of a binucleate population in the meristem of the onion root tip by means of suppression of cytokinesis in the cells undergoing division at the moment of action (GonzilezFernAndes et al., 1966; Gim6nez-Martin et al., 1968). Subsequently, the nuclei of the binucleate cells simultaneously enter into mitosis, forming two independent, nonfusing mitotic figures, so-called bimitosis. The different versions of bimitosis result in binucleate cells of the parent type 2n x 2, as well as mononucleate ones. It is assumed that the specific type of division of hepatocytes, with fusion of the metaphases, is due to the disturbance of reproduction or movement of the centrioles (Nadal, 1970a). In contrast to hepatocytes, artificially induced binucleate onion cells have an intact mitotic apparatus. The next cell class, 4n x 2, is formed in normal growing livers, again by acytokinesis, from 4n cells. In the course of normal development, each of the polyploidizing hepatocytes evidently passes through all the successive stages of the series: 2n, 2n x 2, 4n, 4n x 2, . . . . This is supported by the fact that the appearance of each successive cell class in the parenchyma is preceded by the accumulation of some amount of the previous one (see Carriere, 1969).The formation of high-ploidy 'classes can be explained similarly to that of low-ploidy ones. All transformations of cell classes in postnatal development are the result of mitosis. The sharpest changes in parenchyma composition-the increase in the number of binucleate cells and their replacement by mononucleate cells-occur in baby and young mice and rats during the period of rapid growth and high mitotic activity. Weight stabilization in the animals leads to stabilization of the cell composition as well. The role of mitosis is particularly evident in the accumulation and disappearance of binucleate cells. It has been shown that any effects causing shifts in the number of binucleate cells (nutrition, hormones, partial hepatectomy) primarily affect the mitoses (Nadal and Zaidela, 1966; Wheatley, 1972; Nadal, 1970b, 1973). For instance, artificial growth retardation during the period of ontogenesis, when acytokinetic mitoses take place, reduces the relative number of bi-

312

W. YA. BRODSKY AND I. V. URYVAEVA

"-i *O

t

z 20

s

0

10

20

30

40

50

body weight (gm)

FIG.20. Binucleation in early- and late-weaned rats. Left: Development of binucleate cells in the livers of adolescent rats weaned at different ages. Open circles, Rats weaned at 15 days of age (early-weaned); solid circles, rats weaned at 25 days of age (late-weaned). At least six rats are represented by each time point. Right: Correlation of percentage of binucleate cells in early (open circles) and late-weaned (solid circles) rats with body weight. (Courtesy of Wheatley, 1972.)

nucleate cells as compared with the control animals. If these mitoses are inhibited, which transforms the binucleate cells 2n x 2 into 4n, the relative number ofbinucleate cells increases. One example is shown in Fig. 20. In late-weaned rats, binucleate cells accumulate with a lag relative to early-weaned ones. The reason is the retardation of growth and mitoses in late-weaned rats. The hormonal effect must also depend on the growth period and mitotic status ofthe liver. This is probably the reason for some contradictions in the results of experiments with endocrine effects on liver ploidy (see Carriere, 1969). Of some interest are the results of recent investigations by Nadal (1973,1975; Nadal and Boffa, 1975), who demonstrated that the blood serum of adult rats, unlike that of baby rats, depresses mitoses and induces binucleation. Thus the changing pattern of nucleation and polyploidy can be explained by the particular kind of polyploidizing mitosis. Acytokinetic mitosis does not increase the number of cells; it results in binucleate cells which are polyploid as regards the total sum of the chromosomes. The next mitosis of a binucleate cell increases the number of cells and results in nuclei of the next order of ploidy. The growth models discussed below are based on a sequence of acytokinetic and completed mitoses. Other types of polyploidizing mitoses due to irregular spindle formation or to defects in reproduction and movement of centrioles are evidently manifested only slightly. This can be seen from the good agreement between the values calculated from the model and those observed experimentally.

