METABOLIC REGULATION IN THE CELL CYCLE

5 METABOLIC REGULATION IN THE CELL CYCLE Robert R. Klevecz and Gerald L. Forrest

I. Introduction II. The Methodology of Cell Cycle Analysis (Optimal Conditions, Choice of Cells, and Sampling Intervals) A. Alignment Synchrony B. Selection Synchrony III. Temporal Regulation of Gene Expression A. RNA Metabolism B. Expression of Enzyme Activity IV. Changing Perspectives of the Cell Cycle A. The Mitotic Cycle B. The Chromosome Replication Cycle and Its Diagram C. Serum Factors and the Cell Surface D. The Cell as a Clock References

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If life can be likened to a bouncing ball, then the essence is in the bounce, not the ball. J. Bronowski, April, 1965

I. Introduction As a technique synchrony has been applied with usefulness to basic studies in the molecular biology and biochemistry of cells and is an important tool in the study of certain disease states. Most macromolecular processes have a direction­ ality which may be obscured in a random population of cells. Enzyme levels, for example, are determined both by the rate of synthesis and the rate of degrada-

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tion. In a random exponential population of mammalian cells, enzyme levels appear as constant age-averaged values and the relative contributions of synthesis and degradation cannot be determined independently, and only approximated by resorting to inhibitors or inducers to perturb the steady state. The use of a synchronous population may reveal that the two processes are temporally separated and somewhat sequential or that they occur over a restricted portion of the cell cycle. Senescence and cancer are phenomena expressed by cells at the two extremes of the proliferative state: the transformed cell divides indefinitely, the aged culture nevermore. The questions being asked about these two phenomena are similar and they are inherently cell cycle-related questions. Current research in the field is directed toward determining (1) where or at what point or points in time in the cycle cells exit and enter the proliferative state; (2) if there is a particular ensemble of cellular proteins present and if the biochemical composi­ tion of an arrested or differentiated cell is different from a cycling cell at the same point in the cycle relative to definable markers; and (3) what hormonal or nutritional factors and characteristics of the parochial environment, such as cell density, mediate the exit and entry. As a discipline, the study of the cell cycle is an investigation into the nature of a cellular clock which must ultimately generate the biological clock. The paucity of information on the structure of the cellular timekeeping process may indicate what a dynamic and elusive system it is. Many processes and activities in eukaryotic organisms display overt daily rhythms which have sufficient inertia to persist in the absence of external stimuli. Most workers agree that underlying the circadian rhythm there must be an endogenous and cellularly based clock whose time constants are generated by means of coupled metabolic processes, but identifying the clock with any particular element of organismic chemistry or in the case of mammalian cells even demonstrating clock properties has proved difficult.

II. The Methodology of Cell Cycle Analysis (Optimal Conditions, Choice of Cells, and Sampling Intervals) The techniques of synchronization, because they represent the manipulation of a highly labile biological system must be performed with particular attention to the possibility that such manipulations are perturbing the system. Synchrony is essentially a device to amplify the chemistry and behavior of the one cell, and therefore every effort should be aimed at making the population behave as an individual. In this respect, the likelihood of success may be increased by adhering to the following suggestions: (1) cells with short cycles are preferred

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over those with long cycles; (2) discrete changes in the patterns of synthesis are more easily detected in diploid or cloned aneuploid cells than in heteroploid cells; (3) cells should be as homogeneous as possible with regard to gross parameters such as chromosome number, mass/cell and generation time; (4) given a homogeneous established cell line to start with, the above characteristics can best be maintained by passaging the cells so as to keep them in exponential growth and by avoiding confluency; (5) careful control of temperature is advisable with the possibility that certain temperatures will maintain synchrony in the population better than others; (6) Sampling should be more frequent than many laboratories will find comfortable. Most work in our laboratory would suggest that fluctuations in enzyme activity occur with a 3-4-hour period so that half-hour sampling intervals are often necessary to resolve intermittent processes in the cell cycle of lines such as V79 and CHO. Whether the decay of synchrony is too rapid to justify sampling that frequently in a heteroploid cell line with a 24-hour generation time is a question which should be resolved. Certainly the day has passed when three samples, one each in Gi, S, and G 2 , constitute a cell cycle analysis. Finally, it should be apparent that no means has been found to hold all cellular processes in place. The fact that some methods of synchronization may arrest one obvious event (DNA synthesis) and upon reversal generate the syn­ chronous occurrence of a second event (mitosis) may give a false indication of the quality of synchrony, since other independent events such as enzyme synthesis may continue to be initiated in the absence of continuing DNA synthesis (Klevecz, 1969a; Studzinski and Lambert, 1969). A number of reviews and collections are available which emphasize synchrony methodology. In particular the work by Stubblefield (1968), Nias and Fox (1971), and the Methods in Cell Biology series by Prescott, especially volumes 9 (1975a) and 10 (1975b), can be recommended. Murdoch Mitchison's excellent book "The Biology of the Cell Cycle" contains a useful discussion of methods as well as the best summary of our knowledge of the cell cycle to 1971. A. Alignment Synchrony 1. S PHASE ARREST

Rueckert and Mueller (1960) first arrested cells in S phase with the folic acid antagonists, amethopterin and aminopterin, and with FUdR to achieve a partial synchronization of He La cells. Xeros (1962) attempted a more physiological approach by administering 2 mM thymidine to the cell and exploiting the end product inhibition of aspartate transcarbamylase by thymidine triphosphate. Synchrony using high thymidine was improved by Bootsma et ah (1964), who employed two high thymidine blocks with an interveining reversal of the block.

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Subsequent work revealed that in some cell lines 4-10 mM thymidine was necessary to block DNA synthesis effectively (Tobey et al, 1967) and that the problems of unbalanced growth and chromosome aberration (Yang et al, 1966), associated with folic acid antagonists, are present with high thymidine as well. Thilly (1972) and Thilly et al (1975) employed a regimen of repeated short duration S phase realignments with 0.25 mM thymidine to exploit the fact that the relaxation time for unbalanced growth, as expressed in changing cell volume, is short relative to the length of time between periods of thymidine arrest. Consequently, the cell volume and protein content per cell, which character­ istically increase during prolonged S phase arrest, return to and remain at control levels after four cycles of short term S phase blockage and reversal. This would seem to be the most promising method if thymidine must be used to achieve synchrony. A somewhat similar procedure was applied to human lymphoblasts by Zielke and Littlefield (1974). Unfortunately, these workers blocked the cells for 15 hours which would seem to subvert the main advantage of multiple intermittent blocks. Apart from the problems of unbalanced growth and the chromosome aberrations induced in cells engaged in synthesis at the time of the block, cells treated with high thymidine continue to synthesize DNA (Studzinski and Lambert, 1969) and may not be blocked at the Gi/S boundary but later in S, prior to the replication of the bulk of DNA (Williams and Ockey, 1970; Amaldi et al, 1972; Huberman et al, 1973; Comings and Okada, 1973; Klevecz etal., 1975). Other inhibitors of DNA synthesis have been used with greater or lesser success. Sinclair (1967) used 1 mM hydroxyurea (HU) which is reputed to be a more effective inhibitor of DNA synthesis than the folic acid antagonists or excess thymidine. However, two years earlier Sinclair and Bishop (1964) had observed a selective lethal effect on S phase cells and Kim et al. (1967) used 10~2 M hydroxyurea as a selective lethal agent to synchronize cells. Finally, Regan and Chu (1966) obtained a partial synchronization of hamster cells using 5-aminouracil to arrest cells in S. 2. SELECTIVE LETHAL METHODS

In this approach exponential cultures are treated with a toxic agent for a period of time equal to the generation time less a small increment. This window of survivors becomes the synchronous population. Perhaps the best effort in this area is that of Pfeiffer and Tolmach (1967) who applied vinblastine to He La cells for 1 hour less than the mean generation time, removed the irreversibly arrested mitotic cells, allowed the remaining cells to undergo mitosis, and then reapplied the alkaloid slightly more than one generation later to kill lagging members of the population. A single treatment with vinblastine followed by selection had been previously reported by Kim and Stambuk (1966). Elsewhere Kim et al

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(1967) had applied HU to cells using the same principle but depending on the drug-killing effect on cells engaged in DNA synthesis. Similarly Whitmore and Gulyas (1966) treated exponential cultures with tritiated thymidine to kill all those engaged in DNA synthesis. This latter method was a useful demonstration and warning that radioactive thymidine may not be innocuous when used as a label. The combined use of thymidine and vinblastine has also been applied in one instance (Djordjevic and Tolmach, 1967) to synchronized He La cells. Practical and theoretical objections make it likely that selective lethal meth­ ods will never see wide application in synchrony. The treated population must be separated from the untreated, which can be easily accomplished with vin­ blastine blocked metaphases, but with tritiated thymidine, treated cells must be allowed to die and may therefore linger to contribute label to the untreated window. The window may contain in addition to properly cycling cells a population of aberrant noncycling or slowly cycling cells or a population of cells normally arrested in the prereplicative portion of the cycle. Since this latter population may be a significant fraction of the total in diploid or density-sensi­ tive lines such as WI-38 or 3T3, it follows that this method would probably be applicable only to lines with low density dependence and only if the culture conditions are optimized. 3. MITOTIC ARREST FOLLOWED BY MITOTIC SELECTION

Stubblefield and Klevecz (1965) used 0.06 Mg/ml Colcemid for 2-3 hours to arrest Chinese hamster cells in M. Originally designed as a compromise between S phase arrest methods which provide high yields but produce unbalanced growth, and simple mitotic selection (Terasima and Tolmach, 1961) with its low yields, the method still sees use (Housman and Huberman, 1975). It has the advantages of providing a narrower window of cells than unenhanced mitotic selection, and better detachment of mitotic cells, since during the 2-hour treatment the attachment of mitotic cells to the surface is made more tenuous because the arrested cells continue to swell. It has the disadvantage of distorting transport processes (Meizel and Wilson, 1972). The effects of prolonged Colcemid treat­ ment are severe; smooth endoplasmic reticulum and Golgi-like structures are elaborated by the cell and begin to coalesce around the chromosomes. Multinucleated cells appear after 3-4 hours of treatment and predominate in the population by 10-12 hours (Brinkley et al, 1967; Robbins and Gonatas, 1964). Martin et al. (1969a) and subsequently other workers (Mitchell and Hoogenraad, 1975) overextended the method somewhat by collecting cells for 8-10 hours. Doida and Okada (1967) combined excess (4 mM thymidine) arrest and deoxycytidine reversal followed by Colcemid arrest and reversal to synchronize suspension cultures of L5178Y mouse lymphoma cells. Okada and Shinohara (1974) later automated this method so that the addition of thymidine to block

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the cells, its reversal by deoxycytidine, and the subsequent collection of the mitotic cells with Colcemid could be performed in the small hours of the morning, giving cells in metaphase at the desired time. This double block appears to give excellent synchrony, but the objections commonly leveled at inhibitor methods might be doubled in this case, were it not for the studies of Thilly et al. (1975) showing that unbalanced growth due to thymidine arrest is reversed within a few hours. Unlike the automated mitotic selection synchrony to be discussed below, the method of Okada requires that the investigator attend and sample from the culture at the appropriate intervals. Since the L5178Y cell cycle is but 8-9 hours long, the incentive to automate the cell cycle staging as well as initial synchronization is less than it might be. Finally, Rao (1968) showed that nitrous oxide under pressure could be used to accumulate cells reversibly in mitosis. However the cultures were parasynchronous, indicating the nitrous oxide may inhibit cell cycle traverse at points other than M. 4. TEMPERATURE SHOCKS

