Influence of the existence of a resting state on the decay of synchronization in cell culture

General relationships between the distribution of cell doubling times and the growth pattern of an initially synchronized cell population are applied to the model proposed by Smith and Martin (1973) in which the mitotic cycle or "B" phase is preceded by a random-exit resting "A" state. Results show that culture synchronization decays so rapidly as to be virtually unobservable unless the time spent by a cell in the B phase is at least equal to that spent in the A state. If synchronization persists over several mitotic cycles, the growth pattern is determined to a much greater extent by variation in the duration of the B phase than by the probability of exit from the A state. Accordingly the growth pattern of a cell population, like the doubling time distribution which governs the pattern, is of limited usefulness in detecting the existence of a resting state.     

Cell cycle analysis with bromodeoxyuridine pulselabeling in asynchronous hybridoma cultures

Summary The cell cycle kinetics of a mouse hybridoma was examined by immunocytochemical staining of incorporated bromodeoxyuridine in an asynchronous culture. The cell cycle phase traverse times were extracted from a time series of bivariate distributions of incorporated bromodeoxyuridine and total DNA content; the G₁, S and G₂/M phase traverse times were 7, 9 and 4 h, respectively in the exponential growth phase.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

Synchronization of the Human Promyelocytic Cell Line HL 60 By Thymidine

Cultures of the promyelocytic cell line HL 60 were synchronized with thymidine. A concentration of 0.05 mM thymidine and an exposure time of 24 hr was found optimal for blocking about 90% of the cells in S phase. Following release from the thymidine block the cell cultures were followed intermittently over 40 hr for fluctuation in cell numbers, labelling with radioactive thymidine and nuclear DNA distributions. Mathematical evaluation of the results revealed a cycling time of 18.6 hr and a duration of specific cell phases of 8.6 hr, 7.1 hr and 2.9 hr for G₁, S and G₂+ M, respectively. the doubling time was 26 hr and the growth fraction was estimated as 1.     

Variation In G1 Transit Time Relative to the Cycloheximide and Actinomycin D Drug Restriction Points

The transit time distribution at various points in the cell cycle of synchronized Chinese hamster ovary cells was determined from the mitotic index, [³H]thymidine labeling index and increase in cell number monitored at regular intervals after mitotic selection. Variation in G₁ transit time compared with that for the total cell cycle indicates that variation in cell cycle transit time occurs mainly during G₁ phase. the cycloheximide (5.0 μg/ml) and actinomycin D (3.0 μg/ml) restriction points occur 0.2 and 1.7 hr prior to entry into S phase, respectively. the transit time distributions are further characterized by the moments of the distributions. the variance (2nd moment about the mean) of the transit time distribution at the actinomycin D restriction point is similar to the variance of the transit time distribution at the G₁/S border, thus variation in cell cycle transit time originates earlier than 1.7 hr prior to entry into S phase (i.e., the first 3/4 of G₁). If G₁ transit time variability and cell cycle control are related, then the results presented here indicate that the major regulatory events do not occur during late G₁ phase.     

Cell elongation and division probability during the Escherichia coli growth cycle.

A new method is presented for determining the growth rate and the probability of cell division (separation) during the cell cycle, using size distributions of cell populations grown under steady-state conditions. The method utilizes the cell life-length distribution, i.e., the probability that a cell will have any specific size during its life history. This method was used to analyze cell length distributions of six cultures of Escherichia coli, for which doubling times varied from 19 to 125 min. The results for each culture are in good agreement with a single model of growth and division kinetics: exponential elongation of cells during growth phase of the cycle, and normal distributions of length at birth and at division. The average value of the coefficient of variation was 13.5% for all strains and growth rates. These results, based upon 5,955 observations, support and extend earlier proposals that growth and division patterns of E. coli are similar at all growth rates and, in addition, identify the general growth pattern of these cells to be exponential.     

Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations.

A computer simulation routine has been made to calculate the DNA distributions of exponentially growing cultures of Escherichia coli. Calculations were based on a previously published model (S. Cooper and C.E. Helmstetter, J. Mol. Biol. 31:519-540, 1968). Simulated distributions were compared with experimental DNA distributions (histograms) recorded by flow cytometry. Cell cycle parameters were determined by varying the parameters to find the best fit of theoretical to experimental histograms. A culture of E. coli B/r A with a doubling time of 27 min was found to have a DNA replication period (C) of 43 min and an average postreplication period (D) of 22 to 23 min. Similar cell cycle parameters were found for a 60-min B/r A culture. Initiations of DNA replication at multiple origins in one and the same cell were shown to be essentially synchronous. A slowly growing B/r A culture (doubling time, 5.5 h) had an average prereplication period (B) of 2.3 h; C = 2.4 h and D = 0.8 h. It was concluded the the C period has a constant duration of 43 min (at 37 degrees C) at fast growth rates (doubling times, less than 1 h) but increases at slow growth rates. Thus, our results obtained with unperturbed exponential cultures in steady state support the model of Cooper and Helmstetter which was based on data obtained with synchronized cells.     

