Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells

Most cell lines that lack functional p53 protein are arrested in the G₂ phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G₂ phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G₂ phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D₀ value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G₂ phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D₀=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5–7post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.     

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines

Abstract.   Objectives : This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G₂/M phases and consecutive cell survival. Materials and methods : Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. Results : By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G₂ phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G₂ accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly ( P <  0.01) increased the fraction of radiation-induced G₂ arrested cells 30–56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by γ-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. Conclusions : These results indicate that separase has additional targets involved in regulation of G₂ to M progression after DNA damage. Prolonged G₂ phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.     

Additive effects of radiation and docetaxel on murine SCCVII tumors in vivo: special reference to changes in the cell cycle

The purpose of the present study was to investigate the effects of a combination of docetaxel and irradiation in vivo with special reference to docetaxel-arrested G(2)/M-phase cells. At 24 and 48 h after intraperitoneal administration of docetaxel (90 mg/kg), tumor-bearing mice were irradiated with (60)Co gamma rays. Cell cycle distribution was analyzed by a DNA-Ki-67 double staining method using flow cytometry. An accumulation of cells in the G(2)/M phase of up to approximately 40% was observed 24 h after administration of docetaxel. Between 24 and 72 h, the percentage of cells arrested in G(2)/M phase that expressed Ki-67 decreased from 37.2% to 13.8%, in accordance with the increase in the Ki-67-negative G(2)/M-phase fraction. More than half of the cells arrested in G(2)/M phase lost their expression of Ki-67 protein between 24 and 72 h. The G(1)-phase fraction decreased from 28.4% to 8.6% at 24 h after docetaxel treatment; this remained unchanged at 72 h. These flow cytometry data suggested that docetaxel-arrested G(2)/M-phase cells did not enter the next cell cycle and were killed by docetaxel alone. Our data showed that arrest of cells in G(2)/M phase does not contribute to the synergism that has been reported for combinations of docetaxel and radiation in in vivo tumor models.     

DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with ³H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G₂ + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G₂ phases. These observations provide evidence against the existence of a specific “restriction point” in G₁ or at the G₁/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G₂ phases retain the capacity to complete the cell cycle in more favorable culture environments.     

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.     

Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives

The mevalonate pathway is tightly linked to cell proliferation. The aim of the present study is to determine the relationship between the inhibition of this pathway by lovastatin and the cell cycle. HL-60 and MOLT-4 human cell lines were cultured in a cholesterol-free medium and treated with increasing concentrations of lovastatin, and their effects on cell proliferation and the cell cycle were analyzed. Lovastatin was much more efficient in inhibiting cholesterol biosynthesis than protein prenylation. As a result of this, lovastatin blocked cell proliferation at any concentration used, but its effects on cell cycle distribution varied. At relatively low lovastatin concentrations (less than 10 microM), cells accumulated preferentially in G(2) phase, an effect which was both prevented and reversed by low-density lipoprotein cholesterol. At higher concentrations (50 microM), the cell cycle was also arrested at G(1) phase. In cells treated with lovastatin, those arrested at G(1) progressed through S upon mevalonate provision, whereas cholesterol supply allowed cells arrested at G(2) to traverse M phase. These results demonstrate the distinct roles of mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, both being required for normal cell cycling.     

