Duration Of Cell Cycle Phases Research Articles

Identifying mechanisms of chronotolerance and chronoefficacy for the anticancer drugs 5-fluorouracil and oxaliplatin by computational modeling

We use an automaton model for the cell cycle to assess the toxicity of various circadian patterns of anticancer drug delivery so as to enhance the efficiency of cancer chronotherapy. Based on the sequential transitions between the successive phases G1, S (DNA replication), G2, and M (mitosis) of the cell cycle, the model allows us to simulate the distribution of cell cycle phases as well as entrainment by the circadian clock. We use the model to evaluate circadian patterns of administration of two anticancer drugs, 5-fluorouracil (5-FU) and oxaliplatin (l-OHP). We first consider the case of 5-FU, which exerts its cytotoxic effects on cells in S phase. We compare various circadian patterns of drug administration differing by the time of maximum drug delivery. The model explains why minimum cytotoxicity is obtained when the time of peak delivery is close to 4 a.m., which temporal pattern of drug administration is used clinically for 5-FU. We also determine how cytotoxicity is affected by the variability in duration of cell cycle phases and by cell cycle length in the presence or absence of entrainment by the circadian clock. The results indicate that the same temporal pattern of drug administration can have minimum cytotoxicity toward one cell population, e.g. of normal cells, and at the same time can display high cytotoxicity toward a second cell population, e.g. of tumour cells. Thus the model allows us to uncover factors that may contribute to improve simultaneously chronotolerance and chronoefficacy of anticancer drugs. We next consider the case of oxaliplatin, which, in contrast to 5-FU, kills cells in different phases of the cell cycle. We incorporate into the model the pharmacokinetics of plasma thiols and intracellular glutathione, which interfere with the action of the drug by forming with it inactive complexes. The model shows how circadian changes in l-OHP cytotoxicity may arise from circadian variations in the levels of plasma thiols and glutathione. Corroborating experimental and clinical results, the simulations of the model account for the observation that the temporal profiles minimizing l-OHP cytotoxicity are in antiphase with those minimizing cytotoxicity for 5-FU.

Implications of circadian clocks for the rhythmic delivery of cancer therapeutics

The circadian timing system (CTS) controls drug metabolism and cellular proliferation over the 24 hour day through molecular clocks in each cell. These cellular clocks are coordinated by a hypothalamic pacemaker, the suprachiasmatic nuclei, that generates or controls circadian physiology. The CTS plays a role in cancer processes and their treatments through the downregulation of malignant growth and the generation of large and predictable 24 hour changes in toxicity and efficacy of anti-cancer drugs. The tight interactions between circadian clocks, cell division cycle and pharmacology pathways have supported sinusoidal circadian-based delivery of cancer treatments. Such chronotherapeutics have been mostly implemented in patients with metastatic colorectal cancer, the second most common cause of death from cancer. Stochastic and deterministic models of the interactions between circadian clock, cell cycle and pharmacology confirmed the poor therapeutic value of both constant-rate and wrongly timed chronomodulated infusions. An automaton model for the cell cycle revealed the critical roles of variability in circadian entrainment and cell cycle phase durations in healthy tissues and tumours for the success of properly timed circadian delivery schedules. The models showed that additional therapeutic strategy further sets the constraints for the identification of the most effective chronomodulated schedules.

Preface

Preface

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample

BackgroundPresently available flow cytometric methods of bromodeoxyuridine (BrdUrd) labelling do not provide information on the cell cycle time (TC) and the growth fraction (GF). In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling.MethodsThe proposed method is based on two assumptions: (1) the number of labelled cells traversing the cell cycle per unit time is constant and (2) the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture.ResultsThe suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 ± 1.1 h and 21.6 ± 0.9 h, respectively). GF in tumours at day 10 was lower than GF at day 4 (54.2 ± 7.7% vs. 79.2 ± 5.9%, p = 0.0003). Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively.ConclusionThe proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed method might also prove suitable for measurement of cell kinetics in human tumours. Development of suitable software enabling more objective interpretation of the DNA profile in this method would be desirable.

Analysis of the cell cycle and a method employing synchronized cells for study of protein expression at various stages of the cell cycle.

Study of protein expression during the cell cycle requires preparation of pure fractions of cells at various phases of the cell cycle. This was achieved by the development of methods for cell synchronization. Successful cell synchronization requires knowledge of the duration of all phases of the cell cycle. So, in the present review these interrelated problems are considered together. The first part of this review deals with basic methods employed for analysis of duration of cell cycle phases. The second summarizes data on treatments used for cell synchronization. Methods for calculation of percent of cells at various stages of the cell cycle in fractions of synchronized cells are considered in the third part. The fourth part of this review deals with a method of study of protein expression during the cell cycle by means of immunoblotting of synchronized cell fractions. In the Appendix, basic principles are illustrated with practical examples of analysis of the cell cycle, synchronization, and study of expression of some proteins at various stages of the cell cycle using synchronized XL2 (Xenopus laevis) cells.