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.     

Regulation of NIH-3T3 cell G1 phase transit by serum during exponential growth

The proliferation rate of mammalian cells is regulated normally in the G₁ phase of the cell cycle. During this phase, it is convenient to assign positive and negative roles to the molecular programs that regulate the duration of G₁ and the phase transition from G₁ to S phase. Density-dependent inhibition of cellular proliferation results in an increase in the duration of G₁. This form of regulation is due to both secreted factors and cell—cell contact. Serum is mitogenic to a variety of mammalian cell types. Because quiescent cells enter S phase as a result of serum addition to culture media, serum is usually regarded as a source of positive regulatory growth factors. We have measured the length of the G₁, S and G₂+ M phases of NIH 3T3 cells during exponential growth as a function of cell density and serum concentration. The G₁ length increases during exponential growth as a function of density while S and G₂+ M are relatively constant. Further, this increase in G₁ phase time, or density mediated negative regulation, is inhibited by increasing serum concentration. This phenotype is saturable between 10% to 20% serum. Serum concentrations above 2.5% are able to increase the rate of cell cycling (decrease the G₁ phase time) by inhibiting density dependent negative regulation of NIH 3T3.     

A model of tumor growth based on cell cycle kinetics

This work describes a mathematical model of growth based on the kinetics of the cell cycle. A traditional model of the cell cycle has been used, with the addition of a resting (G₀) state from which cells could reenter the reproductive cycle. The model assumes that a growth regulatory substance regulates the transition of cells to and from the resting state. Other transitions between the phases of the cycle were modeled as a first order process. Cell loss is an important feature of growth kinetics, and has been represented by a general but tractable mathematical form. The resulting model forms a system of ordinary nonlinear differential equations. Analytic methods are employed first in the study of this system. Simplifying assumptions regarding cell loss give rise to special cases for which equilibrium solutions can be found. One special case, which assumes first order loss from all cell cycle phases at equal rates, is presented here. For small time values, approximations corresponding to exponential growth were developed. The equations describing an intrinsic growth rate were derived. Simulation methods were used to further characterize the behavior of this model. Parameter values were chosen based on animal tumor cell cycle kinetic data, resulting in a set of 45 model simulations. Several tumor treatment protocols were simulated which illustrated the importance of the intrinsic growth rate and cell loss concepts. Although the qualitative behavior regarding absolute and relative growth is reasonable, this model awaits data for model fitting, parameter estimation, or revision of the equations.     

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures

Cell cycle kinetics of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G₀) of mammalian cells is characterized by a 2c-DNA content whereas G₀-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2′-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.     

STUDIES ON THE DIVISION CYCLE OF MAMMALIAN CELLS : V. Modifications of Time Parameters by Different Steady-State Culture Conditions

In cultures of murine neoplastic mast cells, the duration of different phases of the division cycle (G₁, S, G₂, and mitosis [M]) was determined under optimal and several well-defined suboptimal growth conditions. Two methods of evaluation were applied to the same culture system: first, the relative number of G₁, S, G₂, and M cells was determined by pulse labeling of samples with thymidine-³H and subsequent radioautography in conjunction with a microfluorometric technique permitting rapid measurements of cellular DNA content; second, after pulse labeling with thymidine-³H, the variations with time of the mitotic labeling index were analyzed. Suboptimal culture conditions were obtained by reducing the concentration of single essential medium components (leucine, glucose, or serum) or by the addition of specific metabolic inhibitors (actinomycin D, amethopterin). Growth-limiting culture conditions resulted in increased generation times. Even under control conditions, the cell number doubling time exceeded the generation time, and this difference was more pronounced in suboptimal media. Under most of the suboptimal conditions tested, the increase in generation time was attributable primarily to an extended duration of the G₁ phase. Under certain growth-limiting conditions, however, other phases were also prolonged. In addition, the variabilities of the generation time and of certain cell cycle phases were increased under suboptimal culture conditions. Results obtained by the two methods of evaluation were, in general, in good agreement with each other. Some differences were, however, observed and interpreted in terms of cell death and/or asymmetric frequency distributions of cell cycle parameters.     

Cultured Human Tumour Cells May Be Arrested In All Stages of the Cycle During Stationary Phase: Demonstration of Quiescent Cells In G1, S and G2 Phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9–18%) were located in G₂, phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). to substantiate these findings. cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located ‘somewhere’ in G₁, phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Q_s, and Q_G). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following ‘recruiting’ or ‘synchronizing’ clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells

Human NHIK 3025 cells growing exponentially in 30% or 3% serum had population doubling times of 19.1 and 27.6 hours, respectively. These values were equal to the calculated protein doubling times (17.6 and 26.5 hours, respectively), showing that the cells were in balanced growth at both serum concentrations. Stepdown from 30% to 3% serum reduced the rate of protein synthesis within 1–2 hours, from 5.7% hour to 4.3% hour, while the rate of protein degradation was unchanged (1.7%/hour). In cells synchronized by mitotic selection from an exponentially growing population, the median cell cycle durations in 30% and 3% serum were 17.2 and 23.6 hours, respectively, which were also in good agreement with the protein doubling times. The median G₁ durations were 7.1 and 9.6 hours, respectively. Thus the duration of G₁ relative to the total cell cycle duration was the same in the two cases. Complete removal of serum for a period of 3 hours resulted in a 3-hour prolongation of the cell cycle regardless of the time after mitotic selection at which the serum was removed. For synchronized cells, the rate of entry into both the S phase and into the subsequent cell cycle were reduced in 3% serum as compared to 30% serum, the former rate being significantly greater than the latter at both serum concentrations. Our results thus indicate that these cells are continuously dependent upon serum throughout the entire cell cycle.     

ANALYSIS OF THE PROLIFERATION KINETICS OF BURKITT'S LYMPHOMA CELLS

The in vitro proliferation kinetics of a cell line derived from a patient with American Burkitt's lymphoma were investigated at three different growth phases: lag (day 1), exponential (day 3) and plateau (day 5). The growth curve, labeling and mitotic indices, percentage labeled mitosis (PLM) curves and DNA content distributions were determined. The data obtained have been analysed by the previously developed discrete-time kinetic (DTK) model by which a time course of DNA distributions during a 10-day growth period was characterized in terms of other cell kinetic parameters. The mean cell cycle times, initially estimated from PLM curves on days 1, 3 and 5, were further analysed by the DTK model of DNA distributions and subsequently the mean cell cycle times with respect to DNA distributions during the entire growth period were determined. The doubling times were 39·6, 31·2 and 67·2 hr, respectively, at days 1, 3 and 5. The mean cell cycle time increased from 23·0 to 37·7 hr from day 3 to day 5 mainly due to an elongation of the G₁ and G₂ phases. A slight increase in the cell loss rate from 0·0077 to 0·0081 fraction/hr was accompanied by a decrease in the cell production rate from 0·0299 to 0·0184 fraction/hr. This calculated cell loss rate correlated significantly with the number of dead cells determined by trypan blue exclusion. Analysis of the number of dead cells in relation to the cell cycle stage revealed that a majority of cell death occurred in G₁ (r= 0·908; P < 0·0001). There was a good correlation between the in vitro proliferation kinetics at plateau phase of this Burkitt's lymphoma derived cell line and the in vivo proliferation kinetics of African Burkitt's lymphoma (Iversen et al., 1974), suggesting the potential utility of information obtained by in vitro kinetic studies.     

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.     

Investigation into the experimental kinetic support of the two-state model of the cell cycle

Five previously published cell generation-time distribution functions have been examined in an effort to elucidate the parameters of the two-state model of the cell cycle. These parameters are the fractional number of cells that bypass the G₀ state, the probability of exit from G₀ and the distribution of traversal times through the active state. To explain observed β-curve behavior of cell populations, it is necessary to define the parameters in terms of pairwise behavior of newborn sister cells. From the β-curve, we demonstrate that at least 50% of the cells must pass through the G₀ state. The α-curve is consistent with any positive fraction of newborn cells passing through the G₀ state, and provides no further information. We explore a possible method for resolving the remaining indeterminacy regarding the number of cells bypassing the G₀ state, namely, examination of the generation-time distribution functions of fast sister cells only. Such an approach, although theoretically attractive, presents formidable experimental difficulties, however. If it should turn out that indeed only 50% of the cells are apparently passing through a randomexiting phase of the cell cycle, then an alternative plausible biological mechanism for the observed variability in generation times is supplied by Prescott's hypothesis: variability is a consequence of the inequality in the metabolic content of sister cells at birth.     

Diverse expression of G-proteins in human sarcoma cell lines with different osteogenic potential: Evidence for the involvement of Gi2 in cell proliferation

Previously, it has been shown that the GTP-binding protein G_i2 is implicated in cellular growth [1,2] and differentiation [2,3]. In the present paper we demonstrate that this is also the case for human sarcoma cells. Six human osteosarcoma and three soft tissue sarcoma clonal cell lines were analyzed for levels of G-protein mRNA and polypeptide expression and effector enzyme (i.e., adenylate cyclase and phospholipase C) activation, which were all compared with individual growth rates. Unexpectedly, it appeared that the various strains exhibited large inter-individual variations in G-protein expression and signaling system activation. However, cell doubling time in the exponential phase of growth was inversely correlated (r = 0.71, P < 0.05) to immunodetected levels of intrinsic G_i2α. Furthermore, cells stably transfected with a retroviral (pZipNeo(SV)X) construct containing the activating or inactivating G_i2α-R179E or G_i2α-G204A point mutations consistently reduced or enhanced individual cell strain doubling time, respectively. It appeared that other parameters investigated, including cellular alkaline phosphatase and monoclonal antibody epitope binding, both being markers of the proliferating osteoblast, did not correlate with cell doubling times. © 1996 Wiley-Liss, Inc.