CELL POLYPLOIDY

313

2. Growth Models According to the scheme of Nadal and Zaidela (1966), acytokinetic mitosis and the subsequent division of the binucleate cell are followed by a series of normal mitoses which increase the number of mononucleate polyploid cells (Fig. 21). In the mouse, which has a higher degree of polyploidy than the rat, the situation is more in accord with the scheme in Fig. 22. It differs from the above-mentioned one in that each cell class, including mononucleate cells, forms from the preceding one without being supplemented by self-maintenance. In the scheme in Fig. 22 each cell class is regarded as the cell clone in the terms of Mintz (1970). It is implied that the cells of each class have the same mitotic history and originate from diploid initiator cells with similar properties. These postulates lead to conclusions which can be verified by experiment. Some of them are shown in Fig. 22. The regularities in the relationship among the increase in the single cell mass and in the tissue mass, and the changes in the number of cells in each successive reproduction cycle, are revealed. These relationships served as a basis for a retrospective analysis of the mass of diploid clonogenic cells, which is initial for liver growth. In calculations, we used the relative frequencies of cell classes and also the weight of the liver at the age of 1, 2, 3, and 4 months and 1.5 and 2 years observed for CBNC57BL mice (Fig. 19). The calculations yielded similar values for the initial mass of the clon-

FIG.21. Progress of cell ploidy classes in rat liver. (From Nadal and Zaidela, 1966a, with permission of Academic Press.)

3 14

W. YA. BRODSKY AND I. V. URYVAEVA

0-

Cell class 2n Number of reproductions 0 Increase o f : cell weight I

211.2

number of cells I

I

clone weight

2

I

4n

411.2

I

2

3

4

5

6

2

2 2 4

4

4 4 16

8

4

8 8

32

64

2 8

8n

81’1.2 16n

FIG.22. Classes of mouse hepatocytes as mitotic progeny of a diploid initiator cell.

ogenic cells, namely, from 2.9 to 3.7 arbitrary units for six cases. Moreover, this mass, calculated from the model in Fig. 22, practically coincided with the real weight of the livers of 2-week-old mice (Fig. 19). It follows from the figure, that this stage is characterized by a sharp transition from division of diploid cells to polyploidization. This may also be an important stage in the beginning of cell specialization and the formation of functionally different clones. These problems are beyond the scope of our survey and are still in the early stages of investigation. One may nevertheless assume that the presently obscure functional significance of liver polyploidy is associated with participation of the polyploidization process in the formation of cell clones. The united genome can facilitate cell regulation on the tissue level.

c.

TRANSFORMATIONS OF CELL CLASSES DUFUNG LIVERREGENERATION In adult animals the liver is a slowly renewing tissue with a low level of DNA synthesis and mitotic activity (Cameron, 1971). Removal of part of the organ or its toxic injury, however, causes proliferation of the remaining cells. Regeneration of the liver after operative removal of two-thirds of its mass, according to Higgins-Anderson, and poisoning with carbon tetrachloride has been studied most extensively. After the latent period, the cells of the remnant of the organ begin to enter gradually into a phase of DNA synthesis until the entire resource of divisible cells is exhausted. Following the wave of DNA-synthesizing cells, a wave of mitosis is recorded.

315

CELL POLYPLOIDY

The formation of high-ploidy nuclei during liver regeneration was noted both in cytophotometric and karyometric studies (see Altmann, 1966; James et al., 1966; Ryabinina and Benyush, 1968). Figure 23 depicts the results of the simultaneous determination of thymidine labeling and the amount of DNA in the nuclei of hepato-

10

I

.&-

0

l 92

10 2

4

8

16

32 DNA

(C)

FIG. 23. Polyploidization of hepatocyte nuclei in adult CBN57BL mice (weight 26-28 gm)during liver regeneration. DNA-Feulgen cytophotometry and autoradiography’ with double (3H and I4C) thymidine pulse label. Each graph represents hepatocytes of one animal at a definite time (from 40 hours to 6 days) after partial (two-thirds) hepatectomy. Hatched histogram: DNA in nuclei labeled with thymidine-14C injected 28 hours after the operation. Solid histogram: DNA in nuclei labeled with t h ~ m i d i n e - ~ H injected into the same mouse 1 hour before killing.Open histogram: Unlabeled nuclei of hepatocytes of the same animal. There were 200 to 300 nuclei for each mouse. Thus the I4Clabel revealed mainly postmitotic and Gzcells. The 3H label revealed cells in S phase. (From Uryvaeva and Brodsky, 1972.)