Morphological adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus

During exponential growth, each cell cycle of the α-purple bacterium Caulobacter crescentus gives rise to two different cell types: a motile swarmer cell and a sessile stalked cell. When cultures of C. crescentus are grown for extended periods in complex (PYE) medium, cells undergo dramatic morphological changes and display increased resistance to stress. After cultures enter stationary phase, most cells are arrested at the predivisional stage. For the first 6–8 days after inoculation, the colony-forming units (cfu) steadily decrease from 10⁹ cfu ml⁻¹ to a minimum of 3 × 10⁷ cfu ml⁻¹ after which cells gradually adopt an elongated helical morphology. For days 9–12, the cfu of the culture increase and stabilize around 2 × 10⁸ cfu ml⁻¹. The viable cells have an elongated helical morphology with no constrictions and an average length of 20 μm, which is 15–20 times longer than exponentially growing cells. The level of the cell division initiation protein FtsZ decreases during the first week in stationary phase and remains at a low constant level consistent with the lack of cell division. When resuspended in fresh medium, the elongated cells return to normal size and morphology within 12 h. Cells that have returned from stationary phase proceed through the same developmental changes when they are again grown for an extended period and have not acquired a heritable growth advantage in stationary phase (GASP) compared with overnight cultures. We conclude that the changes observed in prolonged cultures are the result of entry into a new developmental pathway and are not due to mutation.     

Suppression of growth of highly-metastatic human breast cancer cells by norcantharidin and its mechanisms of action

The effects of norcantharidin (NCTD) on the growth of highly-metastatic human breast cancer cells were investigated by in vitro and ex vivo assays. Our results indicated that norcantharidin inhibited the in vitro growth of human breast cancer MDA-MB-231 cell line in dose- and time-dependent manners after the cancer cells were treated with norcantharidin at the concentrations of 6, 30 and 60 μmol/L for 24, 48 and 72 h. Moreover, the sera from the NCTD-treated rabbits after intravenous injection of NCTD at 15 and 30 min significantly suppressed the growth of the cancer cells ex vivo. The analyses by Hoechst 33258 staining and flow cytometry showed that the typical apoptotic morphological changes appeared and cell cycles arrested at G2/M phase in MDA-MB-231 cells after the cells were treated for 48 h with NCTD. In addition, NCTD down-regulated the expressions of anti-apoptotic protein Bcl-2 and up-regulated the expressions of pro-apoptotic protein Bax, eventually leading to the reduction of Bcl-2/Bax ratio in MDA-MB-231 cells. Furthermore, NCTD at concentrations of 6, 30 and 60 μmol/L dose-dependently reduced the phosphorylation of Akt and NF-κB expression in the breast cancer cell line. Induction of apoptosis and cell cycle arrest as well as reduction of Bcl-2/Bax ratio by NCTD may be the important mechanisms of action of NCTD suppressing the growth of MDA-MB-231 cells, which are associated with inhibition of the Akt and NF-κB signaling. Our findings suggest that norcantharidin may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of human breast cancer.     

Growth and production modeling in hybridoma continuous cultures

Several experimental data on continuous cultures of hybridoma cells show that monoclonal antibody productivity is a decreasing function of dilution rate. It has been suggested that this unusual behavior may be due to the arrest of a fraction of cycling cells at a critical point of Phase G(1). Although this hypothesis has been recently investigated by using population balance models, mathematical analysis has been performed without accounting for the dynamics of the arrested cells properly. In this article, a more general and accurate approach is presented and new specific assumptions are introduced to characterize the arrest and the later progress through the cycle. Two different models (stochastic and deterministic) and two different critical points for the arrest (at the beginning and at the end of G(1)) are considered. The cell cycle parameters are estimated so that data predicted by the model fit those reported in the literature. In particular, the fraction of arrested cells, the cell arrest probability, and the mean cell generation time are computed as functions of the dilution rate. Results so far obtained predict that there is an optimal value of dilution rate for maximizing specific production rate of monoclonal antibody. (c) 1993 John Wiley & Sons, Inc.     

Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. 3. Electrophoretic patters of newly synthesized proteins in synchronized proliferating cells and resting cells

Electrophoretic patterns of newly synthesized proteins have been compared for hamster embryo fibroblasts in asynchronous cultures, mitotically synchronized cultures, and stationary phase cultures. Only proteins with molecular weight between 30,000 and 150,000, comprising 60–70% of the total cell proteins and excluding histones and collagen were included in the comparison. Although no significant differences could be detected between such patterns for cells at different stages of the cell cycle, significant differences were detected between patterns for cells in stationary phase and for proliferating asynchronous or synchronous cells in any stage of the cell cycle. These differences amounted to at least 5% of the newly synthesized cellular proteins. Much larger differences were detected between patterns from a nuclear fraction of proliferating and resting cells. These results indicate that normal cells in stationary phase are arrested in a state distinct from any phase of the normal cell cycle and may provide a biochemical marker for resting cells.     

