Methods in Cell Science 18:75-81 (June 1996) © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Synchronization in mammalian system: An introduction

Arun K u m a r S h a r m a Centre for Advanced Study in Cell and Chromosome Research, University o f Calcutta, Calcutta, India

Synchronization is a unique strategy for securing cell mass in an identical phase. The principle of cell synchronization involves blockage of cells at a specific phase of the cell cycle, achieved through dif- ferent means including the use of cytotoxic agents and depriving the medium of some of the essential constituents needed for division. Synchronized populations are essential for study of the cell cycle related events. It is specially necessary as metabolic processes are quite different at different phases in the cell cycle including the transitional phase [1]. Synchronization has assumed enormous importance in recent years in view of the cancer-related events in the cell cycle. The cycle follows the normal course provided the supply of metabolites and cellular envi- ronment remain unhampered. Conditions restrictive for progression at one stage lead to accumulation of cells at a particular phase, and further resumption of growth is through input of metabolite. Such arrest may occur at any of the phases, though the arrest of the synthetic phase may often lead to cell death. In general, the methodology for cell synchronization should have as prerequisites, the action on a target metabolic phase without damaging effect, and poten- tial of substantial harvesting of synchronized cells. The success and extent of synchronization depend to a great extent on the method adopted for securing synchrony.

However, any discussion on synchronization must be preceded by an introduction to the present concept of cell cycle and checkpoints and their relationship to cancer. This understanding is necessary for the identification of the desired phase prior to induction of synchrony in the target cell mass.

Cell cycle and checkpoints

One of the important discoveries, in recent years, is the cyclin-dependent kinase, necessary for cell cycle. The enzyme kinase which controls the behaviour of other proteins by attaching phosphate groups, and the protein cyclin which triggers the kinase at a critical period bringing about changes in cell cycle, are both essential [2].

Cyclins and kinases control several points in the cell cycle. Cyclin activates Cdcz kinase at mitosis at the time of dissolution of nuclear membrane and chromosomes moving to poles. It is also active in late

GI to start replication. The cyclins generally belong to two distinct classes, Cyclin A and Cyclin B, the mitotic cyclins being of the latter type. The cyclin A may be active in activating kinase at the beginning of 'S', being different from the 'Start' which is active in late GI phase.

Events in the cell cycle require to be completed in a definite sequence [3]. Chromosome replication should precede chromosome segregation and as such the progress of mitosis requires completion of 'S' phase [4]. It is true that an early event may produce a substrate required for the next stage and as such completion of the former is essential for the initiation of the latter. However, it does not neces- sarily follow a typical substrate-product relationship since artificially the two events can be uncoupled. Checkpoints permit monitoring of progression through the succession of essential steps and thus coordinate cell cycle for replication and segregation of components, Mutation in one of the control check- points may allow even a later event to proceed without completion of the former. It shows that normally any disruption delaying the first event may enable the checkpoint to send signal so that the latter event is delayed. The importance of checkpoint control, coordinating cell cycle as also their interac- tion with different extemal and internal signals with relevance to cancer, have assumed enormous signif- icance in recent years. However, checkpoint func- tions also indicate that several of these events can proceed on their own. The understanding of these issues is essential for inducing synchrony with pre- cision.

The complex of cyclins and cyclin-dependent kinases is deeply involved in the transitional path- ways of DNA replication and mitosis [5]. Two mech- anisms operate in the interdependent pathways. The events acting on GI and needed for S phase and events on G2 necessary for mitosis, constitute one pathway. The other process exerts controls on GJS to check premature entry into mitosis, and on G 2 to check reentry into G1 and triggering of new S phase. The failure of any of these mechanisms would lead to alteration in genome dosage. Successive divisions without S phase may lead to reduction in number and often lethality, whereas successive 'S' phases without mitosis cause endoreplication.