In vivo tumor growth and response to radiation therapy: a novel algorithmic description

The imaging (e.g. CT/MRI slices including the definition of the tumor contour), the histologic and the genetic data of the patient are appropriately collected. Labels are assigned to the tumor and the anatomical structures of interest. The output of this procedure is introduced into the 3-D visualization package AVS-Express, which performs the visualization of the region of interest. Subsequently, a 3-D discretizing mesh is superimposed on the same region. Each geometrical cell of the mesh belonging to the tumor contains a number of biological cells “ residing”in various phases within or out of the cell cycle. Within each geometrical cell, a number of classes of biological cells, each one characterized by the phase in which its cells reside at any given instant, are de fined. Sufficient registers are used in order to characterize the state of each geometrical cell and each phase class within it (e.g. the number of biological cells in phase G1). The number of biological cells constituting each phase class is initially estimated according to the position of the geometrical cell within the tumor, the expected vasculature of the geometrical cell and consequently the oxygen and nutrient supply, the metabolic activity in the local area (e.g.based on PET/functional MRI) etc. A simplifying assumption dictates that each geometrical cell of the mesh can ’ normally’accommodate a constant Number of Biological Cells (NBC). In case that the actual number of living and dead (but still morphologically existing) tumor cells contained within a given geometrical cell is reduced to less than NBC/2, the entire geometrical cell is assumed to either disappear from the tumor or to constitute part of its inner necrosed region. As a consequence, the whole tumor shrinks. Tumor expansion is simulated in the opposite way. The geometrical mesh covering the anatomic area of interest is scanned every T units of time. For each phase class of a given geometrical cell, behavior algorithms based on the cell cycle phase durations of the tumor cells, the estimated local blood supply, the radiation survival curves of the tumor cells, the genetic data of the tumor (e.g. wild or mutated p53, bcl-2 genes) etc. determine the updated state. For simplification purposes all biological cells constituting each phase class within the same geometrical cell are assumed to be synchronized. An important aspect of the software is the virtual reality visualization of the anatomical region of interest, before, during and after the treatment course. Such a tool would enhance the doctor’ s spatio-temporal perception of the tumor behavior. The three dimensional progress of a tumor is simulated, initially without the application of any therapeutic schemes. In the case of radiotherapy and at the end of the physical treatment planning procedure, the distribution of the absorbed dose (e.g. in Gy) in the region of interest is provided. This distribution is used in order to “ predict”the most likely spatio-temporal response of the tumor. At the same time, the response of the adjacent irradiated normal tissues is simulated. The visualization procedure described in the beginning of this abstract is followed here too. The software system under development is currently undergoing an extensive validation and optimization procedure based on pertinent clinical data concerning e.g. astrocytoma irradiation.

Analysis of cell division parameters and cell cycle gene expression during the cultivation of Arabidopsis thaliana cell suspensions

Arabidopsis thaliana cell suspension cultures were characterized for the first time in detail in terms of biomass accumulation, cell division rate and cell cycle phase durations. Subsequently, this model system was used to follow the transcription profile of key cell cycle genes during a complete cultivation cycle. According to the calculated changes in the relative division rate over time, the cell cycle genes could be classified into four groups based on their transcriptional expression pattern. These differential patterns of gene expression are discussed with respect to the putative roles of the different cell cycle genes in the division cycle. Analysis of protein levels showed that mRNA levels did not correlate with protein levels in all cases. Results obtained in other systems, such as BY-2 cell suspensions or plants, confirm that cell suspension cultures of A. thaliana are suitable for the analysis of cell cycle regulation.

Cell cycle phase duration in bone marrow cells from malnourished rats during suckling

The generation time and duration of the different phases of the cell cycle were estimated in cultured bone marrow cells from malnourished and well-nourished rats during the lactation period by the percentage labeled mitosis (PLM) technique after a short pulse treatment with tritiated thymidine. The PLM were obtained in sequential analysis after every 3 h from removing labeled thymidine to 27 h of incubation. Results have shown a longer cell cycle time in cells from malnourished rats. The G 1 + 1/2M phases are sensitive to experimental severe protein malnutrition (PCM), while S and G 2 + 1/2M phases did not show any differences in duration between malnourished and well-nourished rats.