Repeated heat shocks, which serve to align Tetrahymena (Zeuthen, 1974), appear to induce parasynchronous growth in mammalian cells as well. In a thorough study of the effects of variable numbers of shocks at temperatures between 41.3 and 41.9°C on mouse L cells, Miyamoto et al (1973) found that two or more 1-hour exposures at 11-hour intervals gave considerable synchroni­ zation of cell division. It would be of interest to know what degree of synchrony could be obtained by heat shocks at shorter (M- hour) intervals (see Section IV, D). Unfortunately, a very early report of parasynchronous cell division following cold shocks has never been improved upon (Newton and Wildy, 1959). 5. PREREPLICATION ARREST

A number of years ago Quastler (1960) confirmed, as many had suspected, that in vivo the cells of many tissues were in a quiescent state between mitosis and the DNA synthesis. Nilhausen and Green (1965) pursued this problem in cultured mouse 3T3 cells and showed that at saturation densities the cells had a Gx DNA content and that upon stimulation by subculturing, 95% of the cells synthesized DNA before dividing. This parasynchronous emergence from Gx was exploited (Stubblefield et al., 1967) to collect a better than an exponential number of mitotic cells with Colcemid and was observed to occur in suspension cultures (Tobey and Ley, 1970) as well as monolayers (Yoshikura et al., 1967). Ley and Tobey (1970) further developed it as a synchrony method by depleting the medium of isoleucine and glutamine and concluded that isoleucine held some special role in the initiation of DNA synthesis. Everhardt (1972), on the other hand, suggests that isoleucine deprivation works because cells are less

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sensitive to its depletion than to the removal of other amino acids such as leucine. Holley and Kiernan (1974) concluded that prereplicative arrest could be accomplished by growing the cells sparsely in low concentrations of a number of essential amino acids as well as low glucose or phosphate ions. A similar role for amino acids in the control of DNA synthesis has been shown to exist in liver (Short etal, 1974). 6. TEMPERATURE-SENSITIVE CELL CYCLE MUTANTS

The usefulness of genetic analysis in the resolution of mammalian cell cycle events has been demonstrated to us by Hartwell and his co-workers (1974) in yeast using temperature-sensitive (ts) mutants defective for functions required for continued traverse of the cycle. Successful isolation of ts cell cycle mutants in mammalian lines is limited at this time to just a few examples. At least two of these mutant lines have been sufficiently well characterized to allow their developers to describe them as Gi mutants (Burstin et al, 1974; Liskay, 1974). Some attempt has been made to "map" the altered functions by testing the ability of mutant lines arrested by isoleucine or serum deprivation (Burstin et al., 1974) or high density (Liskay, 1974) to undergo DNA synthesis upon reversal at the nonpermissive temperature. Since there is some indication of a similarity between the isoleucine and serum deprivation arrest points (Pardee, 1974) as well as some question whether movement through G! can be regarded as a single dependent sequence (see Section IV,D), it is probably premature to discuss map position. Prereplication arrest is a useful approach to problems related to the control of proliferation, since the exit from and entry into the cycle may mimic the in vivo state, but as a synchrony technique it falls short of other methods available today. The interval over which cells enter S or divide following addition of the depleted amino acid seems too broad to justify its use in cell cycle studies. For example, in CHO cells with a 12-hour cell cycle, 10 hours or more are required for 90% of the cells to divide following addition of the depleted amino acid (Ley andTobey, 1970). B. Selection Synchrony 1. MITOTIC SELECTION

The selection of mitotic cells by preferential detachment from a random exponential monolayer was the first synchrony technique successfully applied to mammalian cells (Terasima and Tolmach, 1961, 1963). It is still the most widely used and perhaps the best method for achieving synchrony. In its purest form the method should be most gentle and nonperturbing. However, in attempting to solve the inherent limitation of this approach, its low yield of cells (<0.5% of

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the population), a number of supplemental treatments have been applied. The fraction of cells selected has been enhanced using Colcemid (Stubblefleld and Klevecz, 1965), or medium with lower Ca2+ and Mg2+ concentrations (Robbins and Marcus, 1964) and the size of the collected population has been increased by storing repeated collections from flasks (Sinclair and Morton, 1963) or roller bottles (Lindahl and Sorenby, 1966) at lowered temperatures. Such treatments can affect subsequent cell cycle events (Lett and Sun, 1970) and reduce the number of traversing cells (Tobey et al, 1972). To solve the problem of low yields while avoiding the perturbations likely to be introduced by altered media or lowered temperatures, the mitotic selection technique has been automated and applied to roller bottle cultures (Klevecz, 1972). 2. AUTOMATED MITOTIC SELECTION AND STAGING

The cell cycle analyzer removes mitotic cells from random cultures in roller bottles by the gentle shearing of medium over the surface of the monolayer. Four roller bottles are employed with a total growing surface of 2500-7500 cm2 so that the yields of cells can be quite high (2 X 106—5 X 107 cells/selection). The system functions to partition a random culture of cells into a discontinuous series of synchronous cultures, each separated from the others by an interval equal to the time between selections. Several features of the cell cycle analyzer recommend it as a method of synchronization. The instrument operates without the use of inhibitors, altered medium, or reduced temperatures, which can reduce the number of cells travers­ ing the cycle and introduce artefacts into the results. Perfusion conditions can be maintained prior to and during the selection regime to ensure that the selected populations do not differ with respect to prior exposure to nutrient levels or serum factors. Selection can be optimized so that the maximum yield of cells consistent with a high mitotic index is obtained. Optimum selection conditions can be repeatably obtained from selection to selection and from day to day. Since the instrument functions automatically, there is no need for the investiga­ tor to endure a tedious and sleepless vigil in order to perform cell cycle studies. The instrument can be programmed in the afternoon to operate through the night so that synchronous cultures of cells, arrayed at suitable intervals through the cycle, are available the following morning. The details of operation of the commercial instrument have been described elsewhere (Klevecz et al, 1975). 3. VELOCITY SEDIMENTATION

Differential velocity sedimentation separates cells primarily on the basis of volume. This method has been applied to a variety of cells using linear sucrose gradients (Sinclair and Bishop, 1964), Ficoll gradients (Morris et al, 1967; Warmsley et al, 1969), serum gradients (MacDonald and Miller, 1970) or

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medium (Shall and MacClelland, 1971), with g forces from 1 g upward being used to sediment the cells. Relatively large preparations of cells have been obtained using a Ficoll gradient and zonal centrifugation (Warmsley et ah, 1970; Warmsley and Pasternak, 1970). In some instances only a single fraction, enriched for cells of a particular age, is used (Shall and MacClelland, 1971). In other cases the entire gradient is fractionated and used, with the expectation that cells will be arrayed through the gradient by age (Warmsley and Pasternak, 1970). The technique depends for its success on the assumption that the function (probably volume) being exploited for separation is one that changes linearly, or nearly so, with age. If an inflection occurs in the separation function this method will not work, or rather it may appear to work and give misleading results. Volume has been shown to increase exponentially (Anderson et ah, 1969) and discontinuously in a saltatory fashion with age (Klevecz and Ruddle, 1968). An exponential increase in volume with age would result in some gradient fractions having a broad and some a narrow age distribution of cells. If a major inflection in the function occurs, then some gradient fractions will have quite dissimilar populations with respect to age. 4. ELECTRONIC SORTING

a. Viable Sorting. The cell-sorting instrument developed by Herzenberg and his co-workers (Bonner et al, 1972), as with the flow micro fluorograph (Van Dilla et al., 1969), is based on the separation technique first used by Fulwyler (Fulwyler et al., 1969). Individual cells are pulled in a laminar fashion through a capillary tube transected by a laser. The stream of cells is broken into uniform drops downstream from the laser and fluorescent signals from the cells are used to designate which drops receive an electrostatic charge. Charged drops contain­ ing the desired cells are then separated from the rest and collected. Unlike the FMF, most of the work on this instrument has been directed toward viable sorting (Hulett et al., 1973). No success has yet been reported in applying this instrument to cell cycle analysis but with certain reservations it should be possible. The instrument has the capability of sorting on two parameters such as fluorescence and light scattering, a capability which may be essential to adequate resolution of cells of a particular age. At present the limitation on all such machines is the low processing rate. To obtain a population of cells with a 1 hour wide age distribution equal to that routinely achievable by many syn­ chrony methods (2 X 107 cells) would probably require 15 hours of error free sorting and therefore some form of low temperature storage to maintain reason­ able synchrony in the population. b. Analytical and Preparative Sorting of Fixed Cells. One intended function for flow microfluorometry was the resolution of age distribution of cells in a randomly growing population (Van Dilla et al., 1969). Cells are first fixed and

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stained and subsequently the relative amount of any cellular component which can be fluorescently stained is assayed. The system represents an analytical rather than a preparative approach to the cell cycle. It can be applied to demonstrate quantitatively the degree of synchrony immediately following mitotic selection and at intervals through the cell cycle. To accomplish this, cells are stained for DNA content using the acriflavin Feulgen method (Kraemer et al., 1974) and then analyzed at hourly or half-hourly time increments through the cell cycle (Klevecz et al, 1975). However, in the fluorescent Feulgen assay the DNA content is measured in situ and consequently the possibility must be considered that this staining procedure may be affected by the availability of the DNA. More recently the FMF analysis has been applied to fixed cells stained with propidium iodide (Crissman and Steinkamp, 1973) or mithramycin (Crissman and Tobey, 1974), but as with the fluorescent Feulgen technique, the accessibility of the DNA to bind the stain must be considered. Successful application of preparative sorting to the cell cycle lies in the future. The feasibility of this approach has been demonstrated not by selecting cells of a particular cell cycle age but in the sorting of chromosomes according to size (Gray et al, 1975). In theory it should be applicable to sorting of cells of particular ages once the age distribution of any cellular constituent is known. The one caution that might be invoked is that age resolution based on the changing amount of fluorescence will depend on the fact that the constituent changes linearly or at least without a major inflection as a function of age. If not, then some additional manipulation such as two-parameter sorting will have to be used. Otherwise the objections which hold for gradient sedimentation separation might also apply here.