316

W. YA. BRODSKY AND I. V. URYVAEVA

cytes from a regenerating liver. It is seen that di- and tetraploid nuclei predominate among the nuclei during the phase of DNA synthesis (3H label). The other label, I4C, reveals nuclei that have gone through the reproduction cycle and mitosis. This group contains only tetra- and octaploid nuclei in almost all animals. In another experiment the population of the cells, cumulatively labeled with th~m idine-~H, has been collected during the entire regeneration time (Fig. 24).The di- and tet-

DNA content ( l o g scale)

FIG.24 Results of DNA cytophotometry and autoradiography of the same nuclei in

the liver of four CBNC57BL mice after three partial hepatectomies carried out at 1-month intervals. Autoradiography was performed with double thymidine labels: one of the precursors was used as a pulse and the other as a continuous label. Twenty-four hours before the last operation each mouse was injected with a large dose (3pCUgm) of thymidine-5H. This prelabeling ensured continuous sHmarking of all the cells passing through the mitotic cycle afterward ( x). The cells in the DNA-synthesis phase were labeled with thymidine-I4C (0.8 pCUgm) injected 1hour before killing (open circles). Exposure time was 4 months. The unlabeled cells (solid circles) are those that have not yet entered into the cycle. Note that the diploid nuclei disappear during induced hepatocyte proliferation, owing to lack of self-maintenance of cells with diploid nuclei. DNA synthesis occurs in nuclei of any ploidy, including high-ploidy ones. Polyploidy progresses with every operation because of incomplete mitoses.

317

CELL POLYPLOIDY

raploid nuclei replicate DNA, and after the mitosis transform into tetra- and octaploid nuclei. As a result of this process, the number of nuclei increases by a factor of 1.1at the end of regeneration, and the average ploidy of the nuclei, 1.66 times, as reported by Gerhard et al. (1973).Although cell multiplication does take place, toward the end of regeneration the number of cells increases only 1.44 times, according to these investigators. The polyploidization process also characterizes the increase in the mean ploidy per cell. If each cell of any class is reproduced in the course of growth, this index remains unchanged; if, however, each cell polyploidizes, the mean ploidy of the population must double. In the regenerated, labeled part of the parenchyma the mean ploidy of the cells increases about 1.5 times as compared with the unlabeled cells outside the cycle in the same animal (Uryvaeva and Brodsky, 1972). The changes in the average ploidy of the nuclei and cells reflect the redistribution of the cell classes with a general trend toward a decrease in the number of low-ploidy and an increase in the number of high-ploidy cells. The most characteristic result of regeneration is the reduction in the relative number of binucleate cells (Figs. 25 and 27). Here the class 2n x 2 practically disappears, the frequency of mononucleate 4n and 8n cells sharply increases, and they become predominant. The mechanism of reduction in the number of binucleate cells is clear. As in normal growth, mitosis of these cells gives rise to mononucleate ones. Transformations of other types are not so obvious. Maurer et al. (1973) 2 before ReO$’%+om (P.h.7. I month

1II

..

Ill..

after I st p. h.

I month after 2 n d p.h. I month after 3 r d p.h.

..

11..

111

..

I.

.

3 -

Ill,..

4

1111

5

6

I

.Ill,.

.Ill...

,

111 mllll...Ill,. .I I.