An improved method of cell cycle synchronisation for the heterotrophic dinoflagellate Crypthecodinium cohnii Biecheler analyzed by flow cytometry

The selective attachment method for synchronising populations of Crypthecodinium cohnii Biecheler has been investigated with the addition of a filtration step. Appropiate filter meshes can yield populations enriched with cells at a similar point in the G1 phase of the cell cycle, the degree of synchrony being 90–93% G1 cells. Flow cytometric analysis of DNA content allowed the cell cycle progression of synchronised cells to be followed after the synchronisation event. Results indicate that the additional filtration step did not physiologically affect the cells and the calculated cell-cycle phase duration results are consistent with other published data. The technique has been demonstrated on a scale which would be suitable to provide the large samples required for molecular biology studies such as cell-cycle control.

838 S phase duration in relation to S phase fraction as simulated by a computerized model

For several tumour types it has been found that there is a correlation between clinical outcome and the relative number of tumour cells in S phase as measured with DNA flow cytometry (FCM). From data showing that a high S phase fraction is associated with poor prognosis it has been concluded that these tumours have a high proliferative activity and therefore grow faster. Moreover, the size of the S phase fraction has also been used as an equivalent of labelling index to determine tumour growth fraction in experimental cell kinetic studies. In the present study the relationship between DNA distribution and the duration ofcell cycle phases in tumours was investigated with the aid ofa computerized mathematical model. In our study we found poor correlation between the size of the S phase fraction and the proliferative activity when tumour growth fraction was taken into account. A shortening of the duration of the S phase ie, increased cell production per time unit, led to a decrease in the relative number of S phase cells as measured by FCM.

RB phosphorylation in sodium butyrate-resistant HL-60 cells: cross-resistance to retinoic acid but not vitamin D3.

To examine the potential coupling between inducible cellular changes in RB (retinoblastoma) tumor suppressor protein phosphorylation and ability to G0 growth arrest and differentiate, HL-60 promyelocytic leukemia cells were cultured in incremental sodium butyrate (NaB) concentrations and thereby made resistant to the growth inhibitory effects of sodium butyrate, which normally induces G0 arrest and monocytic differentiation in wild type HL-60 cells. The resistant cells were also unable to differentiate in response to NaB, indicating that a regulatory function controlling both G0 growth arrest and differentiation had been affected. The induced resistance was not genetic in origin since the cells regained the ability to G0 arrest and differentiate after being recultured in medium free of sodium butyrate for only three days. The resistant cells had similar cell cycle phase durations as the original wild type cells. The resistant cells retained the ability to both G0 arrest and differentiate in response to 1,25-dihydroxy vitamin D3 (VD3), normally an inducer of G0 arrest and monocytic differentiation in wild type cells. However, they were cross-resistant to retinoic acid (RA), another ligand for the same steroid thyroid hormone receptor family, which induces G0 arrest and myeloid differentiation in wild type cells. The ability to G0 arrest and phenotypically differentiate in response to RA were both grossly impaired. Unlike wild type cells which undergo early down-regulation and then hypophosphorylation of the RB protein when induced to differentiate, in resistant cells, hypophosphorylation of RB in response to NaB was grossly retarded. These changes in RB protein occurred faster when the cells were treated with VD3. In contrast, the changes in RB phosphorylation occurred significantly slower when the cells were treated with RA. The results suggest a coupling between the ability to G0 growth arrest and phenotypically convert and the ability to hypophosphorylate RB.

The Cell Cycle of Euplotes aediculatus (Ciliophora) at Various Population Densities

The population density in cultures of the ciliate Euplotes aediculatus directly influences the rate of asexual population growth. At continuous exchange of medium and continuous supply of food the cultures grow to a density of 2100 cells/ml ± 100 (standard deviation) that is retained at further cultivation. The mortality rate is not substantially increased at this steady population density, instead the cell cycle time is lengthened from 37 hours to about 500 hours. Changes of the duration of individual cell cycle phases with changing reproduction rates were studied by light microscopy of the morphology of the cells and their macronuclei. The M phase had a duration of 1.9 ± 0.1 hours, independent of the population density. The G 1 phase of the macronucleus increased in relative proportion of the cell cycle from 34% at a low population density (below 100 cells/ml) to approximately 70% at a high population density (above 1500 cells/ml). It increased in length from 13 hours to approximately 350 hours. The S-phase of the macronucleus was shortened in relative proportion from 61% at a low population density to approximately 30% at a high population density. Thus the composition of the cell cycle changed with increasing population density to a prolonged G 1 phase. The absolute duration of the macronucleus S phase increased significantly (from 23 to approximately 150 hours) with increasing population density in contrast to its shortened relative proportion of the cell cycle. Accordingly the S-phase is regulated after its start in E. aediculatus just as the G 1 phase. Apparently at high population density most of the cells of the populations reside in G 1 , possibly they enter a G 0 state. The observations imply a direct regulation of the cell cycle by the population density in this unicellular organism.

Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo.

Skeletal growth depends upon enchondral ossification in growth plate cartilage, within which chondrocytes undergo well defined stages of maturation. We infused IGF-I or growth hormone (GH), two key regulators of skeletal growth, into hypophysectomized rats and compared their effects on growth plate chondrocyte differentiation using qualitative and quantitative autoradiography, stereology, and incident light fluorescence microscopy. Stem cell cycle time was shortened from 50 to 15 and 8 d after treatment with IGF-I and GH, respectively. Proliferating cell cycle time decreased from 11 to 4.5 and 3 d, and duration of the hypertrophic phase decreased from 6 to 4 and 2.8 d. Average matrix volume per cell at each differentiation stage was similar for normal, hormone-treated, and untreated hypophysectomized groups. Mean cell volume and cell height were significantly reduced by hypophysectomy at the proliferative and hypertrophic stages, but were restored to physiological values by IGF-I and GH. In contrast, cell productivity, i.e., increases in cell volume, height, and matrix production per unit of time, did not reach normal values with either IGF-I or GH, and this parameter was inversely proportional to cell cycle time or phase duration. IGF-I and GH are thus capable of stimulating growth plate chondrocytes at all stages of differentiation, albeit to variable degrees with respect to individual cell activities. Although it is generally accepted that GH acts at both the stem and proliferating phases of chondrocyte differentiation, our data represent the first evidence in vivo that IGF-I is also capable of stimulating stem cells.

Flow cytometric study of the type II pneumocyte cell cycle in vivo and in vitro.

The type II pneumocyte cell cycle was studied in vivo and in vitro through bivariate flow cytometric analysis of DNA content vs. incorporated 5-bromo-2-deoxyuridine (BrdUrd). The cell cycle phase durations Ts (7.8 h) and TG2/M (1.1 h), measured in vivo, agreed well with earlier estimates obtained by thymidine labeling. Left unilateral pneumonectomy increased the BrdUrd labeling index of type II cells in the remaining lung from an initial value of 1.9 +/- 0.3% to 4.8 +/- 1.0%, but had no effect on Ts or TG2/M in vivo. In both normal and pneumonectomized animals, BrdUrd-positive cells in vivo rapidly completed mitosis but did not enter a second S-phase. These results demonstrate that proliferating type II cells do not form a continuously cycling population in the normal or regenerating adult lung. When cell cycle parameters were measured in vitro immediately after type II cell isolation, Ts increased 2-fold and TG2/M rose up to 10-fold above the value obtained in vivo. After 2 d of primary culture under customary plating conditions, Ts had returned to the same value as that in vivo, but TG2/M remained elevated. Little variability was observed in the duration of S-phase within each treatment group. In contrast, type II cells exhibited considerable heterogeneity in the rate of G2/M-phase traverse, especially in vitro. These data suggest that the inability of adult rat type II pneumocytes to proliferate in primary culture is related to delayed G2/M-phase transit and imply the existence of pneumocyte subpopulations which differ in susceptibility to growth arrest.

Chromosomal damage in preimplantation mouse embryos and its development through the cell cycle

Cytogenetic damage is usually studied in the first metaphase after exposure to ionizing radiation although it is well known that further chromosomal abberations are also expressed during later mitotic divisions (Bauchinger et al., 1986; Lloyd et al., 1992). For such investigations the preimplantation mouse embryo is very well suited. If irradiation takes place in the 1-cell stage it can easily be followed whether the cells of the embryo have reached the first, second or third mitosis after the exposure. Furthermore, the duration of the various cell cycle phases is well known and therefore radiation effects on the cell cycle, e.g., the G 2 block, can be investigated (Streffer and Molls, 1987). Studies on these two phenomena will be reported here.