III. Temporal Regulation of Gene Expression In prokaryotes and in some unicellular eukaryotes, the genome is available for transcription at all points in the cell cycle (Knutsen, 1965; Mitchison and Creanor, 1969; Donachie and Masters, 1969; Baechtel et al, 1970). Upon replication of a particular gene, there is a doubling in potential for enzyme synthesis (Kuempel et al, 1965). In the case of completely repressed and fully induced or derepressed genes, enzyme synthesis is continuous through the cycle. In normally growing cells subject to control by negative feedback, synthesis is autogenous, and enzymes are made periodically, often, but not rigorously, in register with their location and time of replication on the bacterial chromosome (Masters and Pardee, 1965; Masters and Donachie, 1966). In its simplest form the oscillatory induction-repression model predicts that the concentration of end products or catabolites of particular genes will vary depending on the enzyme concentration and activity and that together the components of the

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system will oscillate out of phase. Support for the oscillatory repression model in unicellular eukaryotes has been provided in Chlorella by Molloy and Schmidt (1970), but Griffith (1968) has questioned whether simple feedback loops would all have the same dynamic properties and whether enzymes regulated by such loops would display maxima at one-generation intervals. He has argued that sustained oscillations, involving the interaction of the gene, one enzyme, and its metabolites, are unlikely and that only by introducing additional steps in the control circuit can such periodicities be produced. Goodwin (1963), who gener­ ated the early thought leading to this model, amplified it by suggesting that periodic enzyme synthesis is entrained to DNA replication by a burst in messen­ ger synthesis (Goodwin, 1966). In its simplest form oscillatory induction-repression model fails as a means of describing mammalian cell enzymes because no accounting is made for what appears to be restricted periods of inducibility in cultured cells. In the few instances in which enzyme activity has been induced in cycling cells it appears to take place predominantly in S phase. In all instances where it has been measured, the induction of enzyme activity, or in some instances, "superinduction" has been shown to be restricted to a limited portion of the cell cycle. In the case of the least restricted induction, the synthesis of tyrosine aminotransferase (TAT) was shown to be induciblele by dexamethasone during two-thirds of the cell cycle in HTC cells (Martin et αί, 1969a, b; Martin and Tomkins, 1970). No inductive effect could be demonstrated from G2 through the first three hours of Gt (Gi-3). The authors have suggested that inducibility in their system requires the synthesis of a posttranscriptional repressor which is the site of action of the inducer (Martin and Tomkins, 1970). In their model both the repressor gene and the structural gene for TAT are transcribed during the inducible periods of the cell cycle. The repressor interacts with the mRNA coding for TAT and promotes its degradation. The presence of the inducer antagonizes the repressor resulting in a stabilization of the TAT mRNA, thereby increasing the amount of TAT. In a series of studies which did not rely so heavily on inhibitors, Palmiter and Schimke (1973) suggested an alternative mechanism to explain superinduction. Using estrogen-stimulated chicken oviducts, they have shown that there is an increase in the rate of elongation of the nascent polypeptides and, since the poly some size remains the same, they argue that there must also have been an increase in initiation. Consequently, increased translation of TAT mRNA may come about as a consequence of differential stability of the induced messenger and hence, a favored competition for rate-limiting factors common to the translation of all messengers. No messenger binding repression is required in the scheme. Acid phosphatase can be superinduced in L5178Y cells with actinomycin D (Kapp and Okada, 1972b), and alkaline phosphatase induced in He La cells with hydrocortisone (Griffin and Ber, 1969), or in Henle cells by prednisolone (Melnykovych et al., 1967). Actinomycin D caused acid phosphatase to increase

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to maximum within an hour after treatment, while alkaline phosphatase activity following hydrocortisone and prednisolone treatment displayed a lag of 12-20 hours before maximum expression. Induction occurred during S phase with pred­ nisolone and hydrocortisone and in late S with actinomycin D. Serine dehydratase was induced in CHO cells using dibutyryl cyclic AMP and testosterone (Kapp et αί, 1973) and again induction was limited to a short period of the cell cycle in late S phase. In the lone exception to S phase restricted induction Martin et al (1969a) reported induction starting at the third hour of Gi in HTC cells, but the method of measuring the onset of DNA synthesis and the fact that synchroniza­ tion was accomplished by 10 hours of Colcemid collection make it equally likely that some cells had begun to replicate DNA by Gx 3 (Amaldi et αί, 1973). If restricted induction in the cell cycle holds, it might be viewed as somewhat paradoxical since in many instances induction of enzyme activity takes place in nondividing, and therefore presumably nonreplicating tissues. One might argue that true induction, the synthesis of specific messengers, requires DNA synthesis and that in these instances induction in steady-state systems is a posttranscriptional event, were it not for the evidence of Palmiter and Schimke (R. T. Schimke, personal communication) who showed that ovalbumin messenger is made in the presence of inhibitors of DNA synthesis. Perhaps we can explain these contradictory findings by paraphrasing Orwell's pigs (Orwell, 1946) that all portions of the cell cycle are inducible, only some are more inducible than others. (One might argue of course, that degrees of inducibility are less arbitrary than degrees of equality.) In this view messenger synthesis following treatment with an inducer occurs to a limited extent no matter where in the cycle the inducer is introduced, but only during replication of the DNA molecule containing the particular gene is induction maximal. Substance is given to this by Geiduschek and his co-workers (Riva et aly 1970) who find that transcription of T-4 late genes requires the presence of a replicat­ ing fork or nick to occur maximally. Also in accord with this is the fact that the response seen in cell cycle induction is rapid, with a two- to tenfold increase occurring in just a few hours, whereas full induction in more quiescent tissues such as chicken oviduct requires 9 days (O'Malley et al., 1967). To return briefly to the oscillatory induction-repression model, it has suc­ ceeded in a nonrigorous way in explaining the regulation of gene products of mammalian cells in the sense that it anticipated the oscillatory behavior of many enzymes. The pattern of changes in mammalian cells is sufficiently complex to determining whether it serves adequately as a model. For example, certain enzymes display multiple maxima in the mammalian cell cycle. Later we will suggest how the cycle might be generated from multiples of a fundamental cycle, G q , so that the multiple maxima in activity may be generated by the same feedback loop which controls cell division. Should this turn out to be the case we would be much closer to a unified view of regulation in the cell cycle.

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A. RNA Metabolism 1. RATE OF TOTAL AND RIBOSOMAL RNA SYNTHESIS

In the early enthusiasm that followed the establishment of synchrony meth­ ods adequate to generate relatively large quantities of cells, a number of investigators set out to measure the rate of total RNA synthesis and specific RNA synthesis by uridine labeling. The results from these experiments fall into two categories. Scharff and Robbins (1965), Enger and Tobey (1969), and Stambrook and Sisken (1972a) found that the rate of total RNA synthesis measured by uridine incorporation in HeLa, CHO, and V79 cells, respectively, increased more or less linearly from the beginning of Gi through to G2 and then decreased as the cells entered mitosis. In contrast to that, Klevecz and Stubblefield (1967) found that the rate fluctuated through the cell cycle of Chinese hamster Don C cells such that there was a minor peak in the rate of RNA synthesis in Gx and one or two major peaks, depending on the cell, in S and G 2 , while the overall rate doubled between Qx and G 2 . The fluctuations appeared to be due to changes in ribosomal RNA synthesis and were somewhat out of phase with maximum DNA synthetic rates. Earlier, Reiter and Littlefield (1964) had found some evidence for fluctuations in rate in FUdR synchronized L cells and Crippa (1966) had found a similar pattern in mitotically synchronized Chinese hamster cells. Pffeifer and Tolmach (1968) found that the rate through Gi was more or less constant and then increased abruptly two- or threefold near the beginning of S phase to give a pattern very similar to that observed by Klevecz and Stubblefield, and Crippa. Using time lapse cinematography and autoradiography of exponential cells to measure the rates of RNA synthesis, Seed (1963) found a discontinuous increase in rate in diploid or near diploid cells and a more nearly linear increase in heteroploid cells. Seed, since he used asynchronous cultures, circumvented the objections regarding perturbations normally leveled at synchronization experiments. Kasten and Strasser (1966) found fluctuations in the rate of nucleolar and nonnucleolar RNA synthesis by autoradiographic analysis of CMP cells and noted that the fluctuations appeared to be reciprocally related to the rate of nucleolus-associated DNA synthesis. Gaffney and Nardone (1968) also measured the total nucleolar and nonnucleolar RNA synthesis in mitotically selected L-929 cells analyzed by autoradiography. They found that there was a two- to threefold increase in the rate of both nucleolar and nonnucleolar RNA synthesis through the cell cycle. It would appear from their curves that this increase took place at the beginning of S phase. They also found what they described as distinctive peaks in nucleolar and nonnucleolar RNA synthesis in late S and G 2 . Fingerhut and Nardone (1973) found that S phase cells incorporated from two to three times as much uridine into 18 and 28 S RNA as did Gx cells. They found no difference in the rate of processing of 18

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and 28 S RNA's as a function of cell cycle position. In agreement with the results of random exponential cells 18 S RNA appeared in the cytoplasm more rapidly than did 28 S. Stambrook and Sisken (1972a) have objected to the observations of discon­ tinuous synthesis, or more correctly, to the large increase in RNA synthesis in S and G2 by suggesting that the variations in rate are inducible by culture conditions. In support of this objection are the results of Cunningham and Pardee (1969), Sander and Pardee (1972), and Plagemann and Roth (1969). Stambrook and Sisken (1972a) found that the rate of incorporation of tritiated uridine into the intracellular pool paralleled the changes in the apparent rate of RNA synthesis. They argue, therefore, that the large increase in apparent rate of RNA synthesis observed by some (Kim and Perez, 1965) are occasioned by pool changes. They indicate that the overall rate of uridine incorporation into the solu­ ble pools varies by as much as tenfold through the cycle. The objections to the use of uridine presented by Stambrook and Sisken are valid. However, in the origi­ nal report Klevecz and Stubblefield (1967) described a twofold increase in total RNA synthesis and in anticipation of transport and pool problems endogenous pools were swamped by the addition of sufficient exogenous thymidine, deoxycytidine, and uridine to ensure that the endogenous pathways were contributing minimally to the total intracellular pyrimidine synthesis. Stambrook and Sisken (1972b) have also done double-labeling studies in which they compared the rate of tritiated uridine uptake to that of adenine. They found that the rate of RNA synthesis as determined from the relative amount of adenine incorporated into RNA to the specific radioactivity of the tritiated ATP pool would indicate that the rate of RNA synthesis doubles, although not discontinuously, during the cell cycle, in at least partial agreement with the original observations. 2. RESUMPTION OF RNA SYNTHESIS FOLLOWING MITOSIS

There is general agreement that the rate of RNA synthesis is greatly dimin­ ished during the division stages (Robbins and Scharff, 1966) and that although processing of ribosomal precursors resumes soon thereafter (Fan and Penman, 1971) little except 4 S and 5 S RNA's (Zylber and Penman, 1971) are made during this portion of the cell cycle. Stewart et al (1968) showed that the re-formation of polysomes following mitosis occurred without the requirement for new RNA synthesis, implying the utilization of preexisting messenger (Hodge et al, 1969). Simmons et al (1973) measured the restitution of RNA synthesis in He La cells reentering interphase following synchronization and found that new RNA synthesis did not require new protein synthesis. However, the mainte­ nance of the normal Gj rate of RNA synthesis did require protein synthesis. Simmons et al (1973) found that most of the new RNA that was made in early Gj was detected initially at the nuclear envelope and peripherally to the

5. Metabolic Regulation in the Cell Cycle

163

chromosome mass. They considered that multiple RNA polymerase activities must persist through mitosis to initiate new RNA synthesis in early telophase. Taube et al. (1974) examined the production of viral RNA's during the cell cycle of adenovirus-transformed rat embryo cells using a thymidine blockade to arrest the cells at the Gi/S boundary. They found that during mitosis viral RNA synthesis was restricted to the same extent as cellular RNA. They conclude that the transformed state can be correlated with a more or less continuous function­ ing of the integrated genome throughout interphase in contrast to the restricted synthesis of many gene products.

