Cell cycle dependency of monoclonal antibody production in asynchronous serum-free hybridoma cultures

The cell cycle kinetics of F3(B6) mouse hybridoma was examined by immunocytochemical staining of bromodeoxyuridine incorporated into the DNA of exponentially growing cells in three different cultures: one supplemented with 10% fetal bovine serum and two adapted to serum-free media, TABIES and BITES. The serum-free cultures, particularly the BITES, had longer cycling times and higher specific antibody production rate. Both observations were correlated to the prolongation of the G₁ phase traverse time and substantiated with a starvation blocking experiment.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

Cell cycle analysis of tumour-derived cultures ofCrepis capillaris: A kinetic analogy with proliferation of animal tumour cells

Summary Analysis of the cell cycle by three methods has revealed unusual kinetics of proliferation in tumour derived suspensions ofCrepis capillaris. The different methods of analysis yield different estimates of cycle phase durations, and such discrepancies have been explained in terms of low growth fractions with rapid total cycle traverse. Specifically, confidence in the estimation of G₂ duration by the fraction of labelled mitosis analysis, and comparison with shorter G₂ estimates obtained by the two other methods, suggests that cells drop out in G₁. However, cells which do not drop out of the proliferative compartment traverse G₁ extremely rapidly. Extremely short cell cycle durations in which the G₁ phase is virtually non-existent are uncharacteristic of plant cell suspension cultures, in which the G₁ phase has previously been shown to be extended as compared with meristematic root tip cells. A model has been proposed in which a central core of rapidly dividing cells continuously loses cells into a subpopulation of resting or G₀ cells with the G₁ DNA content. Similarities between plant and animal tumours with respect to cell growth and division are discussed.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

Noninvasive estimation of cell cycle phase and proliferation rate of human mesenchymal stem cells by phase-shifting laser microscopy

The simultaneous determination of the cell cycle phase of individual adherent mesenchymal stem cells (MSCs) using a fluorescence microscope after staining with 4′,6-diamidine-2′-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by phase-shifting laser microscopy (PLM) revealed that the laser phase shift of cells in the G₂/M phase was markedly higher than that of cells in the G₀/G₁ phase. Even in the synchronous cultures to G₀/G₁ and G₂/M cell cycle phases, the laser phase shift of the cells in the G₂/M phase was markedly higher than that of the cells in the G₀/G₁ phase. The analysis of the cultures of MSCs from different donors with the addition of FGF2 at different concentrations revealed that there was a marked negative correlation between the average phase shift and mean generation time. In conclusion, it is possible to estimate noninvasively the proliferation activity of MSCs population by measuring the phase shift using PLM.     

Modulation of G(2) arrest enhances cell death induced by the antitumor 1-nitroacridine derivative,Nitracrine

Nitracrine (Ledakrin) is an antitumor drug which is activated by cellular enzymes and binds covalently to DNA. Previous studies have shown that covalent binding and crosslinking of DNA is associated with the cytotoxic and antitumor activities of this compound. In this study, cell cycle perturbations, effects on DNA synthesis and the cell death process initiated by Nitracrine were studied in murine leukemia L1210 cells. We show that exposure of L1210 cells to Nitracrine at the IC₉₉ concentration delayed progression through the S phase and transiently arrested cells in G₂/M as found by flow cytometry. Higher drug concentration (2 × IC₉₉) inhibited cell cycle progression in the S phase and induced rapid cell death. Both studied concentrations of the drug produced different effects on DNA synthesis as determined by bromodeoxyuridine incorporation, with a delay in the S phase progression at EC₉₉ concentration and irreversible arrest in early S phase at the higher dose (2 × IC₉₉). At both concentrations of Nitracrine cell death occurred preferentially in the S phase as revealed by the TUNEL assay. When cells treated with the drug for 4 hours were post-incubated in the presence of 1 mM caffeine this led to rapid cell death and suppression of the G₂ arrest. This was associated with a about 10-fold increase in the cytotoxicity of Nitracrine. Similar effects were observed for another DNA crosslinking agent, cis-platinum, and to a lesser extent, for DNA topoisomerase I inhibitor, camptothecin. Together, our studies show that suppression of G₂ arrest induced by Nitracrine greatly enhances its cytotoxicity toward L1210 cells.     