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

Terminal cell differentiation usually results in an irreversible arrest in the G1 phase of the cell cycle and loss of cell renewal ability. Human promyelocytic leukemia HL-60 cells induced with 12-o-tetradecanoylphorbol-13-acetate (TPA) differentiate into monocytes/macrophages and accumulate in G1. We determined the effect of TPA on the growth kinetics of a human leukemia cell line (KOPM-28), which developed several of the characteristics of megakaryocytes in response to TPA, such as the surface antigen complex IIb/IIIa, platelet peroxidase and polyploidy. Cell growth was immediately and completely inhibited by TPA. Flow cytometric analysis of cellular DNA content revealed a gradual decrease in cells in G1 and an accumulation of cells in G2. These data suggest that TPA prolonged G1 and rapidly arrested the cells in G2. Synchronized cells were utilized to further analyze the rapid G2 arrest. Cells arrested with aphidicolin at the G1/S interphase were released, and the effects of TPA (added at different intervals) on cell cycle progression were examined 14 h after release. The results showed that TPA added at the end of the S phase, as well as at the G1/S interphase incompletely but distinctly arrested cells in G2. Moreover, G2 arrest was observed when TPA was added to cells released from a colcemid-induced G2/M block, suggesting that cells already in G2 were inhibited by TPA from moving through M to G1. Since some cells became multi-nucleated in the course of incubation with TPA, this G2 accumulation may have resulted at least in part from a prolongation of the phase or a transient G2 block. These changes in cell cycle progression induced by TPA may be characteristic of and/or related to megakaryocytic differentiation of hemopoietic precursor cells.     

Method for probing cells in radiation-induced G2 arrest: demonstration of potentially lethal damage repair

Chinese hamster ovary cells were arrested in the G2 phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G2, for the investigation of G2 arrest. Comparisons were made of the number of cells retrieved from G2 arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. The retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. The repair curves (surviving fraction vs time) were each described by a single exponential. G2 cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

PMS‐1077, a PAF antagonist,induced differentiation of HL‐60 cells with its novel activity

The cell differentiation‐inducing effect of 2‐N,N‐diethylaminocarbonyloxymethyl‐1 ‐diphenylmethyl‐4‐(3,4,5‐trimethoxybenzoyl) piperazine, hydrochloride (PMS‐1077) was determined in human leukaemic HL‐60 cells with profiling of cell proliferation, analysis of cell cycling, characterization of expression of various CD molecules and determination of phagocytotic activity of differentiated HL‐60 cells. After treatment with PMS‐1077, HL‐60 cells exhibited a decreased cell viability during which cell cycle was arrested in G₀‐/G₁‐phase. Flow cytometric analysis showed CD11b and CD14 were up‐regulated, whereas CD15 was unaffected. Together with the finding that PMS‐1077‐treated HL‐60 cells exhibited activities of differentiation by examining their ability of phagocytosing latex beads, an antiproliferative effect and a differentiation‐inducing role were determined for PMS‐1077 in HL‐60 cells.     

Studies of the cell cycle of in vitro cultured skin fibroblasts in goats: work in progress