B. Expression of Enzyme Activity Several years ago Mitchison (1969) undertook to assemble all available reports on enzyme activity changes in the cell cycle of prokaryotes and unicellu­ lar and multicellular eukaryotes. On the basis of then existing data he considered that four patterns of enzyme behavior—step, peak, continuous linear, and exponential, depending on their modes of synthesis, modulation, and degrada­ tion—would adequately describe the range of possible patterns. This systematiz­ ing has held reasonably well for prokaryotes and simple eukaryotes and the reader is referred to Schmidt (1974) for a more recent discussion of the kinetics of enzyme synthesis in unicellular eukaryotes. It now appears however that metazoan organisms display more complicated patterns in the sense that multi­ ple peak or oscillatory enzyme activity patterns are often seen. The assay of the temporal expression of enzyme activities in the cell cycle has a number of purposes. It may serve to demonstrate unique markers for an otherwise undefined point in the cycle; it may permit the examination of a key enzyme upon which further cell cycle traverse is dependent; or it may be used to reveal the components involved in regulation of enzyme expression generally: the transcription, translation, modulation, and degradation of a particular activ­ ity. Obviously, different patterns will be obtained when different means of expressing enzyme activity are used. The preferred form is as activity in moles of substrate utilized/min/cell. In some cases specific activity has been expressed as activity per μg of protein or per μ% of DNA. Enzyme activity/unit of protein is more appropriate for tissues where a steady state exists. In vitro the cell is the unit of interest and when specific activity/Mg protein is used, real fluctuations in the cell cycle may be obscured and artifactual ones introduced. In this section specific activity means activity/unit of protein and is used perjoratively. The reader is referred to Mitchison (1971) for a thorough discussion of this point. In this section the enzymes have been grouped together according to their intracellular location or their function in metabolism. Table I summarizes almost all of

TABLE I. Method of synchrony0

Mouse L cells Thymidine kinase 1 Thymidine kinase 1 Thymidine kinase 11 or 13 DNA polymerase 7 DNA polymerase 2 DNA polymerase 11 or 13 DNA polymerase 7 Ribonucleotide reductase 2 Deoxycytidine monophosphate deaminase 1 PolyADP-ribose polymerase 8 Mouse myeloma Cl IgG 3 Mouse L5178Y cells Collagen:galactosyl transferase 6 Collagen:glycosyl transferase 6 Uridine diphosphatase 6 Esterase 6 5'-Nucleotidase 6 Glycoprotein:galactosyl transferase 6 Succinic dehydrogenase 6 Monoamine oxydase 6 Fetuin:fucosyl transferase 6 PSM:fucosyl transferase 6 FetuiniTV-acetylglucosaminyl transferase 6 DlypeptideiAf-acetylgalactosaminyl transferase 6 Acid phosphatase 6 Acid phosphatase 6

Enzymes and proteins

SUMMARY OF MAMMALIAN CELL CYCLE ENZYMES AND PROTEINS

Bosmann (1970b) Bossmann (1970b) Bosmann (1970a) Bosmann (1970a) Bosmann (1970a) Bosmann (1971a) Bosmann (1971b) Bosmann (1971b) Bosmann (1971a) Bosmann (1971a) Bosmann (1971a) Bosmann (1971a) Kapp and Okada (1972a) Bosmann and Bernacki (1970)

Byars and Kidson (1970)

Littlefield (1966) Mittermayer et al (1968) Adams (1969) Gold and Helleiner (1964) Turner et al (1968) Adams (1969) Littlefield et al (1963) Turner et al (1968) Mittermayer et al (1968) Colyereiß/. (1973)

Reference

2 a

I

s

s

ON

9 q

6 6 6 6 6 6 6 Mouse P815Y cells 5 5 5 5 5 5 5 Mouse 3T6 1 Rat HTC cells 9 9

Collagen

RatdiploidPR 105 7 Chinese hamster Don C cells 9 Thymidine kinase 9 Thymidine kinase 9 Thymidine kinase (G3) 9 Ribonucleotide reductase 9 Glucose-6-phosphate dehydrogenase

Tyrosine aminotransferase Lactate dehydrogenase Alcohol dehydrogenase Glucose-6-phosphate dehydrogenase

Collagen

NADPH-cytochrome c reductase Uridine diphosphatase Cytochrome c oxidase Succinate-cytochrome c reductase Lactate dehydrogenase Glucose-6-phosphate dehydrogenase Glutamate dehydrogenase

General proteolytic activity Try p sin Cathepsin j3-7V-Acetylglucosaminidase ß-A^-Acetylgalactosaminidase ß-Galactosidase RNA-dependent DNA polymerase (1970) (1970) (1970) (1970) (1970) (1970) (1970)

ei ß/. et al efa/. et al

(1969a) (1969a) (1969a) (1969a)

Stubblefield and Murphree (1967) Klevecz (1969a) Klevecz (1969a) Murphree et al (1969) Klevecz and Ruddle (1968)

Davies ei a/. (1968)

Martin Martin Martin Martin

Davieseitf/. (1968)

Warmsley et al Warmsleyeifl/. Warmsley et al. Warmsley et al Warmsley et al Warmsley et al Warmsley et al

Bosmann (1971c) Bosmann (1971c) Bosmann (1971c) Bosmann and Bernacki (1970) Bosmann and Bernacki (1970) Bosmann and Bernacki (1970) Bosmann (1971d)

{Continued)

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5* S £ £

TABLE I—Continued

Lactate dehydrogenase Malate dehydrogenase

Method of synchrony 0

9 10 3 Chang's liver cells 11 Chinese hamster V79 9 9 1 1 1 or 9 Syrian hamster 12 12

1 1 1 1 1

Chinese hamster Don C cells 9 9 9 9 9 9 9 Chinese hamster ovary (CHO)

Ornithine decarboxylase iS-Adenosylmethionine decarboyxlase Lactate dehydrogenase Hexokinase Tubulin

Adenylate cyclase

Glucose-6-phosphate dehydrogenase Glucose isomerase Phosphoglucomutase 6-Phosphoglucose dehydrogenase Hexokinase Serine dehydratase Tubulin Histones

Lactate dehydrogenase Lactate dehydrogenase Lactate dehydrogenase (G3) Lactate dehydrogenase Thymidylate synthetase Ornithine decarboxylase Tubulin

Enzymes and proteins

SUMMARY OF MAMMALIAN CELL CYCLE ENZYMES AND PROTEINS

Vendrely et al (1970)

Russell and Stambrook (1975) Russell and Stambrook (1975) Klevecz (1972) Klevecz (1972) Forrest and Klevecz (1972)

Makman and Klein (1972)

Blomquist et al. (1971) Blomquist ef A/. (1971) Blomquist et al (1971) Blomquist et al (1971) Blomquist et al (1971) Kapp et al (1973 NolsLndetal (1974) Gurley and Hardin (1968)

Klevecz and Ruddle (1968) Klevecz (1969b) Klevecz (1969b) Conrad (1971) Conrad (1971) Friedman et al. (1972) Forrest and Klevecz (1972)

Reference

3

a r o

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12 14 15 15 Human HeLa cells Thymidine kinase 1 Thymidine kinase 2 Thymidylate kinase 1 DNA polymerase 2 Deoxycytodine monophosphate deaminase 1 Alkaline deoxyribonuclease 1 Acid phosphatase 1 Alkaline phosphatase 1 Alkaline phosphatase 1 Alkaline phosphatase 3 Alkaline phosphatase 4 Ornithine transaminase Polyadenosine diphosphoribose polymerase 2 Polyadenosine diphosphoribose polymerase 3 Tubulin 1 Histones 1 Histones 1 and 3 Histones 2 or 11 Nonhistone nuclear proteins 3 Human KB cells Lactate dehydrogenase 3 Fumarase 3 Human WI-38 Lactate dehydrogenase 1

Glucose-6-phosphate dehydrogenase PolyADP-ribose polymerase Adenylate cyclase Phosphodiesterase

Klevecz and Kapp (1973)

Bello(1969) Bello (1969)

Brent et al (1965) Stubblefield and Mueller (1965) Brent et al (1965) Friedman and Mueller (1968) Gelbardefß/. (1969) Churchill and Studzinski (1970) Churchill and Studizinski (1970) Churchill and Studzinski (1970) Griffin and Ber (1969) Melnykovich et al (1967) Regan (1966) Volpe (1969) Roberts et al (1973) Smulson er 0/. (1971) Robbins and Shelanski (1969) Robbins and Borun (1967) Borun et al (1967) Spalding et al (1966) Bhorjee and Pederson (1972)

Miwaefß/. (1973) Raska (1973) Raska (1973)

{Continued)

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TABLE I.—Continued

Human Henle cells 3 3 3 5 6 11 of 9 11 Human fetal PR100 1

Method of synchrony 0

Davies et al (1968)

Melnykovych et al (1967) Takahashi et al (1969) Takahashi et al (1969) Lerner and Hodge (1970) Buell and Fahey (1969) Ums et al (1974) Millisetf al (1974)

Reference

Key to method: (1) Mitotic selection; (2) amethopterin; (3) double thymidine block; (4) 5-aminouracil; (5) density separation; (6) thymidine-Colcemid; (7) FUdR; (8) hydroxyurea; (9) Colcemid; (10) isoleucine deficiency; (11) single thymidine block; (12) histochemical determination; (13) aminopterin; (14) 5-benzyl-3-(l'anilinoethylidene) pyrrolidine-2,4-dione; (15) G, arrest in 0.5% serum.

a

Collagen

Alkaline phosphatase IgG IgM IgG IgG Adenyl cyclase Phosphodiesterase

Enzymes and proteins

SUMMARY OF MAMMALIAN CELL CYCLE ENZYMES AND PROTEINS

o S3

a.

O

P.