Sensitivity to macrophage-mediated cytostasis is cell cycle dependent

We have examined the sensitivity of proliferating lymphoid cells in different phases of the cell cycle to macrophage-mediated cytostatic activity. These studies evaluated the ability of target cells enriched in individual cell cycle phases to pass into the next phase during brief (2–6 hr) periods of coculture with activated or nonactivated peritoneal macrophages. Both normal (concanavalin A-stimulated spleen cells) and neoplastic (Gross virus-induced thymic lymphoma) cells were analyzed. Spleen cells or lymphoma cells were first separated by centrifugal elutriation into populations highly enriched for G₁, S, or G₂/M phases of the cell cycle and cultured in the presence of nonactivated or activated macrophages for periods of 2, 4, or 6 hr. The cellular DNA content of recovered nonadherent target cells was then analyzed by flow cytometry after staining with propidium iodide. Macrophage contamination of target cell populations was insignificant under these conditions. Nonactivated macrophages did not affect target cell cycle traverse when compared with target cells cultured alone. Activated macrophage mediated cytostatic activity resulted in complete block of the transition of cells in G₁ phase into S phase and of the further accumulation of DNA by cells in early S phase. Cells already in mid to late S phase were able to continue DNA replication at rates nearly equivalent to control cells. There was no inhibition of the passage of cells through G₂ or mitosis. These effects were seen by as early as 2 hr of macrophage-target cell coculture and both normal and neoplastic cells exhibited identical patterns of cell cycle phase sensitivity.     

The effect of potassium on the cell membrane potential and the passage of synchronized cells through the cell cycle

The cell membrane potential of cultured Chinese hamster cells is known to increase at the start of the S phase. The putative role of the cell membrane potential as a regulator of cell proliferation was examined by following the cell cycle traverse of synchronized Chinese hamster cells in the presence or absense of high exogenous levels of potassium. An increase in external potassium levels results in a depressed membrane potential and a reduced rate of cell proliferation. A potassium concentration of 115 mM was used in experiments with synchronized cells since at that level cell proliferation is almost completely halted, recovery of growth is rapid and complete, and the membrane potential is reduced to a level well below that normally found in cells in the G₁ phase. A mitotic population was divided into four aliquots and plated in either control medium or medium containing 115 mM K⁺. Cells placed directly into high K⁺ medium were retarded in their exit from mitosis and displayed a delayed and abnormal entry into the S phase. If control medium was added after two hours, cell cycle traverse was normal, but delayed by two hours compared to control cells. If the mitotic cells were plated directly into control medium and two hours later were shifted to high K⁺ medium, the cells entered the S phase in the absence of the normally observed increase in membrane potential and proceeded to the next mitosis normally. It was concluded that the increase in membrane potential observed at the start of the S phase in isolated synchronized cells is not a requirement for the initiation of DNA synthesis. In addition, sensitivity to the high potassium regimen was found at two different times during the cell cycle. In one case, cells were impeded in their transit through mitosis. Such cells displayed an altered chromosome structure which may account for the partial mitotic block. In the second case, synchronized cells displayed a sensitivity to the high potassium regimen in early G₁ which appeared to be separate from the block in mitosis and independent of a change in the membrane potential.     

Autoradiographic studies on the incorporation of3H-amino acids into chromosomes during the mitotic cell cycle ofHaplopappus gracilis

The synthesis of chromosomal proteins and the incorporation of labelled proteins into chromosomes in the mitotic cell cycle ofHaplopappus gracilis, 2n=4, were traced autoradiographically with³H-arginine,³H-lysine, and³H-tryptophane. The duration of the mitotic cell cycle in the root tip cells was determined by³H-thymidine autoradiography and was measured to be 13.0 hr (G₁ 1.3 hr, S 6.5 hr, G₂ 3.8 hr and M 1.4 hr).³H-arginine labelled proteins which were synthesized at S and G₂ were found to be incorporated into chromosomes to a greater extent than proteins which were synthesized either at G₁, at the transition phase from late S to early G₂, or at the mitotic phase. Such varied incorporation was also found in³H-lysine labelled proteins, but not in³H-tryptophane labelled proteins. These findings indicate that the chromosomal proteins are synthesized mainly at S and G₂. Some of the³H-arginine labelled proteins which were synthesized during the first mitotic cell cycle, were found to be incorporated into the chromosomes of the second mitotic cell cycle. The incorporation of the proteins synthesized at one stage of the mitotic cell cycle was found to occur locally in some regions of the chromosomes, while the pattern of incorporation was observed to be similar between euchromatic and heterochromatic regions.     

Flow cytometric BrdUrd-pulse-chase study of X-ray-induced alterations in cell cycle progression

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd)-pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G₂, i.e. the fraction of BrdUrd-negative G₂ cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G₂ and in S are equally delayed. S phase transit of cells irradiated in S or in G₁ did not appear to be affected. There were apparent changes in flow of cells out of G₁, which could be explained by the delayed entry of G₂ cells into the compartment because of G₂ arrest. Thus, in asynchronous cells the method was able to detect G₂ delay in those cells irradiated in S and G₂ phases and demonstrate the absence of cell-cycle delays in other phases.     