1

.Ill;*. llL -I

FIG.25. Progression of polyploidy in the mouse liver after repeated hepatectomies. Each column represents samples of the liver of the same animal removed by sequential operations. Height of the vertical bars in each graph shows the relative frequency of a hepatocyte class. From left to right: 2n, 2n x 2,4n, 4n x 2 , 8 n , 8n x 2, 16n, 16n x 2, 32n, 32n x 2,64n. (From Faktor and Uryvaeva, 1975.)

318

W. YA. BRODSKY AND I. V. URYVAEVA

proposes a model of cell transformations for regeneration of the mouse liver after carbon tetrachloride poisoning, based on the results of an investigation of cell kinetics (see Fig. 26). According to this model, all mononucleate cells undergo the usual mitosis and double in number, and all binucleate cells go through the mitotic process, forming partly two mononucleate cells and partly one binucleate cell with nuclei of the next degree of ploidy. A comparison of the actual results of transformations of hepatocytes on the sixth day of regeneration in adult mice with those calculated with the aid of the Maurer model is shown in Fig. 27. It can be seen that, on the whole, polyploidization after partial hepatectomy proceeds in the same way as after carbon tetrachloride poisoning. The divergence between the calculated and actual data relates primarily to classes 2n and 8n. The reduction in relative frequency of diploid cells 2n is a common result of regeneration after partial hepatectomy in adult mice (Figs. 25 and 27). It has been demonstrated by experiments with labeled thymidine (Figs. 23 and 24) that only some of the diploid cells entering into the mitotic cycle undergo normal mitosis and are thus used for self-perpetuation, while the other part of this population polyploidizes. The mechanism of their polyploidization, like their further fate, is not clear. In some animals practically no self-maintenance of the diploid cells has been revealed. This phenomenon has also been observed in rats by other investigators (Fujita et al., 1974). It is further seen from Fig. 27 that many

I

32%

FIG.26. A model of mouse liver regeneration after carbon tetrachloride poisoning. The left and right columns represent the measured percentage of mononuclear and binuclear cells at the beginning (30 hours) and the end (10 days) of the regeneration. I,

Mononucleates increase in number by means of normal mitoses; .II, binucleates transform by a mitotic process into the other classes of nuclear ploidy-into mononuclear cells with nuclei of high ploidy (IIa) and into binucleate cells, (IIb), with a probability 0.33 and 0.67, respectively. (Courtesy of Maurer et al., 1973.)

319

CELL POLYPLOIDY

mouse 2

A

B

C

A

B

C

U m o o a . @ B

2n 2n.2 4n 41-1'28n 8n.2 16n FIG. 27 Formation of cell classes during liver regeneration after partial hepatectomy. Comparison of experimental data with those expected from the model of Maurer et al. (1973; see Fig. 26). Two-thirds of the liver was removed from three adult CBNC57BL mice, and cell classes were determined in the extracted part (A). Sixth day after operation, at the end of the regeneration, the mice were killed and cell classes were again determined (B). Predicted cell classes (C) were calculated according to Maurer's model.

more high-ploidy cells are actually formed than would be expected from the Maurer model. Evidently, besides acytokinetic mitoses, which constitute half of the total number in the given scheme, other polyploidizing mitoses also take part, for instance, the monopolar, multipolar, and c mitoses described by Klinge (1968). These mitoses may cause transformation of class 4n into 8n and 8n into 16n, bypassing the intermediate stage of binucleation. Gerhard (1975) assumed such transformations for rat liver after partial hepatectomy. The question arises as to what extent the cell transformaions in reparative growth are similar to those undergone by hepatocytes in postnatal growth. In other words, does regeneration speed up natural transformations or does it induce special mechanisms? A sharp decrease and, not infrequently, complete disappearance of binucleate cells after regeneration is not typical of any stage of normal growth. One may have an impression that acytokinetic mitoses can undergo normalization during regeneration. Thus, according to Nadal's observation (1970a), one-third of the mitoses in the liver of young rats proceed without cytotomy. Conversely, if part of the liver is removed from these rats, all the mitoses in the stimulated cells end in cytotomy. A similar example is given in Table 111.A possible cause of the temporary normalization of the mitotic cycle in a regenerating liver is the