<

HS

σ4

00

ON

5. Metabolie Regulation in the Cell Cycle

169

the enzymes analyzed in the mammalian cell cycle according to cell type and method of synchrony through August, 1975. 1. PYRIMIDINE BIOSYNTHESIS AND METABOLISM

Thymidine kinase (TK) has been among the most studied of enzymes related to DNA synthesis. The effective use of thymidine in determining the time of DNA synthesis and its use in synchronizing large populations of mammalian cells by inhibiting DNA synthesis must have stimulated a number of studies on this enzyme. The increase in TK activity, prior to DNA synthesis, originally led to speculation that TK might be the key enzyme or "trigger" for the initiation of DNA synthesis. However, analysis of many different mammalian systems using a variety of different synchronizing methods has shown that the major peak of TK synthesis occurs in late S or G2 after DNA replication, and that cells are able to replicate DNA normally in the absence of this scavenger pathway. In mouse L cells, a single late S/G2 phase peak was observed in TK specific activity by Littlefield (1966), by Mittermayer et al (1968), and by Adams (1969). Brent et al (1965) and Stubblefield and Mueller (1965) using HeLa, and Stubblefield and Murphree (1967) using Don C, also found a late S/G2 peak. Klevecz (1969a) found two peaks in a diploidlike Chinese hamster Don C line, one during early S, and a second in late S/G2, while in a heteroploid G3 hamster line, only a broad single peak in G2 was evident. Klevecz has ascribed these differences to the degree of heteroploidy or diploidy of a cell line. Deoxycytidine monophosphate deaminase is activated by dCTP and inhibited by dTTP (Gelbard et al, 1969). Gelbard et al (1969) found that this enzyme was synthesized through S phase and reached a maximum in late S/G2 in HeLa cells. A similar pattern was observed in mouse L cells (Mittermayer et al, 1968) and again, maximum activity occurred after maximum DNA synthesis. Ribonucleotide reductase has been assayed in synchronous Don C cells (Murphree et al, 1969) and in L cells (Turner et al, 1968). In L cells specific activity was measured and was maximum during S phase. In Don C cells ribonucleotide reductase activity was at a minimum in Gi; it then increased through S phase and reached its highest level in late S (Murphree et al, 1969). The increase in Don C cells appeared to be continuous but sampling was too infrequent to confirm or deny a continuous pattern. Thymidylate synthetase, the enzyme that converts dUMP to dTMP, has been analyzed in synchronous Don C cells by Conrad (1971), who found two activity maxima, one in late Gx/S, and a second in late S/G2. Early and late enzymes of pyrimidine biosynthesis have been assayed by Mitchell and Hoogenraad (1975) in synchronized rat hepatoma (HTC) cells. Carbamylphosphate synthetase, aspartate transcarbamylase, and orotate phosphoribosyltransferase activities were analyzed in cells synchronized by mitotic

170

Robert R. Klevecz and Gerald L. Forrest

selection following treatment with Colcemid for 8-10 hours. The first two enzymes were found to increase rapidly through early Gx with a peak in late G x . The activity increased again to give a major peak in S phase and then fell rapidly to give little detectable activity in G2 and M. The last enzyme, orotate phosphoribosyltransferase, rose slowly during Gi, then increased rapidly during G^S to give a peak at mid to late S. No activity was found in G2 or M. There also appeared to be three minor activity peaks in Gi at early, mid, and late Gi/S superimposed on the overall pattern. The comparison of early and late enzymes may be especially significant since carbamylphosphate synthetase and aspartate transcarbamylase are the initial enzymes in pyrimidine synthesis and have been found together in an enzyme complex, while orotate phosphoribosyItransferase has been found associated in a separate complex with the terminal enzymes of pyrimidine synthesis. 2. DNA SYNTHETIC ENZYMES

DNA polymerase was assayed in L cells (Littlefield et αί, 1963; Gold and Helleiner, 1964) and in He La cells (Friedman and Mueller, 1968). In L cells, Littlefield et al showed that two forms of polymerase were present, one in a particulate form and one in a soluble form. There was a drop in DNA polymer­ ase specific activity of the soluble form and an increase in specific activity in the particulate form prior to and for 3 hours after thymidine release. The authors suggested that the soluble polymerase became particulate at the time of DNA synthesis. Gold and Helleiner (1964) also found a decrease in the specific activity of the soluble DNA polymerase when DNA synthesis began. The specific activity of DNA polymerase was lowest when DNA content per cell was maximal. The same results were also observed when DNA polymerase activity was expressed per cell. In He La cells, Friedman and Mueller (1968) assayed a nuclear and a cytoplasmic DNA polymerase. The nuclear polymerase was corre­ lated with DNA synthesis while the cytoplasmic polymerase was not. The nuclear polymerase needed a cytoplasmic factor, associated with cytoplasmic polymerase, for activation. Only the nuclear polymerase was correlated with nuclear DNA synthesis, the cytoplasmic or soluble polymerase appeared to have a cell cycle pattern similar to dCMP deaminase and ribonucleotide reductase. Some evidence for an RNA-directed DNA polymerase has been reported in mouse lymphoma L5178Y cells (Bosmann, 197Id). The specific activity in­ creased continuously through the cell cycle in contrast to several other enzymes which are synthesied as peak enzymes in this cell line. 3. POLYAMINES

Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase are the only two enzymes in polyamine biosynthesis that have been analyzed in the

5. Metabolie Regulation in the Cell Cycle

171

mammalian cell cycle. Russell and Stambrook (1975) found that ornithine decarboxylase and S-adenosylmethionine decarboxylase were both S phase en­ zymes in synchronized V79 Chinese hamster cells ODC activity increased tenfold from Gx to S phase and showed an exceptionally sharp maximum. S-Adenosylmethionine decarboxylase began to increase 2 hours later in the cycle than ODC and gave a broader activity curve. Friedman et al (1972) measured ODC activity in Don C Chinese hamster cells and found three peaks of activity corresponding to G1 /S, late S, and G2 or mitosis. These later results are similar to our data for ODC activity in Chinese hamster ovary (CHO) cells (G. L. Forrest and R. R. Klevecz, unpublished) in which three peaks of ODC activity occurred at G! /S, late S and G 2 . 4. CHROMOSOMAL PROTEINS

This topic, one of the more exciting areas of inquiry, cannot be adequately reviewed in so little space. The reader is referred to Elgin and Weintraub (1975) for a more thorough discussion. Poly [adenosine diphosphate ribose (ADPR)] polymerase is a chromatinbound enzyme that has been assayed in several synchronous cell lines (Colyer et al, 1973; Roberts et al, 1973; Smulson et al, 1971; Miwa et al, 1973). The enzyme catalyzes the transfer of the ADPR groups of NAD to nuclear proteins to form a homopolymer of ADPR residues attached to nuclear proteins (Nishizuka et al, 1967). In transformed hamster cells, the specific activity of poly ADPR polymerase was highest in G2 and lowest in S phase (Miwaeftf/., 1973), while in HeLa cells maximal specific activity occurred during Gl and the lowest activity occurred during S (Smulson et al, 1971). Stimulation of DNA template activity for DNA polymerase following modification by poly ADPR polymerase was greatest with G2 nuclear fractions in HeLa cells. Mouse L cells gave a pattern of poly ADPR polymerase activity with a maxima occurring at two points in S phase (Colyer et al, 1973). In the later report, activity was expressed per μg of DNA whereas the activities in hamster and HeLa cells were expressed as activity per μg of protein. In HeLa cells, Robbins and Borun (1967) showed that histone synthesis occurred only during S phase. Inhibition of DNA synthesis with cytosine arabinoside resulted in a decrease in histone synthesis and in the destruction of histone-synthesizing polysomes. In other HeLa cell systems, histone synthesis was correlated with DNA synthesis but inhibition of DNA synthesis with 5-fluorodeoxyuridine, amethopterin, or thymidine did not block histone syn­ thesis (Spalding et al, 1966; Chalkley and Maurer, 1965). Gurley and Hardin (1968) have analyzed all five histone fractions in synchronized CHO cells. Synthesis occurred at all times during the cell cycle for all five histones, but there was an increase in the rate of synthesis during S phase. Cells blocked at the Gx /S boundary by thymidine continued to synthesize all five histone fractions.

172

Robert R. Klevecz and Gerald L. Forrest

Nonhistone chromosomal proteins were isolated from synchronized HeLa cells and analyzed on polyacrylamide gels (Bhorjee and Pederson, 1972). Twenty-two distinct bands were observed ranging in molecular weight from 15,000 to 180,000 daltons. Only a few proteins were found to fluctuate through the cycle. Band 15 was reduced 50% in mid S and G 2 . Reductions in bands 4 and 17 occurred during G2 and band 11 decreased in early S but reappeared in midS. 5. CARBOHYDRATE METABOLISM

Hexokinase, the first enzyme in carbohydrate metabolism, gives two peaks, one at the Gi/S boundary and a second in late S phase in Chinese hamster V79 cells (Klevecz, 1972). In synchronous CHO cells, hexokinase activity gave three maxima; in late G x , mid S, and in late S/G2 (G. L. Forrest and R. R. Klevecz, unpublished). A more linear increase in hexokinase through the cell cycle was described by Blomquist et al. (1971) in CHO cells. We have sampled at 30-minute intervals in order to show the reliability of the oscillatory pattern. More frequent sampling than has heretofore been performed will probably be needed in cell cycle analysis in order to detect rapid fluctuations in the activity of enzymes and other proteins. Glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway, has been studied in a number of cell lines. In Chinese hamster Don C cells Klevecz and Ruddle (1968) have shown large fluctuations in activity through the cell cycle. Peaks occurred at the end of G x , in mid S, and again in late S. Similarly in CHO cells, three maxima were observed; in Gi, early S, and late S (G. L. Forrest and R. R. Klevecz, unpublished). Again, these results are in contrast to those of Blomquist et al. (1971) where a linear increase was described. Vendrely et al (1970) have assayed G6PD in three different hamster lines using histochemical techniques and have derived results showing marked fluctuations in activity with maxima in S and G 2 . Warmsley et al. (1970) reported a linear increase in the specific activity of G6PD through the cell cycle in P815Y mouse cells. Martin et al. (1969a) showed that G6PD specific activity decreased slightly through the cell cycle in rat HTC cells. The second enzyme in the pentose pathway, 6 phosphogluconate dehy­ drogenase (6PGD), also fluctuates through the cell cycle in Chinese hamster V79 cells (R. R. Klevecz, unpublished) where peaks were observed at the Gi/S boundary and in late S. In CHO cells, Blomquist et al. (1971) reported a more continuous increase for 6PGD. More recently we have assayed 6PGD in CHO cells and have found three maxima in Gx, early S, and late S (G. L. Forrest and R. R. Klevecz, unpublished). Blomquist et al. (1971) also described a continuous increase for glucose phosphate isomerase and phosphoglucomutase, but in examining their data it appears that in order to draw a straight line through the

5. Metabolie Regulation in the Cell Cycle

173

data, the standard deviation for some time points would have to be in excess of 50% of the mean. Lactate dehydrogenase (LDH) has been studied in many synchronous cell lines with differing results. Oscillatory changes in activity have been observed in Don C cells (Klevecz and Ruddle, 1968; Klevecz, 1969a,b), in V79 cells (Klevecz, 1969a, 1972), in CHO cells (Kapp et al, 1973), and in WI-38 cells (Klevecz and Kapp, 1973). In Don C and WI-38 three maxima were observed in activity. It was also shown in Don C that the periodicity in LDH activity represented changes in the amount of LDH protein as assayed by specific antibody (Klevecz, 1969b). Vendrely et al (1970) have reported fluctuations in LDH activity in EHB, BHK, and normal hamster cells using histochemical methods. Bello (1969) has described a continuous increase in LDH activities and fumarase in human KB cells. However, sampling was infrequent, occurring at 4-hour intervals. Since LDH activity can fluctuate rapidly with a period of 3-4 hours (Klevecz, 1969a) it seems quite possible that oscillations, even if they occurred, would have been missed. Continuous increases in the specific activity of LDH have been described in mouse P815Y cells (Warmsley et al, 1970) and possibly in rat HTC cells (Martin et al, 1969a). 6. MITOCHONDRIAL ENZYMES