Down-regulation of statin,a nonproliferation-specific nuclear protein,and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G₁ phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G₁ phase in the apoptotic process is an abortive one, with the end result of cell demise. © 1995 Wiley-Liss, Inc.     

Reversible accumulation of plant suspension cell cultures in g(1) phase and subsequent synchronous traverse of the cell cycle

The induction of DNA synthesis in Datura innoxia Mill. cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G₂ phase, and underwent cell division simultaneously. Only 10% of the cells remained in G₀ or G₁. Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G₀ + G₁/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G₂ phase. However, reversal of G₁ arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G₀ + G₁ phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G₀. The frequency of cells entering G₀ was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium.     

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G₁ phase of the cell cycle instead of G₀. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G₀ phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G₁, 12% were in S and 37% in G₂ phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P <0.01) [³H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P <0.01) [³H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G₁ phase of the cycle to enhance IGF-I effects in cell proliferation.     

A METHOD FOR COMPARING EFFECTS OF DIFFERENT SYNCHRONIZING PROTOCOLS ON MAMMALIAN CELL CYCLE TRAVERSE : The Traverse Perturbation Index

After treatment of Chinese hamster cells (line CHO) with various protocols for synchrony induction, the subsequent ability of cells to traverse the cell cycle (i e., to perform, an essential cell cycle process) has been determined by measurement of the DNA distribution pattern among cells in large populations with the Los Alamos flow microfluorometer In the cultures prepared by the various synchronizing techniques the vast majority of cells traversed the cell cycle in a normal fashion; however, in all cultures examined there remained small subpopulations which, though remaining viable for several days, could not carry out normal traverse. After reversible inhibition of DNA synthesis by means of a double-thymidine blockade, approximately 17% of the cells were unable to complete genome replication. After reversal of G₁ arrest resulting from cultivation of cells in isoleucine-deficient medium, 12 4% of the cells commenced synthesis of DNA but were unable to complete the S phase. Cells prepared by mitotic selection yielded a subpopulation (5 5% of the total cells) with a G₁ DNA content which remained viable but noncycling for at least 5 days. We propose a term "traverse perturbation index" which is defined as the fraction of cells converted to a noncycle-traversing state as the result of experimental manipulation. A knowledge of the perturbation index will allow direct comparison of effects on cell cycle traverse of various synchrony-induction protocols     

Threshold expression of cyclin E but not D type cyclins characterizes normal and tumour cells entering S phase

Complexes of cyclin-dependent kinases (cdk) and their partner cyclins drive the cell through the cell cycle, each such complex phosphorylating a distinct set of proteins at a particular check-point or phase of the cycle. Immunocytochemical detection of cyclins combined with measurement of cellular DNA content by flow cytometry makes it possible to relate expression of each of these proteins with the actual cell cycle position, without the necessity of cell synchronization. In the present study, we have investigated expression of E and D type cyclins in G₁ cells and in cells entering S phase, in eight different human hematopoietic and solid tumour cell lines (two leukaemias, a lymphoma, three breast carcinomas, a colon carcinoma and a bladder transitional cell carcinoma) during their exponential phase of growth, as well as in normal mitogen stimulated lymphocytes. In all the cell types studied, the average level of D type cyclin expression was invariable throughout the cell cycle. A great intercellular variability, in particular of the G₁ cell subpopulations, and the presence of a large fraction of G₁, S and G₂+ M cells that were cyclin D negative (20–40% in tumour cell lines and about 80% among lymphocytes), were other characteristic features of D type cyclin expression. In contrast to D type cyclins, the expression of cyclin E was discontinuous during the cycle, peaking at the time of cell entrance to S. Also, a well defined threshold in expression of cyclin E characterized cells that were entering S phase, and virtually no cyclin E negative cells were seen during the early portion of S phase. The data indicate that while cell entrance to S phase is unrelated to expression of D type cyclins (at the time of entrance), accumulation of cyclin E up to critical level is a prerequisite for initiation of DNA replication. The great intercellular variability in expression of D type cyclins and their invariant average level across the cell cycle suggest that these cyclins, in addition to their acknowledged function in promoting cell progression through mid- to late-G₁ may have other role(s), related or unrelated to the cell cycle progression. The presence of a large number of D type cyclin negative cells in all phases of the cycle suggests that during exponential growth the cells may not express this protein and yet may traverse the cycle, including G₁ phase.     