Two important events are clearly involved in eukaryotic gene replication (1) at the end of mitosis

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and (2) at the beginning of 'S' phase [6]. The initi- ation competence at G1 is capable of triggering 'S' phase and initiation competence at G 2 phase. The ini- tiation competence is lost in 'S' phase with the onset of replication. The further reversal of competence starts from G2 to G1 through the M phase. The com- petence evidently is established much before 'S' phase, though not adequate to initiate replication. At G1/S boundary, the process reaches its culmination, triggering S phase and DNA replication. With repli- cation, there is again loss of competence. Further studies on protein kinases and phosphatases have shown that transitions for GJM and M/G1 respec- tively initiate early and late mitotic events. Moreover, most of the mitotic events have been suggested to be dissociable and governed by different factors, despite their simultaneous functioning [7].

Implication of damage at different points

In the study of synchrony, an understanding of check- points and their response to agents is essential. In mammalian cells, the damage in G1 does not permit entry into S phase and damage in G2 leads to no mitosis [8]. The cyclin-dependent kinase (Cdk) inhibitor plays an important role in G 1 arrest. With damaged DNA, there is increased level of P53 tumor suppressor which in turn accelerates the formation of inhibitors. It has been suggested that such inhibitors directly interact with cyclin Cdk complex [9-12]. The exact mechanism through which unreplicated DNA causes arrest of cell cycle, the implicit need for inhibitor for arrest, as well as the clear identifica- tion of genes responsible for P53 induction following DNA damage are yet unsettled issues.

Unreplicated and damaged DNA may also enter mitosis, such as noted in caffeine. With unreplicated DNA, prevention of tyrosine phosphorylation permits cells to enter mitosis in vertebrates - whereas pre- vention of dephosphorylation may allow entry as noted in yeast [13]. Leaving aside DNA replication and damage repair, cells treated with topoisomerase II inhibitors do not allow the cells to enter into mitosis, and caffeine may reverse the block [14].

Spindle assembly and arrest

Several authors have shown that entry into mitosis proper is caused by mammalian promoting factor along with complex of Cdc2 kinase and cyclin B. Assembly of spindle is a landmark point. The acti- vation involves breakdown of nuclear membrane, condensation of chromatin and formation of spindle. The termination involves a reverse cycle - cyclin B degradation and inactivation of maturation promoter [15]. Moreover, protein degradation has been found to be a component of cell cycle regulation. The

protein ubiquitination and deubiquitination mediated by several enzymes are involved in cell cycle progression [16, 17]. The mechanics of cyclin B destruction is claimed to be ubiquitination-mediated proteolysis [14].

The monitoring point in the spindle may be attach- ment of microtubules to kinetochore, dynin on the centromeric DNA, a long range mechanism so that kinetochore can monitor the distance to the pole and a combination system [4, 18, vide 7]. The treatment of spindle with microtubular poisons like benonyl or mocodazole blocks progression and mitosis.

In mammalian system, monoclonal bodies recog- nize specific phosphopeptide epitope, and only the unattached or lately attached kinetochores to micro- tubules [19]. It has been shown that kinetochore associated protein which attach to antibodies is a component of spindle assembly checkpoint. The other evidence is obtained through the use of 2- aminopurine which overcomes mitotic arrest induced by taxol, preventing microtubule depolymerization. The nature of the molecular mechanism between microtubule and cortex is not fully known [20].

In addition, the other checkpoint is to monitor chromosome alignment and other aspects of spindle assembly. In tissue culture of sea urchin embryo, single kinetochore unattached to microtubule can delay the onset of anaphase [21]. Drugs binding with microtubules in low doses - such as anticancer vin- blastine - show a decrease in microtubules attached to each kinetochore [22]. Antikinetochore antibody or mutant centromeric DNA sequence, delaying com- pletion of mitosis, shows kinetochore as the check- point.