320

W. YA. BRODSKY AND I. V. URYVAEVA

TABLE I11 DISAPPEARANCE OF BINUCLEAR CELLSDURING REGENERATIONOF LIVERIN YOUNG CBNC57BL MICE

THE

Percent in each cell class 2n

2n x 2

Control" Average 8.9 48.4 Range 4.2-12.4 37.2-55.6 40 hours after partial hepatectomyb Average 16.4 2.1 Range 9.3-22.0 0.4-6.5

4n

4n x 2

8n

8n x 2

19.6 12.1-39.2

21.6 15.8-26.4

1 .o 0-1.7

0.5 0-1.5

78.9 68.0-85.3

1.1 0-1.8

1.5 0.4-1.8

-

-

" Eight mice, weight

14-17 gm. Four mice, weight 14-16 gm; selected population of labeled postmitotic cells. The H when the wave of labeled mimice were killed 15 hours after t h ~ m i d i n e - ~injection toses had already passed. For technique see the footnote to Table I.

switching over of metabolic processes to the promotion of fast proliferation, and this affects the specialized functions (see Section IV,C,2). D. IRREVERSIBILITY O F POLYPLOIDY Polyploid hepatocytes cannot be the source of formation of diploid or lower-ploidy cells. Further transformations of a cell that has gone through the first polyploidizing mitosis (2n x 2) can only'be directed toward an increase in ploidy (Figs. 21 and 22). In a stable, nongrowing organ the cell classes do not change either; growth stimulation causes cycling of the cells and their shift to the right in the ploidy series. Thus, in repeated hepatectomies each consecutive operation enhances cell polyploidy (Figs. 24 and 25). The irreversibility of polyploidization is also obvious in species with a high degree of genome multiplication, which occurs simultaneously throughout the entire population. These polytene and polyploid cells are usually terminally differentiated and are incapable of yielding nuclei able to support development. A case is known of somatic reduction-metamorphosis of the mosquito intestine (Grell, 1946). In the tissue cultures of plants some investigators have also observed a reduction in DNA content down to the diploid level by successive mitotic divisions without intervening DNA synthesis (Rasch et al., 1959; Patau and Das, 1961). These cases involve a reduction in nuclei which have previously gone through several successive polytene cycles of DNA synthesis without mitosis.

CELL POLYPLOIDY

32 1

A similar phenomenon was assumed with respect to hepatocytes (Perry and Swartz, 1967; Swartz, 1967). It was suggested that, along with polyploid cells, the liver may contain cells that withdrew from the mitotic cycle prior to mitosis in the G , phase, similar to the G, population described in skin epithelium (Gelfant, 1963). The hypothesis of a G , population can b e verified with the model of a regenerating liver. It is necessary to note the important properties of this model. The proliferative pool in young and adult (not yet old) mice and rats is close to 100%.This is confirmed by the total labeling of hepatocytes using continuous t h ~ m i d i n e - ~labeling H (Stocker et al., 1972; Schultze et al., 1973; Uryvaeva and Faktor, 1974). With a 100% proliferative pool, G, cells must inevitably enter into the mitotic cycle. Also, one of the following three possibilities must be realized. The G , cells enter into mitosis, bypassing DNA synthesis and forming nonradioactive mitoses. Or the G2cells go through the next endoreduplication cycle and then undergo, or do not undergo, mitosis. The realization of these three possibilities may lead, respectively, to the appearance of unlabeled mitoses during continuous labeling or of labeled mitoses with diplochromosomes or, in the latter case, to an excess in the number of S phases over that of subsequent mitoses in the regenerating liver. In this connection it is important to note that during liver regeneration all cells undergoing DNA synthesis then enter into mitosis without creating an excess of S phases. This is indicated both by a comparison between the curves of the labeling index and those of the mitotic index in rats (Grisham, 1962; Polishchuk, 1967; Fabrikant, 1968) and by special calculations of the total number of S phases and mitoses during regeneration of carbon tetrachloridepoisoned livers in mice (Schultze et al., 1973). Very few hepatocytes go through two reproduction cycles; most of them proliferate just once (Fabrikant, 1967; Liosner and Markelova, 1971; Maurer et al., 1973). However, all mitoses are preceded by DNA synthesis. None of the investigators who have studied liver regeneration in experiments with continuous labeling with thyn~idine-~H has found unlabeled mitoses (Stocker, 1966; Brodsky et al., 1969; Helpap et d.,1971; Schultze et al., 1973). A special study carried out on a large number of mice during different periods after one or several operations did not reveal nonradioactive mitoses or mitoses with diplochromosomes (V. M. Faktor, unpublished). Consequently, during the formation of nuclear and cell classes of hepatocytes, only disturbances during the late stages of mitosis, after the separation and drifting apart of sister chromosomes, 'take place. Therefore DNA classes in the liver are the true ploidy classes. The polytene or endomitotic cycle does not play a role here.