Bosmann (1971b) compared the specific activities of two mitochondrial enzymes in synchronous L5178Y cells. Monoamine oxidase, an enzyme of the outer mitochondrial membrane, was elevated in S phase and was expressed in two peaks, one in mid S and one in late S. Succinic dehydrogenase, an enzyme of the inner mitochondrial membrane, gave a single maximum in early S phase. In contrast to these results Warmsley et al (1970) assayed the specific activity of cytochrome c oxidase and succinic-cytochrome c reductase (two inner mito­ chondrial enzymes) in P815Y mouse neoplastic mast cells and observed a gradual increase in specific activity as DNA content increased. Warmsley et al (1970) suggested that differences in patterns of activity might be due to the method of synchrony, since they had previously observed periodic synthesis in using inhibi­ tors of DNA synthesis (Bergeron et al, 1969, 1970). While the gradient separa­ tion methods used in the later work of this group might be a less perturbing method of synchronization, the possible collection of artifacts associated with this method (see Section II,B,3) should be borne in mind. Glutamic dehydrogenase, a soluble enzyme of the mitochondrial matrix showed fluctuations in P815Y cells (Warmsley et al, 1970). Specific activity decreased through G x , then increased during DNA synthesis. Vendrely et al (1970) using histochemical and autoradiographic techniques, measured malate dehydrogenase in three different hamster lines; BHK, EHB, and normal embryonic hamster cells. Randomly growing cells were assayed for

174

Robert R. Klevecz and Gerald L. Forrest

enzyme activity and for position in the cell cycle. Their results suggested fluctuations in malate dehydrogenase activity with a maximum occurring in S phase. 7. AMINO ACID METABOLISM

Ornithine transaminase is a regulated enzyme with a role in proline biosyn­ thesis and in the interconversion of proline, glutamate, and arginine. Ornithine transaminase was analyzed in synchronous HeLa cells where the specific activity showed a single maximum apparently in G2 or M (Volpe and Strecker, 1968; Volpe, 1969). Serine dehydratase activity has been analyzed in synchronous CHO cells (Kapp et al, 1973). It appeared to increase starting in late Gx and reached a plateau in early S, one which was maintained through S phase. Glutamic dehydrogenase was studied in mouse P815Y cells (Warmsley etal., 1970). The specific activity of this enzyme decreased through G2 then gradually increased through G 2 . 8. MEMBRANE AND ASSOCIATED ENZYMES

Several enzymes that are found tightly bound to cell membranes have been analyzed through the cell cycle in L5178Y mouse cells (Bosmann, 1970a). Enzymes that were assayed from the smooth endoplasmic reticulum were UDPase, glycoprotein: galactosyltransferase, glycoprotein:fucosyltransferase, PSM: fucosyltransferase, polypeptide:7V-acetylgalactosaminyltransferase, and fetuin:7V-acetylglucosaminyltransferase. UDPase was described as an exclusively S phase enzyme with essentially no detectable activity in Qx or G2/M. The glycoprotein transferases were all S phase enzymes, but variations in specific activity patterns were evident (Bosmann, 1971a). The glycoproteinigalactosyland fetuin:fucosyltransferase enzymes displayed late S maxima. PSM: fucosyl­ transferase peaked 1 hour before fetuin fucosyltransferase. Fetuin: acetylglucosaminyltransferase gave maximum activity in mid S, 2 hours earlier than fetuin: fucosyltransferase. Polypeptide:7V-acetylgalactosaminyltransferase displayed the most complex pattern, showing possibly three bursts of activity. These occurred in late Gj /S, mid S, and late S. Nonspecific esterase activity was found associated with the rough endo­ plasmic reticulum and was largely an S phase enzyme (Bosmann, 1970a). Its activity increased rapidly in early S, peaked in mid S, and then decreased rapidly. Several plasma membrane enzymes that have been studied through the cell cycle include 5'-nucleotidase (Bosmann, 1970a), collagen: galactosyl- and col­ lagen: glucosyltransferase (Bosmann, 1970b), and alkaline phosphatase (Churchill

5. Metabolie Regulation in the Cell Cycle

175

and Studzinski, 1970; Melnykovych et aly 1967; Griffin and Ber, 1969; Regan, 1966). The maximum specific activity of the transferase enzymes occurred 1-2 hours prior to the peak in 5'-nucleotidase activity. In addition, 5'-nucleotidase displayed a late S shoulder of activity which was absent from the transferase enzymes. The specific activity of alkaline phosphatase has been studied in synchronous HeLa cells (Churchill and Studzinski, 1970; Griffin and Ber, 1969; Regan, 1966) and in human Henle cells (Melnykovych et al, 1967). In human Henle cells specific activity displayed a minimum of G t /S boundary. Regan (1966) did not find any significant change in the specific activity of alkaline phosphatase in the HeLa cell cycle. Griffin and Ber (1969) also found little change in cell cycle specific activity using HeLa cells. Churchill and Studzinski gave a profile for cell cycle activity that was similar to that observed for other plasma membrane proteins in L5178Y cells. Monoamine oxidase, which is found on the outer mitochondrial membrane, shows a single S phase peak in synchronized L5178Y cells (Bosmann, 1970a). Monoamine oxidase activity increased rapidly to a maximum in early S, then decreased rapidly through the remainder of S phase to reach a minimum level in G 2 . Succinic dehydrogenase is present in the inner mitochondrial membrane. Its specific activity increased in late Gx /S to give a peak in mid S and late S, then decreased into mitosis (Bosmann, 1971b). 9. LYSOSOMAL ENZYMES

Bosmann and Bernacki (1970) have compared the specific activities of several lysosomal enzymes in synchronous L5178Y cells. j3-7V-Acetylglucosaminidase, ß-Af-acetylgalacotosaminidase, and acid phosphatase gave a linear increase through the cell cycle. /3-A^-Acetylgalactosaminidase did not begin to increase until S phase, while the other two enzymes began increasing immediately after removal of the Colcemid block. Although there was a general linear increase, small reproducible oscillations were evident. Cathepsin D activity gave a much different pattern of activity in L5178Y than that of galactosidase activity (Bosmann, 1971a). Peak activities were observed in the S and M phases of the cell cycle. We have also looked at cathepsin D-like activity in Chinese hamster V79 cells and found maxima during late Gx/S and in late S phase. One clone of V79 also had in addition high activity in mitosis (G. L. Forrest and R. R. Klevecz, unpublished). Kapp and Okada (1972a), using L5178Y cells, found that acid phosphatase activity oscillated through the cycle with peaks in early S and late S while Churchill and Studzinski (1970) showed that the specific activity of acid phosphatase in HeLa cells was maximal in early G x , decreased during late Gx, and then increased again through S and to a maximum in G2 phase. In Chinese

176

Robert R. Klevecz and Gerald L. Forrest

hamster cells (V79 and CHO) acid phosphatase oscillated through the cell cycle with the main peak of synthesis in S phase (G. L. Forrest and R. R. Klevecz, unpublished data). jß-Glucuronidase appears to be an S phase enzyme in CHO and V79 cells (G. L. Forrest and R. R. Klevecz, unpublished). Activity decreased during G!, it then increased to a maximum in mid S, and decreased through G2 with a pattern similar to acid phosphatase activity. 10. PROTEOLYTIC ACTIVITY

Proteolytic activity has been assayed in L5178Y mouse lymphoma cells (Bosmann, 1971c) and in V79 Chinese hamster cells (G. L. Forrest and R. R. Klevecz, unpublished). In L5178Y cells, general proteolytic, cathepsin, and trypsin activities showed similar patterns with maxima in S phase and in mitosis. In V79 cells, three peaks of trypsinlike activity were present, occurring in mitosis, late Gx/early S, and in mid S. Cathepsinlike activity maximized in mitosis, in early S, and in late S giving a different pattern from the trypsinlike activity. Bosmann (1971c) has speculated that proteolytic degradation is most needed in S phase where proteins and glycoproteins are synthesized at the highest rate (Bosmann and Winston, 1970) and that degradation would account for the rapid turnover of proteins and glycoproteins found in the cell. He has also suggested that high activity in mitosis may be necessary for the remodeling of membrane proteins during division. The proteolytic activity in S phase in V79 cells also occurs at a time where protein degradation appears to be at a maximum (Forrest and Klevecz, 1972), and when many enzyme activities are decreasing (Klevecz et al, 1975). In view of the known effects of proteolytic enzymes on the stimula­ tion of protein synthesis and cell division (Lundblad, 1949; Mano, 1966; Monroy et al, 1965; Rubin, 1970; Burger, 1970; Mallucci et al, 1972), it seems possible that the first peak (Gx/S) of proteolytic activity could activate the new translational processes in the cell and might represent an endogenous mechanism for overcoming prereplication arrest. 11. COLLAGEN, TUBULIN, AND THE IMMUNOGLOBULINS

Collagen is synthesized in many fibroblastic cell lines and can account for 10% of the total protein (Davies et al, 1968). Collagen levels were determined in the cell cycle of mouse 3T6 cells and rat aorta PR105 cells (Davies et αί, 1968). Synthesis increased in S phase and gave a peak in early S in 3T6 cells, while in rat aorta collagen increased during S and gave a plateau in late S phase. Immunoglobulin synthesis has been assayed in synchronous human lymphoid and mouse myeloma cell lines (Takahashi et αί, 1969; Lerner and Hodge, 1970;

5. Metabolic Regulation in the Cell Cycle

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Byars and Kidson, 1970; Buell and Fahey, 1969; Yagi, 1970). Maximum syn­ thesis occurred in early S phase in all of the cell lines studied and Yagi (1970) and Takahashi et al. (1969) found two maxima in immunoglobulin synthesis, one during S and a second during G 2 . However, some differences were noted in the time of immunoglobulin secretion. Byars and Kidson (1970) found the maximum secretion occurred during G2 and mitosis in mouse myeloma cells while Buell and Fahey (1969) reported that secretion was greatest during S phase in human lymphoid cells. Tubulin was assayed by two different methods in synchronized Chinese hamster cells (Forrest and Klevecz, 1972). Extrapolation of three-point, colchicine-binding assays to initial binding activity or coelectrophoresis of a tritiumamino acid-labeled extract with purified, iodinated tubulin gave similar patterns. In Don C cells, maxima were observed in mid S and G 2 , while in V79 cells maximum tubulin levels were found in G 2 . Colchicine-binding activity using single-point binding assays showed relatively more binding activity in the early portion of the cycle. Binding activity by this assay decreased as the cells entered S and then increased again to give a S and G2 pattern similar to that observed with other assay methods (Klevecz and Forrest, 1975). Noland et al. (1974) also found tubulin levels changed periodically in CHO cells and reported maximum binding in S and again in late G2 similar to that reported by Forrest and Klevecz (1972). Robbins and Shelanski (1969) found what they described as a con­ tinuous increase in mitotically synchronized HeLa cells, but only five samples were taken from HeLa cells with a 20-hour generation time. The pattern of binding activity was similar to that observed by Robbins and his co-workers for total RNA synthesis and protein synthesis (Scharffand Robbins, 1965). 12. CYCLIC NUCLEOTIDES

Cyclic AMP effects on cell proliferation have been studied in some detail and the levels of cAMP, adenylate cyclase, and cAMP phosphodiesterase have been assayed as a function of cell cycle position. Adenylate cyclase activity was found to be high in Gx and then decrease dramatically in S by Raska (1973). Adenylate cyclase activity has also been observed to fall in S phase following reversal of a thymidine blockade and then increase in late G2 and M (Makman and Klein, 1972). Millis et al. (1974) found that adenylate cyclase, phosphodi­ esterase activities, and cAMP levels were correlated in a rational manner. Both adenylate cyclase and phosphodiesterase activities were high early in Gx in Colcemid-synchronized cells and high in G2 and low in M in thymidinesynchronized cells, while phosphodiesterase activity increased in G2 and was maximal in M. Burger et al. (1972), in a population of 3T3 cells that were parasynchronous at best, found that cAMP levels were constant and high through most of the cycle, dropping only in M.