Checkpoints and cancer

The elimination of damaged cells is an alternative to repair [3]. However, mammals apply two methods to deal with damaged cells: (1) cell cycle arrest till repair, and (2) permitting death of damaged cells. DNA damage, unreplicated DNA or depolymeriza- tion of spindle can all lead to death. Evidences indicate that initial states of metabolism leading to arrest and cell death are common to both.

The ability of c-myc genes for abnormal prolifer- ation inducing P53 dependent cell death, suggests similarity of cellular response to replication-segre- gation on the one hand, and signals controlling growth and development on the other [23]. The signals controlling proliferation and checkpoints interact. The P53 plays a role in preventing malig- nancy through: (1) checkpoints altering ceils to arrest and repair, (2) killing of damaged cells, and (3) killing of cells which have undergone mutation to unbalance the normal coordination between signals controlling cell proliferation. Cancer at one phase is a disease of cell cycle control.

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Studies have also revealed that a group of small proteins bind to kinases and inhibit cell progression [24]. In fact, Cdk inhibitor proteins have become one of the key factors in cell cycle control in eukaryotes. The linkage of such proteins to tumor suppressors, provide with an insight into the relationship of cell cycle and cancer.

The product of retinoblastoma tumor suppressor gene, otherwise termed a PRB, generally influences the two thirds of the G1 phase of the cell cycle and more or less it operates in the midst of all cycle clock [25]. Cells at the point of entrance to G1 from mitosis, continuously need serum mitogens till the triggering of S phase, after which they become serum inde- pendent. This transition point from serum dependent to serum independent phase is termed as the Restriction point (R) [26]. After the cell passes through 'R' point, there is a commitment to growth through 'M' phase [27]. As such it is a critical tran- sition point. The retinoblastoma protein (PRB) shows certain changes at a period close to 'R' point transi- tion. At the initial phase of G~, it is underphosphory- lated, which later undergoes hyperphosphorylation - a state being maintained in the remainder of the cell cycle. It becomes dephosphorylated again after 'M' phase. During oncogenesis, oncoproteins of tumor viruses stop PRB functioning by binding. Moreover, phosphorylation leads to inactivation of growth inhibitory functions of PRB. The factors causing phosphorylation of PRB protein accelerate cell proliferation.

The mechanism of correlation of checkpoint action and cell cycle arrest is not fully known. Lesions in the checkpoint detecting DNA damage play a role in tumor progression. The loss of one copy of chro- mosome 13, as the common cause of loss of hetero- zygosity for the retinoblastoma gene, suggests that lesions in spindle assembly checkpoint play a role in the generation of cancer. The analysis of the extent to which internal and external signals are integrated with cell cycle progression at the molecular level, the molecular basis of stimulation of proliferation, and inhibition of proliferation by mitogens and antiproliferative signals, are indeed challenging problems.

Methodology for synchronizat ion

Physical methods. For synchronization, several tech- niques are in vogue, both physical and chemical. Most of the physical methods are based on the gradient separation excepting the detachment tech- nique which is of wide use. The detachment method is based on principle of detaching the cells, initially done through enzymes and microscopic detection of cells at tt, e maximum frequency of division. It is followed by separation of mitotic cells by shaking and later plating. The detachment can be facilitated

through the use of calcium free medium which decreases cell adherence. The synchrony can be eval- uated by continuous tritiated thymidine followed in successive cycles. Such systems undoubtedly permit the study of G~ events but post S phase events often require further treatment with certain chemicals.

Mitotic detachment and selection are widely adapted because of no toxicity and high potential for harvesting, but limited only by the fact, that optimum harvesting is possible only with monolayer culture, which must be ensured.

As compared to mitotic detachment, other methods are principally based on gradient, depending on dif- ferential volume and density which are used as para- meters for selection of cell cycle. A volume change in different phases of the cell cycle is however assumed, a premise which is debated. The fact that cell volume becomes double in mitotic cycle is correct but it does not imply that the specific volume can be used as criteria for all the intermediate stages of the cell cycle. Through gradient method such as Ficoll, a good percentage of synchronized cells can be harvested though not as high as one could get through continuous detachment.