322

W. YA. BRODSKY AND I. V. URYVAEVA

E. PROLIFERATIVEPROPERTIESO F POLYPLOID AND AGING HEPATOCYTES It may be assumed that the irreversible process of polyploidization

brings the cell, after a certain number of mitotic cycles, to a definite degree of ploidy and a position in the series (Fig. 22). Thus the place in the ploidy series indicates the cell age, as it were. In this respect a hepatocyte ploidy series resembles a terminal differentiation series. The resemblance, however, relates not to the expression of differentiation (whose criteria for hepatocytes are difficult), but to the decrease in proliferative ability. The ploidy level achieved by the cell, no matter how high, does not in itself prevent it from going through the next mitotic cycle. The use of the combined method of autoradiography and cytophotometry in the same nucleus can establish DNA synthesis in cells of classes 16n, 1% X 2, and even 32n, to say nothing of the lower-ploidy types 8n and 8n X 2, whose reproduction is a common phenomenon (Fig. 24). A decrease in proliferative ability with polyploidization is revealed when evaluating the response of hepatocytes to a mitotic stimulus. After partial hepatectomy, the wavelike increase in DNA synthesis is due to the entry of different classes of cells into the S phase. The level of proliferative response can be expressed by the index of DNAsynthesizing cells (Table IV). It was found that this index, which characterizes the inducibility, or mitotic reactivity, of the hepatocyte, decreases smoothly with an increase in the degree of ploidy (Uryvaeva

EXAMPLEOF FOR

TABLE IV

LABELING INDEXCALCULATION CELLS OF DIFFERENTPLOIDY A

Number ofcells Relative frequency up to partial hepatectomy, A" Thymidine-3H-pulselabeled 23 hours after the operation, Bb Labeling index B/A, (%)