Effects of cortisol, 17beta-estradiol and thyroliberin on prolactin and growth hormone production, cell growth and cell cycle distribution in cultured rat pituitary tumour cells.

Prolactin and growth hormone production were measured in a rat pituitary tumour cell strain (GH₃) after treatment with cortisol (5 × 10^?6 M), thyroliberin (2.5 × 10^?6 M) and 17β-estradiol (10^?6 M). The changes in hormone production were related to alterations in cell growth rate and cell cycle distribution. Cortisol inhibited prolactin production, stimulated growth hormone production and reduced the cellular growth rate measured two days after start of treatment (maximum about 40% inhibition). Flow-micro fluorometric analysis of DNA distributions showed that cortisol treatment reduced the relative number of cells in S phase (maximum effect about 50%) with a compensatory increase of the proportion of cells in G₁ phase. The lack of inhibition of prolactin production after three days of cortisol treatment may partly be related to the increased number of cells in G₁ phase. Thyroliberin and 17β-estradiol did not significantly affect cell growth after six days of treatment, although the fraction of cells in S phase was reduced by approximately 40% with a corresponding increase of cells in G₁ phase. For thyroliberin and 17β-estradiol, the stimulatory effect on prolactin production and the inhibitory effect on growth hormone production witin a period of treatment of six days cannot be explained by a shift in cell cycle distributions. None of the three hormones influenced the growth fraction which was equal to unity. In conclusion, thyroliberin and 17β-etradiol are able to change prolactin and growth hormone production without altering the cell cycle distribution. However, the effects of cortisol on prolactin and growth hormone production may partly be due to an alteration in cell cycle traverse resulting in an increased number of cells in the G₁ phase.     

An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine-labelled cells, using FCM-derived data

Abstract. Chinese hamster ovary cells in vitro were pulse-labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd-labelled cells in relation to that of G₁ and G₂ cells, was calculated. RM was then used to calculate DNA synthesis time (T_S), at all post-labelling times (t). Since labelled cells in G₂ and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G₂ and M (T_g2+M) phases is finite, a non-linear relationship exists between RM and post-labelling time. Because of this, the use of a linear formula in the calculation of T_S yields results that are affected by t. We found that RM data can be corrected with regard to T_G2+M resulting in the derivation of a non-linear T_S formula. This non-linear T_S formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G₁, S and G₂+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd-labelled cells in each window at every post-labelling time. Plots of the fraction of BrdUrd-labelled cells v. post-labelling time were then made for each window. T_S obtained in this way was in agreement with T_S obtained with the corrected RM method. In conclusion, we present a method to calculate T_s which theoretically first makes the determination of RM independent of T_G2+M, and secondly compensates for the non-linear function of RM with post-labelling time caused by accumulation of BrdUrd-labelled cells in G₂+ M.     

The differentiation of T-lymphocytes. III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture.

Seven human cultured lymphoblastoid cell lines (CLL) were divided into two major groups based on studies of their cell cycle characteristics and surface Ig. CLL I (lines CL, MW, HH and TM) had generation times ranging from 25–40 hr, S phase times of 10–12 hr, G₂ + M times of 6–8 hr, and demonstrated sharp differences between the percentage of SIg(+) cells in different phases of the cell cycle. Line TM was particularly discordant with the highest percentage of SIg(+) cells in G₂ + M. CLL II (lines PS, JR and HT) demonstrated generation times ranging from 18–21 hr, S phase times of 7–10 hr and G₂ + M phase times of 2 hr. In this second group, two of the three CLLs had no differences between cells taken from different points of the cell cycle. DNA synthesis and cell density could not be correlated with either of the above major parameters, i.e. cell cycle times or SIg expression. The results suggest that human CLLs fall into subgroups in which specific patterns of cellular and immune functions may predominate.     

A Rapid Automated Stathmokinetic Method For Determination of In Vitro Cell Cycle Transit Times

To provide a rapid method for examining cell cycle dynamics, we utilized continuous exposure of Chinese hamster ovary cells and human colon cancer cells to colcemid to block cycling cells in metaphase, suppressing re-entry into G₁. Changes in cell cycle compartment distribution were monitored by DNA flow cytometry. Analysis of the rate of G₂+ M compartment accumulation after addition of colcemid permitted calculation of all cycle transit parameters. These compared favorably with data in the same cell lines determined by the fraction of labeled mitoses technique. Serial assessment of DNA flow cytometry after addition of colcemid permits rapid quantitation of cycle traverse rates.