The physical method of 'Elutriation' using elu- triator rotor in the centrifuge, utilizes differential cell size as the principal factor in distinction, G1 cells being smaller than those in other phases of the cell cycle [28, 29]. There are several advantages of elutriation. It permits rapid separation of asynchro- nous cells in different phases of the cycle excepting mitosis. Moreover, the procedure is carried out in room temperature and does not involve any toxic compounds.

In cell sorting, no specific stain or chemical iden- tification is used as the parameter. The parameters are mostly biophysical - such as, cell size, surface markers or nuclear cytoplasmic ratio. The technique however suffers from some limitation of overlapping, often making distinction between phases difficult. It may however be utilized for enrichment of G~ phases.

Cell sorting of synchronized cells, using fluores- cence and electronic cell sorter, has opened up a com- pletely different line of approach. The problem of automated cell sorting is that it involves staining with fluorochromes which may affect viability or may cause mutagenesis or DNA damage [30-32]. The sophisticated computer backup is also a serious limitation. It is a good method but the instrument is to be provided for, along with the fluorescent dye. If properly carried out, the sorting fidelity can reach even up to 98% level with the sample flow rate as 2.5 × 103 cells/sec.

Chemical methods. Of the chemical methods, both isoleucine and hydroxyurea are widely used. The deprivation of isoleucine in the medium leads to arrest and does not permit the cells to pass from Go to G1 phase. The medium without isoleucine often

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induces rapid arrest from Go to G1, initially done on Chinese hamster [33, 34]. The viability of the stationary phase could be maintained even upto 60 hours or more following recovery, the division being highly synchronous. This method has the advantage of securing synchrony in suspension, permitting a large number of cells to come under the orbit of synehrony. But possibly because of the genomic diversity of different cell lines with regard to their response to isoleucine, it is not necessarily univer- sally applicable.

One of the most widely used methods for syn- chronization is serum restriction and use of hydroxy- urea. The procedure is based on the principle of exposing the cells to serum deficient medium, to be grown later in medium containing serum followed by addition of concentrated hydroxyurea. The medium is normally removed after a long period of blocking. The transfer to medium later with hydroxyurea results in entry of cells in S phase. In this method, the choice of hydroxyurea concentration is of prime importance. The lower dosage would reduce the effect at G1/S boundary whereas toxicity may result in high concentrations.

For induction of synchronization in Chinese hamster, Tobey et al. [34] developed a three step procedure involving presynchronization in early 'S' phase with isoleucine and hydroxyurea, growth in medium with topoisomerase inhibitor and finally culturing in drug free medium.

In addition to isoleucine and hydroxyurea, a number of other chemicals are also being used though not so effective as the other two. The thymidine in high concentration can almost inhibit the pace of cell division but even then DNA synthesis occurs in a few percent of cells. Normally, the concentration needed for synchronization is much lower than that required to produce G1/S arrest. As such, certain cells enter into S phase and heterogeneity sets in. In order to mitigate this limitation, a second thymidine treat- ment after a certain period is needed. This double thymidine block yields comparatively better results, though it has its own limitation because of partial synchronization.

In order to secure blockage of ceils at specific stage, several chemicals including decreased con- centration of calcium, excess of thymidine, inhibitors of polymerases and synthetases, folic acid analogue, and amethopterin causing thymidine deficiency, have been applied. Out of all these, the decreased amount of calcium leading to quiescence followed by increase in calcium concentration has been found to be very effective. The blocking, if induced, occurs just prior to S phase, so that there is a burst of synchrony following recovery. The effects of some of the mutagenic compounds such as EMS and NMS have also been shown to lead to 'S' phase delay, a common effect of toxicity and sister chromatid exchange [35].