Percent in each cell class

2n 2n x 2

4n 4n

X

2 8n 8n

X

2 16n Total

353

2.0

28.4

27.5

32.0

7.1

2.5

0.5

100

116

1.6

15.0

13.3

14.4

1.1

0.5

-

46

-

80

53

48

45

15

21

0

-

~~~

~

Ploidy classes were estimated in the removed part of the liver. Same mouse; t h ~ m i d i n e - ~ H was injected 1 hour before killing. For technique see the footnote to Table I. When identifying the ploidy classes in the DNA-synthesis phase, sizes of nuclei were taken into account (see Fig. 16).

323

CELL POLYPLOIDY

x

loot

1

2n

217.2

4n

4n.2

8n

cell classes En-2

FIG. 28. Decrease in proliferative response by polyploidization of mouse hepatocytes. Six mice were subjected to partial hepatectomy. Twenty-three hours after the operation mice were give t h ~ m i d i n e - ~1Hhour before killing. The label index was determined as shown in Table IV. Each curve represents one mouse.

and Marschak, 1969; Watanabe, 1970; Brodsky and Uryvaeva, 1974; Fig. 28). The low labeling indexes for high-ploidy classes during the first synchronous wave of DNA synthesis do not mean that these classes do not take part in regeneration. They have a longer lag period and begin DNA synthesis later. The trend toward a decrease in proliferative ability with polyploidization was noted long ago on the basis of investigations into this phenomenon in plants, where polyploidy was associated with the state of differentiation (Mazia, 1961; see also Fig. 29). In hepatocytes, too, the gradient of the proliferative function evidently reflects the physiological differences in the ploidy series, depending on parallel specialization processes. A reduction in proliferative ability with polyploidization seems to be associated with a change in the surfacelvolume ratio. However, the

Time

(hours)

FIG.29. Proliferative response of diploid cells (0) (en) as compared with tetraploid

ones ( 0 )(4n) to stimulation of mitoses in pea root segments cultured on medium containing auxins and kinetin. (From Matthysse and Torrey, 1967, with permission of Academic Press.)

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W. YA. BRODSKY A N D I. V. URYVAEVA

differences in the surfacelvolume ratios of 2n, 4n, 8n, and 16n cells obtained in a culture by means of Colcemid did not affect the increase in the growth rate of these sublines (Harris, 1971). The proliferative potentials of hepatocytes depend not only on their ploidy, but also on the age of the animals. It was noted long ago that, after partial hepatectomy in aged mice or rats, the liver is restored more slowly and less completely than in young animals (see Bucher, 1963).With age, the growth fraction of the liver parenchyma decreases (Fabrikant, 1969; Stocker et al., 1972; Schultze et al., 1973; Uryvaeva and Faktor, 1975).In young animals the growth fraction includes practically all the hepatocytes (99.8%); in adult animals 90-93% of the cells are capable of reproduction; and in aged animals a maximum of 70-77%. In young and adult mice, hepatocytes of any ploidy proliferate after partial hepatectomy. The growth fraction of aged (1.5- to 2-year-old) animals is found to contain mainly tetraploid cells, while many octaploid cells and those of higher ploidy do not enter into the cycle at all (Uryvaeva and Faktor, 1975). Deterioration of cell functions (proliferative in this case) with the animal’s age is a well-known phenomenon. But the causes of this deterioration are far from clear. Is the age of the cell determined exclusively by the number of divisions it has undergone, and its aging, by the exhaustion of the proliferative potential inherent in the cells (Hayflick, 1965; Orgel, 1973)? In the case of hepatocytes this is obviously not quite so. Proliferative potentials decrease with polyploidization. But cells of the same ploidy that have passed through the same number of mitoses, for instance 8n, respond differently to the proliferative stimulus in the parenchyma of young and aged mice. Harrison (1973)has suggested that senile changes in cell properties may largely be due to the deteriorated medium of the aged organism. In verifying this idea with regard to hepatocytes, it is important to remember that they belong to those few cell types that form the medium of the organism. In any case, it is obvious that polyploidization of hepatocytes is not an indication of aging of the tissues. Most cell polyploidization occurs in youth, and the functional properties of the cells increase with polyploidization, often proportionally to genome growth.

VII. General Conclusion A diploid set of chromosomes is the usual composition of the eukaryotic genome. This state is ensured by the reproduction and division of chromosomes during the mitotic cycle. Polyploidy or polygenomy, or multiplication of the genome of the somatic cell results

CELL POLYPLOIDY

325