The effect of serum starvation and olomoucine treatment on the cell cycle and apoptosis of goat skin fibroblasts cultured in vitro is reported in this paper. The cells were obtained from the ear of a female goat 1.5 years of age. Analysis of cell cycle distribution by fluorescence-activated cell sorting (FACS) showed that 3.4, 60.8 and 15.1% of normally cycling cells were at G1, G0 and S phase, respectively. Serum starvation for 1, 3 and 5 days arrested 70.1, 70.2 and 83.4% cells, respectively, at G0/G1 phase. Seventy-eight percent of confluent cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Of cells not deprived of serum, 73.6% were arrested at G1/G0 when treated with 100 microM olomoucine for 9 h compared to 85.5% of cells that had been starved of serum for 2 days (co-inhibition) (P<0.01). After co-inhibition, 45% of cells entered S phase when re-cultured in normal medium for 5 h, indicating that the inhibition was reversible. Under normal culture conditions, 1.2% of cells underwent apoptosis. Serum starvation for 1, 2, 3, 5 and 10 days caused apoptosis in 1.7, 3.9, 4.5, 11.7 and 90.3% of cells, respectively. Treatment with 100 microM olomoucine for 9h did not increase the number of apoptotic cells significantly (1.9%, P>0.05). When cells were co-inhibited, 4.1% of cells underwent apoptosis. In conclusion, although serum withdrawal for 5 days or more effectively arrested cells at G0/G1 stages, it increased apoptosis of cells significantly. However, co-inhibition by serum withdrawal and olomoucine treatment was found to be an appropriate treatment to obtain more healthy G0/G1 cells based on the low percentage of apoptotic cells after treatment.     

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 (Qs and QG2). 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.     

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.     

Effects of etoposide on the proliferation of hexaploid H1 (ES) cells

Etoposide is a specific inhibitor of topoisomerase II, which is an enzyme that enables double-stranded DNA to pass through another double-stranded DNA. Topoisomerase II is a major constituent of chromosome scaffold, existing at appreciable amounts in cells. To examine the effects of etoposide on the cell cycle, hexaploid H1 (ES) cells (6H1 cells) were used with diploid H1 (ES) cells (2H1 cells) as a control. Exponentially growing 2H1 and 6H1 cells were exposed to etoposide at various concentrations, and cultured for about 60 days in L15F10 medium with leukemia inhibitory factor. With a high concentration of etoposide (1 μM), the DNA histograms showed G(2)/M accumulation, suggesting that etoposide arrested the cell cycle at the G(2)/M phase. With a low concentration of etoposide (50 nM), the cell proliferation was suppressed with a doubling time of 98.4 h for 2H1 cells and 51.6 h for 6H1 cells, and without significant alteration in DNA histograms. Time-lapse videography revealed that 6H1 cells survived in the medium containing 50 nM etoposide had a cell cycle time of 18.8 h, which was equivalent to 19.2 h of the doubling time for the 6H1 cell population in drug-free medium, suggesting that a part of the cell population died and was excluded from the cell system. It was concluded that etoposide affected the cell cycle at a wide range of concentrations.     

Concentration- and stage-specific effects of nitrite on colon cancer cell lines

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Nitrite in cured meats is thought to contribute to increased incidence of colon cancer. We sought to determine the effect of nitrite on human colon cancer cell lines at different stages. Our results indicate nitrite has no effect on proliferation of stage 1 SW116 colon cancer cells, while nitrite inhibits proliferation of stage 2 SW480 at 10 nM–100 μM and inhibits stage 3 HCT15 proliferation at 100 nM–1 μM, but promotes a significant increase in proliferation on stage 4 COLO205 cells at 100 μM. Furthermore, nitrite inhibited invasion into Matrigel® of stage 3 SW480 colon cancer cells in a concentration-dependent manner. However, it significantly promotes the invasion of stage 4 cells at 100 μM. Our FACS data demonstrated that nitrite decreased cell cycle progression in SW480 and HCT15 with arrested G2/M transition and delayed G1 phase entry in a concentration-dependent manner. However, 100 μM nitrite promoted cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas.     

Effect of cell cycle on the regulation of the cell surface and secreted forms of type I and type II human tumor necrosis factor receptors

The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and democolcine lead to accumulation of cells primarily in G₁/S, S, S/G₂/M, G₂/M, and M stages of the cell cycle, respectively. Whilie no significant change in TNF receptors occurred in cells arrested in G₁/S or S/G₂ stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G₁ and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response. © 1995 Wiley-Liss, Inc.     

Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts

Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.