The other target for inducing synchrony, is the M phase checking spindle assembly. Of the different spindle poisons, colehieine is considered to be the most potent one, inhibiting spindle formation through its effect on spindle proteins [vide 36, 37]. The use of colcemid is being applied on a large scale in mam- malian cell lines to secure breakdown of spindle assembly, metaphase arrest and subsequently syn- chronized cells. The advantage of using colcemid is that it does not affect the biochemical pathways in G1, S and in other phases of mitosis. The effect being reversible, the cells recover from a narcotized stupor and show a high degree of synchrony on recovery. If colcemid treatment is associated with repeated harvesting through monolayer shaking and har- vesting, high frequency of synchronous cells can be assured. The physical detachment of cells coupled with colcemid treatment undoubtedly is very advan- tageous, with very little toxicity. Colcemid is less toxic than colchicine and it is used in leucocyte culture on a large scale. In bone marrow too, the effect is on metaphase arrest. The cells accumu- lated in metaphase can be induced to divide syn- chronously following the application of medium without colchicine. Similarly, okadaic acid and topoisomerase inhibitors allow premature condensa- tion and derangement in spindle formation [38]. Lately, a number of other compounds has also been worked out inhibiting the normal cell cycle including lavostatin, saurosporine, mimosine, noco- dazole, sodium vanadate, polyamine inhibitors and others, some of which have been dealt with in this treatise.

Evaluation

Several methods have, however, been adopted for evaluation of synchrony. A very ample technique is thymidine incorporation and autoradiography. Though time consuming, it is the simplest method not only for evaluation of replicating cells but also for cells in mitosis. The other method is liquid scintillation counting of radioactive compounds. However, as is well known, this method though quick, does not give enough information as in autoradiography.

The blocking at checkpoints also helps evaluation through cell cycle analysis, upto multiparametric level. The use of anticyclin antibodies, anti BrdUrd antibodies, have all been applied for evaluation of synchrony. The procedure permits quantitation of DNA, RNA and protein in different phases of the cell cycle.

Of the fast methods for cell sorting and evalua- tion, flow cytometry (FCM) is now widely used [31, 32]. The technique is based on the principle of quantitation of DNA by flow microfluorometry which permits the study of cell cycle with a small

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sample of cells. It clearly differentiates between cells with G 1 o r G~ and M content of DNA. A variable graph is obtained depending on the position of the cell in different stages of the S phase. Its advantage of rapid mass screening scores over its disadvantage of expensive instrumentation.

A comparison of the several methods for syn- chronization and evaluation was carried out by Grdina et al. [39]. Cell dispersion with progression has been universal and requirements of line and cell number differ in different cell lines. In general, mitotic selection followed by hydroxyurea blocking can yield a very high frequency of S phase cells.

Comparative assessment

However, none of the methods is free from limita- tions. The mitotic selection undoubtedly causes least disturbance, and can yield high frequency of G~ cells but its response in only selected cell lines and low recovery rate are its serious limitations. The method of cell elutriation has the advantage of being very rapid, and can be executed at room tem- perature. The automated cell sorting and use of flow cytometer, though expensive, permit the use of different parameters for sorting and analysis as well.

Of the different methods of cell synchronization, mitotic detachment and selection have a compara- tively wide application. It causes least perturbation and cells can be synchronized in M, G~ and S phase if enriched with hydroxyurea. This method, however, can only be applied to monolayers of selected cell lines and not in suspension. In order to have syn- chrony at S or G2, it is desirable to combine mitotic detachment with serum deprivation followed by stimulation and hydroxyurea treatment. Such modi- fications lead to a good harvest. For some of the cell lines in suspension culture, isoleucine deprivation yields moderate amount of G~ or S cells. Sorting uti- lizing sorters, no doubt may yield G1 cells - but with heterogeneity in terms of late and early G1 phase. Moreover, it is desirable to study the effect of hydroxyurea on progression, viability, and biochem- ical pathways along the cell cycle.