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In contrast, Zielig et al. (1972) measured cAMP levels in thymidine-synchronized He La cells and found that upon release of the block, cAMP levels decreased to a plateau in S phase followed by a second decrease to a minimum in G 2 . Following mitosis there was an increase in Gx and another decrease prior to the beginning of S phase. Adding a further complication is the fact that adenylate cyclase is higher in transformed than nontransformed lines (Sheppard and Bannai, 1974) even though the level of cAMP is lower (Burk, 1968; Otten et al, 1972; Sheppard and Bannai, 1974). In most instances treatment of cells with cAMP or with hormones or analogs which elevate intracellular cAMP has resulted in arrest in either Gx or G2 (Pastan et al, 1975). For a more thorough discussion of this topic the reader is referred to Pastan et al. (1975) and Makman et al (1974), and Chapter 8 of this volume.

IV. Changing Perspectives of the Cell Cycle A. The Mitotic Cycle From the earliest days of cytology onward to the middle of this century, the control of cell growth and division was commonly held to be synonymous with the mechanism of mitosis. Even E.B. Wilson (1896), who viewed so many problems clearly, never phrased the question of cell growth as a cyclic process. This division-oriented view of the cycle was understandable; the light microscope was the instrument of investigation and interphase from a microscopist's point of view is dull when compared to the organization and equipartition of the chromosomes at mitosis. Even when efforts were put into investigating inter­ phase events (Bullough, 1952; Swann, 1957) it was often with the idea in mind of discovering the source of energy for mitosis. At the end of this period a number of laboratories had begun investigating events in the cell cycle of mammalian and nonmammalian systems (Glinos and Gey, 1952; Zeuthen, 1953; Mitchison, 1957; Scherbaum, 1957) so that Mazia (1960) was able to summarize by saying that there were a number of parallel pathways occurring in interphase, each necessary but none sufficient, leading to cell division. B. The Chromosome Replication Cycle and Its Diagram Howard and Pelc (1953), who presented a relatively simple picture of the cell cycle and the now familiar Gi, S, G2 terminology, are usually credited with defining the substages within interphase, but several years earlier Swift (1950)

5. Metabolie Regulation in the Cell Cycle

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had already shown that the doubling in DNA content took place as an event in interphase and not at mitosis. Nevertheless, the contributions of Howard and Pelc stand because new techniques, isotopic labeling and autoradiography, were applied to the previously refractory interphase. At this juncture the use of the classic cell cycle diagram and the Gi, S, G2 terminology results in a picture of the cell cycle which is too rigidly determinis­ tic to serve as a useful model for understanding cell proliferation. It can only be accurate in the limit where all of the cells which undergo mitosis begin DNA synthesis after an interval equal to Gi. This situation is approximated by some established cell lines which display little density dependence, but uncertainty develops when a significant fraction of the cells that complete mitosis either fail to enter S or enter with a variance which is greater than 20% of the mean. The problem becomes one of describing the state of the noncycling fraction. Are the cells which enter S later than the minimum predicted time moving through Gx at a slower pace, are they transiently arrested at some point or points in Gi, or are they in an alternate pathway or cycle? Lajtha (1963) attempted to reconcile the diagram with reality by proposing a separate state, G 0 , to account for the fact that cells which could and would ultimately divide were spending more than a Gx period of time in the prereplica­ tive phase. The distinction between possible states for these cells is more than semantic as can be illustrated with a hypothetical example. Cells are said to be arrested at the Gx /S boundary by excess thymidine but generally protein and RNA synthesis continue in the presence of thymidine so that Gj /S arrest refers only to the chromosome replication cycle. Subsequent release of the block and the measurement and description of some constituent such as an immunoglobulin as being synthesized in mid-S phase, is only sensible if it can be shown that immunoglobulin synthesis is dependent on DNA replication and similarly arrested by high thymidine. If it occurs independently then synthesis may have occurred during the thymidine arrest. If after release it is synthesized again when half the S phase is completed this may only be coincidental and dependent on the elapsed time between the block and release. In consequence there may be considerable benefit in devising a system for describing the dependent and independent sequences of events in the prereplicative phase (and the remainder of the cycle) so that they may be uniquely defined in time. C. Serum Factors and the Cell Surface The question of greatest current interest is the nature and in some cases the existence of a quiescent state distinct from Gi arrest. Since the demonstration by Todaro et al (1965) that cell lines maintained in a resting state can be stimulated to divide by the addition of serum, a number of laboratories have

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developed this system as a means of examining the transition from a quiescent to a proliferative state (Wiebel and Baserga, 1969; Temin et aL, 1974; Pardee, 1974). Later Ley and Tobey (1970) exploited nutritional deprivation as a means of arresting cells in this same stage. In some instances prereplication arrest has been considered to occur at a single point, R (Pardee, 1974), and an attempt has been made to map the arrest points for known effectors such as cAMP, low serum, and low isoleucine, relative to the beginning of S phase as defined by hydroxyurea arrest. In this one instance all these effectors appeared to act at the same point, but other workers (Liskay, 1974; Burstin et αί, 1974) have attempted to map their ts mutants relative to these same points and have found that the mutation maps between isoleucine and serum deprivation (Burstin et aL, 1974). In WI-38, the time of entry of cells into S following stimulation has been shown to display considerable heterogeneity by Augenlicht and Baserga (1974). In part the problem may be due to the inherent heterogeneity in the individual arrest points so that it may be erroneous to regard cells deprived of isoleucine or grown in low serum as being at a point in the cycle. Indeed the original reports of arrest points (Ley and Tobey, 1970; Pardee, 1974) revealed considerable variance. The nature of the arrest and its identity or not with a point in the cycle of cycling cells is a problem of immediate interest and can be approached in other ways. Temin et al (1974) viewed the heterogeneity of the prereplication period as being generated in the G ^ or IGi state which is approximately equal to G 0 , and suggested that virally transformed and tumor cells, unlike their normal counter­ parts, are unable to enter this retreat state and consequently die when deprived of essential nutrients. Temin distinguishes between normal and RSV-transformed chick flbroblasts on the basis of their ability to recognize a nonpermissive environment. RSV cells according to him continue cell cycle traverse and often die. He believes that selective pressures may have been adequate to generate cell lines with the capability of producing their own multiplication stimulation factors. Since many of the cells used were derived from cancer tissue, there is a likelihood that such selection may have been begun in vivo. Pardee's (1974) restriction point is equivalent to Temin's IGi disjunction, and it is viewed as a switching point which enables normal cells to shift to minimal metabolism under suboptimal growth conditions. In concurrence with Temin he suggests that malignant cells have lost their restriction point control. Baserga and his co-workers (1973) build a case for considering arresting normal cells as being in a state different from Gx arrest. They consider that cells that do not require serum for stimulation and that do not respond to serum with an increase in chromatin template activity as being in Gx and not in G 0 . It appears that those cells which are capable of entering G0 first arrest in Gi and then enter into progressively deeper G0 states. In support of a distinct G0 state

5. Metabolie Regulation in the Cell Cycle

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is the work of Becker and Stanners (1972) and Becker et al. (1971). Becker and Stanners used mitotically synchronized, asynchronous, and arrested cells and compared the electrophoretic pattern for proteins between 30,000-150,000 MW. Using analysis of variance they showed greater differences between expo­ nential and stationary phase cultures than between cultures in various stages of the cell cycle, with the greatest difference occurring between nuclear prepara­ tions of exponential and stationary cultures. Further evidence for a resting state distinct from Gi has been provided by Green and his co-workers, who used 3T3 and 3T6 cells arrested in 0.5% serum, and found that 12 hours are required following stimulation for DNA synthesis to begin. Upon stimulation, rRNA and rRNA precursors are synthesized (Mauck and Green, 1970) the amount of poly A-containing-mRNA from hnRNA in­ creases (Johnson et al.y 1975) and the message content of cytoplasm increases (Johnson et al., 1974). Unexpectedly, the turnover of 28 S and 18 S RNA was found to be more rapid in resting than in growing cells, while messenger RNA was equally stable in either case (Abelson et al, 1974). After withdrawal of brief serum stimulation 12-18 hours are required for total RNA to reach the level of that in unstimulated cells (Mostafapour and Green, 1975). D. The Cell as a Clock In examining the synchrony literature it is apparent that certain cell cycle times occur with much greater frequency than others. Many cells have cycle times of 11-12, 15-16, 19-20, and 24 hours. A systematic compilation of cell cycle times from published synchrony experiments has been collected and arranged graphically in Fig. 1. With a few exceptions only mitotic selection synchronies showing the subsequent mitotic wave were included. The distribu­ tion of generation times forms a series with a repeating interval best estimated as between 3.5 and 4 hours, with many cell lines capable of expressing more than one generation time. V79, for example, divide with cell cycle times of 8.5, 11.5, and 16 hours, He La with cell cycle times centering around 15.5, 19.5, 22.5, and 24-25 hours, and L cells with cell cycles of 15.5-16, 19, 22.5, and 26 hours. Even though a variety of cell lines growing under variable conditions were considered, the intervals between preferred generation times appeared to be similar. It seemed therefore that there might exist preferred generation times in mammalian cells which were quantized in multiples of 3-4 hours. We found that the distribution of cell cycle times from synchronous or random exponential cultures of V79 could be influenced by cell density and temperature. In clones growing under low density conditions the variance in generation time of individ­ ual cells about the mean was small in the first three generations following

182

Robert R. Klevecz and Gerald L. Forrest

Tg =nGq+ 2Gq

6 \18 x 20/

29 31 29 \30 \ 3 2 ,

10 11

\\\l//

1 23

\WI//

if G q =3.75hr

n=5H n=3—I -n=4—1

-n=2—I

10

15

20

-n = 6—I

25

30

Generation Time (hr) Fig. 1. Quantized variation in generation times of mammalian cell lines. Generation times were determined from the published data on cells synchronized by mitotic selection or from time lapse cinematography of random cultures. The list is not exhaustive but represents a sampling of papers published between 1961 and the present in which the stated generation time could be directly confirmed in the data. Wherever possible modal generation times were obtained; that is, the time of maximum mitotic index rather than the center of mass of the mitotic wave. The calculation of possible generation times uses the simple expression T g = «G q + 2G q . To obtain a precise fit to polymodal distribution of generation times, values for the probability of emergence from G q and the density function describing dispersion will have to be determined. T g = generation time in hours. G q (quantized G) = incremental increase in generation time in hours. Numbers above each line in the figure are explained in the following tabulation: Number on figure

Cell

τ.

7.7

J. M. Brown, Rad. Res. 4 3 , 627 (1970).

2 3

Squamous cell carcinoma in vivoa V79-M6-39 0 V79

7.8 8.5

4

L5178Y

8.5

R. R. Klevecz, Proc. Natl. Acad. Sei. 73, (1976). R. R. Klevecz, B. A. Keniston and L. L. Deaven, Cell 5, 195 (1975). L. N. Kapp and S. Okada, Exp. Cell Res. 7 2 , 4 6 5 ( 1 9 7 2 ) .

1.