from incomplete mitotic cycles. The reduction occurs in the processes following the phase of DNA synthesis. Depending on which division phases are bypassed, we speak of a polytene (endoreduplication) or endomitotic cycle, or of a reduction in the program of mitosis itself as a type of incomplete cell cycle. In the case of omission of mitosis entirely, the DNA replication cycles follow one another. In Diptera, such polytene cycles are the basis for the formation of giant multistranded chromosomes. Other cases are also known in which the polytene cycle does not affect the nuclear structure on accumulation of even very large amounts of DNA. Phenomena interpreted as endomitosis involve specific nuclear morphology in this process. In definitive tissues of mammals and also of some animals and plants, the mechanism of genome multiplication corresponds neither to the polytene nor to the endomitotic m e c h a n i ~ mPolyploid .~ cells are formed as a result of aberrations in the mitotic process during the later phases, after the separation of the chromosomes. In this case the nucleus does not divide at all, or the daughter nuclei fuse together, or a binucleate cell is formed on failure of the cytoplasm to cleave. Such mitoses are polyploidizing, and the mode of formation of polyploid nuclei is termed mitotic. One may speak of certain advantages of an incomplete cell cycle over a complete one. They are evident at extreme degrees of incompleteness (polytene and endomitotic cycles) and involve the possibility of rapid growth due to the shortening of the reproduction cycle without an interruption in function. Sometimes another aspect is decisive, namely, cell gigantism based on polyploidy, which is also attained through incomplete cell cycles. The significance of polyploidy developing in adult mammals (the liver and other glands, megakaryocytes, vegetative ganglia) can be only partially explained from the same standpoint. The moderate degree of genome multiplication provides no basis for cell gigantism. The simplification of the cell cycle on omission of only the final phases of mitosis is also insignificant and does not ensure any gain in the form of growth acceleration. One can suggest that the united polyploid genome facilitates cell regulation at the tissue level. Nevertheless the transition of cells from diploidy to polyploidy entails only quantitative changes and does not appear to be a change in quality. This point of view is supported by the following considerations. Cell polyploidization is not an absolutely essential factor for After this survey had been accepted for publication, valuable data on plant cell polyploidy was published by W. Nag1 (1976,Annu. Reo. Plant Physiol. 27,39).

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complete differentiation; it does not determine the specificity of a cell type. Thus adult hepatocytes may be diploid or polyploid in related species of animals, and diploid megakaryocytes as well as polyploid ones, are capable, of producing blood platelets. One is also impressed b y examples of the formation, during induced growth, of polyploid cells in tissues in which they normally are not typical, as is known for the atrial myocardium of rats. I n the liver of rodents, and evidently also of humans, polyploid cell classes are formed on the basis of acytokinetic mitosis and a subsequent particular kind of mitosis of binucleate cells. During normal growth, a rather regular sequence of such mitosis and, respectively, of the states of mononucleation and binucleation is found in the life of each cell. This appears to be a realization of the development program. Indeed, the postnatal growth of the liver is due mainly to cell polyploidization. This scheme, however, is easily broken down by the intervention of reparative growth during normal postnatal growth. The cause of modification of the mitotic cycle may be the interaction of cellular functions and their effect on providing mitosis syntheses. Transition from a complete mitotic cycle to an incomplete one seems not to be a change in program, but a regulatory process which modifies the course of mitosis. The proposed hypothesis is as follows. The metabolic resources of the cell are not infinite, therefore the performance of some function or another may be inhibited at the epigenome level. This situation arises on explantation of differentiated tissues in a culture in which the cells reversibly lose specific phenotypic traits. The interaction of the proliferative and tissue-specific functions has features of mutual balancing, as a result of which an increase in one of them entails the weakening of the other. In the cases investigated it was found that the molecular basis of this phenomenon was the competition of different metabolic pathways for the lacking metabolite, or translational competition. Polyploidizing tissues are characterized by the retention of proliferative ability in maturing and mature cells. Then proliferative and specialized functions show signs of mutual balancing, as demonstrated for hepatocytes, cells of the pigment epithelium of the retina, the myocardium, and the salivary glands. Possibly, it is the effect of the specific functions that causes quantitative displacement of proliferation and modifications in the mitotic cycle, with the resulting polyploidy. The reciprocal relationships of the mitotic and the tissue-specific functions are most probably one of the manifestations of the general principle of quantitative balancing of functions not vitally essential to the cell. The phenotype of a differentiated cell capable of several different specialized syntheses may depend on their competition.

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