The method of centrifugal elutriation, if coupled with blocking by hydroxyurea, cell separation in various phases of the cell cycle can be obtained. It is more rapid than cell sorting and mitotic selection, which require considerable period for processing.

The serious limitation of the use of chemicals is the disturbance caused in the chemical equilibrium of the cells and subsequent phases of division. Moreover, due to differential susceptibility of dif- ferent phases to the chemicals, often synchronization is not homogenous. The physical methods like cen- trifugation or elutriation are free from these dis- advantages.

Conclusion

It is pertinent to enquire as to what extent overall synchronization is a reality. It is true that harvesting of synchronized cells can reach to a level of 98% or so with mitotic selection and use of blocking agents. Such a procedure permits a comparatively accurate analysis, despite the fact that it represents only a fraction of the ceils initially present. It does not imply absolute synchronization of all cells.

The discovery of checkpoints in the cell cycle has undoubtedly facilitated a precise understanding of the landmark stages in Go, G1, S and M phases of the cell and the crucial transition points for triggering of actions.

With the proper understanding of checkpoints and monitoring through cyclin protein kinase complexes, theoretically, growth can be arrested at any phase, namely Go, G1, S, G2 and mitosis, with effects on the transition points in particular.

Arrest in a phase followed by synchronization, may in theory, appear to involve a homogeneous mass of cells. But such an assumption ignores the predetermined generation time with specific period earmarked for each phase. A cell arrested at G1 phase for a long period by a blocking agent, and another cell just completing G1 through a normal cycle, may not necessarily complete the cycle at the identical period. Just as the period taken by the cell to complete the cycle is predetermined, so also the period for each phase. Prolonged arrest of cells at G~ may upset the overall generation time of the cell, which may need the completion of the cycle at a rapid rate, as compared to the those arriving later at G1 phase. However, concrete evidences of such unalterable timing cycle of each phase and cell are yet to be obtained. A knowledge of the precise duration of each event in the overall cycle of mam- malian cells is of signal importance. It is true that okadaic acid brings about premature condensation and spindle derangement, but what is more important is the long term effect, if any, on the duration of sub- sequent Go and successive cycles. Research on the duration and the control of different phases in relation to synchronization is assuming special significance with special reference to cancer, which clearly at one stage, is a disease of the cell cycle. The application of synchronization aids in working out this duration and the response of the nuclei and their metabolic pathways to therapeutic drugs. Of the methods so far applied, the chemical ones, however effective they may be, suffer from the serious limitation of their possible effect including even mutagenicity. Such disadvantage assumes enormous significance in studies on oncogenesis where mutation p e r se plays an important rote. As far as practicable, physical techniques combining selection and elutriation coupled with multiparametric evaluation through flow cytometry may yield the best possible results.

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The methods in vogue for high synchronization may serve the purpose for the present, with limita- tions of chemical blocking being inherent. However, by the term absolute synchrony, the implication is that once synchronized it should be perpetuated through the different cycles. Once arrested, it should remain synchronized, throughout. Normally, effects of synchronization are not permanent in that sense. The principal reason for such predisposition to asyn- chrony, is the programmed cell cycle and life cycle of an organism, as discussed. Any alteration mani- fested in arrest through physical and chemical agents would in the long run, affect the overall timing cycle, manifested later in asynchrony. The visible effect of such genetically programmed system may be delayed, and the transient effect can only be stabi- lized through genetic alteration of regulatory gene.

The above discussion is an attempt to introduce the subject of synchronization as it stands to day, in the light of recent developments. There have been remarkable refinements in techniques capable of providing solutions to different problems through synchronization. It is hoped that this Special Issue would be able to stimulate researches on synchro- nization as it cuts across unexplored avenues of biological and medical research.

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Address for correspondence: Arun K. Sharma, Centre for Advanced Study in Cell and Chromosome Research, University of Calcutta, 35 Ballygunge Circular Road, Calcutta 700019, India