Reference

183

5. Metabolie Regulation in the Cell Cycle Number on figure

Cell

τ

5

L5178Y

8.75

6

V79-753B C

9

7

V79-28513

11

8 9

V79-M6-34* Don

11.25 11.5

10

BHK

11.5

11

B14-FAF

11.5

12

Rat sarcoma**

13

CHO

11.9 12.8 12

14

DonC

12

15

CHO

12.5

16

ELD Ehrlich as cites

15

17

HeLa

15.5

18

HeLa d

19 20

L-929 L-60T

15.5 19.5 15.5 16

21 22

V79-325 HeLa

16 19

23

HeLa-S3

19

24

Ham d

19.4

25

WI-38

19.5

26

L-949 d

19.5

27

L-132 human lung

19.5

Reference H. B. Bosmann and R. J. Bernacki, Exp. Cell Res. 6 1 , 3 7 9 ( 1 9 7 0 ) . M. M. Elkind and E. Kano, Rad. Res. 44,497(1970). J. Kruuv and W. K. Sinclair, Rad. Res. 3 6 , 4 5 (1968). Klevecz, Proc. Natl. Acad. Sei. 73, (1976). W. C. Dewey, S. C. Furman and H. H. Miller, tod Res. 4 3 , 561 (1970). C. Vendrely, The Cell Cycle and Cancer, In "The Biochemistry of Disease," (R. Baserga, ed.), vol. 1, pp. 227, Marcel Dekker, (1971). D. L. Steward and R. M. Humphrey, Nature (London) 212, 298 (1966). K. B. Dawson, H. Madoc-Jones and E. O. Field, Exp. Cell Res. 38, 75 (1965). R. A. Walters and R. A. Tobey, Biophys. J. 10,556(1970). D. L. Steward, J. R. Shaeffer and R. M. Humphrey, Science 161, 791 (1968). R. R. Klevecz, B. A. Keniston and L. L. Deaven, Cell 5, 195 (1975). C. B. Lozzio, Int. J. Rad. Biol. 14, 133(1968). B. Lesser and T. P. Brent, Exp. Cell Res. 6 2 , 4 7 0 ( 1 9 7 0 ) . G. Marin and M. A. Bender, Exp. Cell Res. 4 3 , 4 1 3 (1966). R. R. Klevecz, (unpublished) J. E. Till, G. F. Whitmore and S. Gulyas, Biochem. Biophys. Acta 72, 277 (1963). W. K. S i n c l a i r , / ^ . Res. 39, 135 (1969). J. R. Churchill and G. P. Studzinski, /. CellPhysiol 75, 297 (1970). M. D. Scharffand E. Robbins, Nature (London) 208, 464(1965). J. E. Sisken and L. Morasca,/. Cell Biol. 25, 179 (1965). R. R. Klevecz and L. N. Kapp, /. Cell Biol. 5 8 , 5 6 4 ( 1 9 7 3 ) . D. Killander and A. Zetterberg, Exp. Cell Res. 3 8 , 2 7 2 ( 1 9 6 5 ) . F. Kelley and M. Legaten, Mut. Res. 10,237(1970). (Continued)

184

Robert R. Klevecz and Gerald L. Forrest

Number on figure

Cell

28

HeLa-S3

20

29

22.5 24.5 21.0 21.3

31

KL<* Uamd Ham d Human Kidney HeLa-S3

32 33

L-929 HeLa-S3 e

22.5 23.8

34

HeLa

24

35

HeLa

24.5

36

HeLa

25

37

HeLa**

25

38

L-5

26 29

39

CMP

28.5

40

D983

30

30

Reference

τ,

21.8

M. J. Griffin and R. Ber, /. Cell Biol 40, 297 (1969). J. E. Sisken and R. Kinosita, Exp. Cell

Res. 22,521(1961). G. Galavazi and P. Bootsma, Exp. Cell Res. 41, 442 (1966). P. N. Rao and J. Engelberg,/« "Cell Synchrony: Studies in Biosynthetic Regulation" (Cameron and Padilla, eds.). Academic Press, New York (1966). A. B. Rickinson, Cell Tis. Kinet. 3, 355 (1970). W. G. Thilly, T. S. Nowak, G. N. Wogan, Bioengineering and Biotechnology 16, 149 (1974). T. P. Brent, J. A. V. Butler and A. R.

Cizüvom, Nature (London) 210, 393 (1966). C. S. Lange, Int. /. Rad. Biol. 17,61(1970). J. H. Kim and B. K. Stambuk, Exp. Cell Res. 44, 631(1966). T. C. Hsu, Texas Report. Biol. Med.

18,31(1960). T. Terasima, Y. Fumiwara, S. Tanaka, M. Yasukawa, Cancer cells in culture, Proc. Int. Conf. on Tis. Cult, and Cancer Res., (H. Katsuta, ed.). Tokyo University Press (1968). F. H. Kasten and F. F. Strasser, Nature (London) 211, 135 (1966). B. Djordjevic and O. Djordjevic, Afaft/re (London) 205, 165 (1965).

a

Labeling index in vivo. Video tape microscopy of synchonous cultures. c Slope of curve from random exponential cell cultures. ^Cinematography of random culture. ^Multiple brief high thymidine blocks. ö

selection, but as the culture reached higher densities the generation times of cells in the clone showed a variance which was not normally distributed. Rather, it appeared that modal generation times of 7.5, 11.2, and 15.5 predominated. Using synchronous cultures of mitotically selected cells and time lapse video tape microscopy it is possible to show that V79 cells have a 7.5-hour generation time at temperatures from 37° to 40°C while at temperatures from 36.5° down to 33.5°C the generation time is 11.5 hours.

5. Metabolie Regulation in the Cell Cycle

185

In order to satisfy these observations, I proposed a subcycle, G q , which has a traverse time equal to the period of the clock (Klevecz et al, 1975). The period appears to be fixed at close to the same value in all somatic cells. The timekeeping mechanism appears to be temperature compensated since the time required to traverse Gq is constant at temperatures between 34° and 39°C. Cell cycle time increases at lower temperatures because the number of required rounds through Gq increases. Differences in the length of Gi in cells of the same culture can be envisioned as being a consequence of the gated entry of cells into S. The exit from Gq into S is probabilistic in the sense that it depends on the environment with respect to cell density, nutrition, and the presence of mitosisstimulating factors. Should the environment be nonpermissive when the cell reaches i, the cell enters or reenters G q . Gi arrest (or G 0 ) can be viewed as a consequence of a more permanently unfavorable environment leading to an indefinite number of Gq cycles in which certain cell functions continue while those which are dependent upon the initiation of DNA synthesis cease. Whether this situation in which multiple rounds through Gq occur, and which may lead to a condition not unlike unbalanced growth, can explain the differences in composition between G0 or quiescent cells and normally cycling cells is prob­ lematical. Figure 1 bears some superficial resemblance to the cell cycle diagram drawn by Temin (1971). Gq differs from IGi in having a specified time interval and in its identification with the cellular clock. It enables one to describe the cycle and specify its duration in terms of multiples of the single constant G q . There may also be a similarity of the point i at the disjunction of Gq and S + G2 + M to Pardee's restriction point, R, the arrest point for isoleucine or serum deprivation and cAMP arrest. In their work, both Pardee (1974) and Temin (1971) found an arrest point and in both cases it appeared to occur some hours before the beginning of S phase. This model provides a means of explaining the hetero­ geneity of Gi as it is expressed by serum-starved or nutritionally depleted (Ley and Tobey, 1970) cells upon refeeding; this heterogeneity can be thought of as arising from the gated entry of cells into S, depending on their physiological state and position in the Gq cycle at the time of stimulation. It is worth pointing out that the interval between preferred generation times is the same as the interval between enzyme activity maxima (Klevecz and Ruddle, 1968) or between bursts in thymidine incorporation (Klevecz et αί, 1975). This later observation is taken as indicating that initiation occurs in discrete temporal and spacial clusters within S phase (Hand, 1975). As an interesting sidelight one should mention that a solution to the tempera­ ture compensation problem has been proposed by Njus et al. (1974), who hypothesize that the source of temperature compensation for circadian clock involves ion flux and ion transport changes in the membrane. This model rests most strongly on the fact that membrane-bound enzymes and ion flux are temperature compensated by lipid adaptation.

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Unlike the biological or circadian clock, the cellular clock has no clearly identifiable time cues. In the circadian system the time cue is light and we can only assume that in the cells of a metazoan organism this time cue would be mediated by a hormonal factor, as indeed Axelrod (1974) has observed, and ultimately by some internal second messenger such as cAMP (Cummings, 1975). We should probably begin to look among the known serum factors for one which fits the criteria of a Zeitgeber and to develop cell culture systems in which the clock can be discerned more clearly. REFERENCES Abelson, H. T., Johnson, L. F., Penman, S., and Green, H. (1974). Changes in RNA in relation to growth of the flbroblast. II. The lifetime of mRNA, rRNA, and tRNA in resting and growing cells. Cell 1, 161-164. Adams, R. L. P. (1969). Phosphorylation of tritiated thymidine by L929 mouse fibroblasts. Exp. Cell Res. 56, 49-54. Amaldi, F., Carnevali, F., Leoni, L., and Mariotti, D. (1973). Replicon origins in Chinese hamster cell DNA. Exp. Cell Res. 74, 367-374. Anderson, E. C , Bell, G. I., Peterson, D. F., and Tobey, R. A. (1969). Cell growth and division. IV. Determination of volume growth rate and division probability. Biophys. J. 9, 246-263. Augenlicht, L. H., and Baserga, R. (1974). Changes in the GO state of WI-38 fibroblasts at different times after confluence. Exp. Cell Res. 89, 255—262. Axelrod, J. (1974). The pineal gland: A neurochemical transducer. Science 184, 1341-1348. Baechtel, F. S., Hopkins, H. A., and Schmidt, R. R. (1970). Continuous inducibility of isocitrate lyase during the cell cycle of the eucaryote Chlorella. Biochim. Biophys. Acta 217,216-219. Baserga, R., Costlow, M., and Rovera, G. (1973). Changes in membrane function and chromatin template activity in diploid and transformed cells in culture. Fed. Proc, Fed. Am. Soc. Exp. Biol 32, 2115-2117. Becker, H., and Stanners, C. P. (1972). Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. /. Cell Physiol. 80, 51—61. Becker, H., Stanners, C. P., and Kudlow, J. E. (1971). Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. /. Cell Physiol. 77, 43-50. Bello, L. J. (1969). Studies on gene activity in synchronized cultures of mammalian cells. Biochim. Biophys. Acta 179, 204-213. Bergeron, J. J. M., Warmsley, A. M. H., and Pasternak, C. A. (1969). The timing of phospholipid synthesis in neoplastic mast cells. FEBS Lett. 4, 161-163. Bergeron, J. J. M., Warmsley, A. M. H., and Pasternak, C. A. (1970). Phospholipid synthesis and degradation during the life-cycle of P8154 mast cells synchronized with excess of thymidine. Biochem. J. 119, 489-^192. Bhorjee, J. S., and Pederson, T r (1972). Nonhistone chromosomal proteins in synchronized HeLa cells. Proc. Natl. Acad. Sei. U.S.A. 69, 3345-3349. Blomquist, C. H., Gregg, C. T., and Tobey, R. A. (1971). Enzyme and coenzyme levels, oxygen uptake and lactate production in synchronized cultures of Chinese hamster cells. Exp. Cell Res. 66, 75-80.

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