Cell cycle kinetics in malignant lymphoma studied with in vivo iododeoxyuridine administration, nuclear Ki-67 staining, and flow cytometry

Cell cycle kinetics of malignant lymphoma were investigated using in vivo labeling with iododeoxyuridine (IdUrd) and subsequent flow cytometry (FCM) of IdUrd/DNA and Ki-67/DNA. This approach provides an extensive cell kinetic profile from only one single tumor biopsy, including data upon the percentage of S-phase cells, the IdUrd labeling index (LI), Ki-67-derived growth fraction, duration of the S-phase, duration of the G1-phase, potential doubling time, cell production rate, and total cell cycle time. Tissue samples from 33 patients were studied: non-Hodgkin's lymphoma (NHL; n = 22), Hodgkin's disease (HD; n = 7), and reactive hyperplasia (n = 4). In NHL, the percentage of S- phase cells, LI, growth fraction, duration of the S-phase, and cell production rate were significantly correlated with the histologic malignancy grade according to the Working Formulation (P < or = .02). Data found in HD were not essentially different from those in low-grade NHL and reactive hyperplasia. Remarkably, the duration of the S-phase, the duration of the G1-phase, and the total cell cycle time appeared to be rather independent of histologic malignancy grade within the NHL category. A significant correlation was observed between the IdUrd LI and the percentage of S-phase cells, the growth fraction, the potential doubling time, and the cell production rate (P < .001), but not with the duration of the separate cell cycle phases (P > .05). Our data show (1) that it is feasible to obtain detailed information on the in vivo growth characteristics of malignant lymphoma; and (2) that the transition time through the different cell cycle phases widely varies, even within distinct histologic subgroups.

Cell Cycle Kinetics in Malignant Lymphoma Studied With In Vivo Iododeoxyuridine Administration, Nuclear Ki-67 Staining, and Flow Cytometry

Cell cycle kinetics of malignant lymphoma were investigated using in vivo labeling with iododeoxyuridine (IdUrd) and subsequent flow cytometry (FCM) of IdUrd/DNA and Ki-67/DNA. This approach provides an extensive cell kinetic profile from only one single tumor biopsy, including data upon the percentage of S-phase cells, the IdUrd labeling index (LI), Ki-67-derived growth fraction, duration of the S-phase, duration of the G1-phase, potential doubling time, cell production rate, and total cell cycle time. Tissue samples from 33 patients were studied: non-Hodgkin's lymphoma (NHL; n = 22), Hodgkin's disease (HD; n = 7), and reactive hyperplasia (n = 4). In NHL, the percentage of S-phase cells, LI, growth fraction, duration of the S-phase, and cell production rate were significantly correlated with the histologic malignancy grade according to the Working Formulation (P < or = .02). Data found in HD were not essentially different from those in low-grade NHL and reactive hyperplasia. Remarkably, the duration of the S-phase, the duration of the G1-phase, and the total cell cycle time appeared to be rather independent of histologic malignancy grade within the NHL category. A significant correlation was observed between the IdUrd LI and the percentage of S-phase cells, the growth fraction, the potential doubling time, and the cell production rate (P < .001), but not with the duration of the separate cell cycle phases (P > .05). Our data show (1) that it is feasible to obtain detailed information on the in vivo growth characteristics of malignant lymphoma; and (2) that the transition time through the different cell cycle phases widely varies, even within distinct histologic subgroups.

Simian virus 40 large T-antigen expression decreases the G1 and increases the G2 + M cell cycle phase durations in exponentially growing cells.

The effect of simian virus 40 large T-antigen (Tag) expression on the cell cycle of exponentially growing, established, mouse NIH 3T3 fibroblasts was examined by using a sensitive flow cytometric assay to analyze nonselected cells immediately after infection with a Tag-encoding recombinant retrovirus. Tag expression resulted in reduced percentages of G1-phase cells and increased percentages of S- and G2 + M-phase cells compared with cell populations infected with a control virus not encoding the Tag gene. Cell cycle-blocking drugs were used to examine the exit rate for each of the cell cycle phases, G1, S, and G2 + M, for Tag-expressing and Tag-nonexpressing cells growing in the same cell culture dish. As a result of Tag expression, the duration of the G1 phase was decreased (average G1-phase exit duration decreased by 18%) and the duration of the G2 + M phase was increased (average G2 + M exit duration increased by 29%). The duration of S phase was unaffected by Tag expression.

Cell cycle simulation for flow cytometry

A program has been implemented on a VAX computer that simulates the progression of cells through the cell cycle and generates data similar to those obtained in flow cytometry with different techniques. Features of the program are general applicability and flexibility, options including consideration of (a) mean duration of cell cycle phases, (b) their inter-cell distribution, (c) a first order commitment from G0 into G1 phase, and (d) a total or partial block of the output from any phase. Examples are given of simulated flow cytometric experiments of drug-induced cell cycle perturbation and of bromodeoxyuridine pulse labeling. This program should help to acquire a correct understanding of the relationship between kinetic features and flow cytometric data.

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population.

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.