Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G₁/S interface. Cells were arrested at the G₁/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G₁/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G₁ to G₂cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G₁ phase of the cell cycle are less susceptible to the perforin pathway than cells in G₂and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425–435, 1998. © 1998 Wiley-Liss, Inc.     

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.1.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G₁ phase cells, but increased many-fold during the S and G₂ phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G₂ + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.     

Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KGl), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G₁ were resistant; the sensitivity of G₂ or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G₂ or M, to these inhibitors. The data show that γ-irradiation (0.5–15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G₁ cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G₂ either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G₁, G₂ or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.     

Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells

DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G₂ and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G₁/G₀ (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G₁/G₀ cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G₁/G₀ and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.     

Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Bcl-2 delays cell cycle through mitochondrial ATP and ROS

Bcl-2 inhibits cell proliferation by delaying G₀/G₁ to S phase entry. We tested the hypothesis that Bcl-2 regulates S phase entry through mitochondrial pathways. Existing evidence indicates mitochondrial adenosine tri-phosphate (ATP) and reactive oxygen species (ROS) are important signals in cell survival and cell death, however, the molecular details of how these 2 processes are linked remain unknown. In this study, 2 cell lines stably expressing Bcl-2, 3T3Bcl-2 and C3HBcl-2, and vector-alone PB controls were arrested in G₀/G₁ phase by serum starvation and contact inhibition, and ATP and ROS were measured during re-stimulation of cell cycle entry. Both ATP and ROS levels were decreased in G₀/G₁ arrested cells compared with normal growing cells. In addition, ROS levels were significant lower in synchronized Bcl-2 cells than those in PB controls. After re-stimulation, ATP levels increased with time, reaching peak value 1–3 hours ahead of S phase entry for both Bcl-2 cells and PB controls. Consistent with 2 hours of S phase delay, Bcl-2 cells reached ATP peaks 2 hours later than PB control, which suggests a rise in ATP levels is required for S phase entry. To examine the role of ATP and ROS in cell cycle regulation, ATP and ROS level were changed. We observed that elevation of ATP accelerated cell cycle progression in both PB and Bcl-2 cells, and decrease of ATP and ROS to the level equivalent to Bcl-2 cells delayed S phase entry in PB cells. Our results support the hypothesis that Bcl-2 protein regulates mitochondrial metabolism to produce less ATP and ROS, which contributes to S phase entry delay in Bcl-2 cells. These findings reveal a novel mechanistic basis for understanding the link between mitochondrial metabolism and tumor-suppressive function of Bcl-2.     

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 of human pancreatic carcinoma cell line (MIA PaCa-2) to Bisantrene and Theprubicin in vitro

Summary We tested the effect of Bisantrene (BS) and Theprubicin (THP-ADR) on cell growth of a human pancreatic carcinoma cell line (MIA PaCa-2). After 1 h exposure ID₅₀ of BS or THP-ADR was 3×10⁻⁷ and 5×10⁻⁸ M, respectively. Increasing the exposure time from 1 h to continuous exposure for 5d resulted in 11-fold decrease in ID₅₀ for BS and a 6-fold decrease for THP-ADR. Both drugs inhibited [¹⁴C]thymidine incorporation to the same extent and caused an accumulation of cells into G₂+M phase of the cell cycle. This work is supported by a gift, from Mr. Issam Fares and U.S. Public Health Service Grants AM 07114 and CA 19182.     

Cell cycle parameters of 3T3 cells cultured as aggregates

Summary BALB/c 3T3 cells cultured as aggregates were examined by two independent techniques to determine whether or not cells accumulated at a specific point in the cell cycle, and if so to determine the point at which they accumulate. Replating cells onto dishes followed by pulse labeling with [³H]thymidine and autoradiography indicated that aggregate-cultured cells were in the same phase of the cell cycle as cells cultured as confluent monolayers. Flow microfluorometry confirmed that 75% of the aggregate-maintained cells were arrested in G₀ or G₁, with 25% distributed throughout the rest of the cell cycle. Labeling and mitotic indices of cells in aggregates were also consistent with about 20 to 25% of the cells being in S+G₂=M phases of the cell cycle at any time. This work was supported by PHS Grant CA20323 and NSF Grant PCM 74-15092 to H. G., who is also a Harry H. Pinney Cancer Scholar.     

Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

Calmodulin and cell proliferation

The calmodulin content of synchronized Chinese hamster ovary (CHO-K₁) cells was determined at each phase of the cell cycle. The calmodulin content was minimum in the G₁ phase, increased after the cells entered S phase and reached the maximum level at the late G₂ or early M phase. When 30 μM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G₂ or early M phase. The addition of W-7 also prevented the morphological changes caused by cholera toxin. These results suggest that calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis.     

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.     

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.     

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

Abstract. In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G₂ phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6–12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine ([³H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [³H]TdR labelling was simulated. The presence of two distinct sub-populations in G₂ phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G₁ phase is suggested. The sizes of the slowly cycling fractions in G₁ and G₂ showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3–5%) was arrested in G₂ phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G₂ cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

Occurrence of two cell subpopulations with different cell-cycle durations in the central and peripheral zones of the vegetative shoot apex of Sinapis alba L.

The cell-cycle duration and the growth fraction were estimated in the vegetative shoot apical meristem of Sinapis alba L. The length of the cell cycle was about 86 h, i.e. 2.5 times shorter than the cell-doubling time (M. Bodson, 1975, Ann. Bot. 39, 547–554) and the growth fraction was between 32 to 41%. These data demonstrated that the cell population of this meristem was heterogeneous, including one subpopulation of rapidly cycling cells and one subpopulation of non-cycling cells, i.e. cells with a very long cell cycle compared with that of the rapidly cycling cells. Non-cycling cells had no particular localization within the meristem. Both the central and peripheral zones of the meristem were mosaics of rapidly cycling and non-cycling cells.Abbreviations G₁ pre-DNA-synthesis phase - G₂ post-DNA-synthesis phase - GF growth fraction - M mitosis phase - PLM pulse-labelled-mitoses method - S DNA-synthesis phase - T cell-cycle duration - TdR thymidine     

Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up-regulation of oncogenes in S-49 cyc− cells

Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c-AMP in prostaglandin mediated cell cycle arrest, we use the-49 lymphoma variant (cyc^?) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A₁ (dmPGA₁) inhibits DNA synthesis and cell growth in cyc^? cells. DNA synthesis is inhibited 42% by dmPGA₁ (50 μM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the α,β unsaturated ketone ring. Dimethyl PGA₁ is most effective in inhibiting DNA synthesis in cyc^? cells, with prostaglandins PGE₁ and PGB₁ being less potent inhibitors of DNA synthesis. DmPGE₂ caused a significant stimulation of DNA synthesis. S-49 cyc^- variant cells exposed to (30–50 μm) dmPGA₁, arrested in the G₁ phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S-49 cyc^? cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G₁, S, G₂, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S-phase DNA synthesis from the G₁ cell cycle block. The S-49 cyc^? cells are known to have a G₁/S boundary through M phase transition time of 14.8 h, making the location of the prostaglandin cell cycle arrest at or very near the G₁/S interface. The oncogenes, c-fos and c-myc which are normally expressed during G₁ in proliferating cells have a 2–3 fold enhanced expression in prostaglandin G₁ arrested cells. These data using the S-49 variants demonstrate that dmPGA₁ inhibits DNA synthesis and arrests the cell cycle independent of cAMP-mediated effects. The prostaglandin arrested cells maintain the gene expression of a G₁ synchronous cell which suggests a unique molecular mechanism for prostaglandin action in arresting cell growth. These properties indicate that this compound may be an effective tool to study molecular mechanisms of regulation of the cell cycle.     

Effects of light and nitrogen limitation on the cell cycle of the dinoflagellate Amphidinium carteri

Cell cycle phase durations of cultures of Amphidinium carteriin light- or nitrogen-limited balanced growth were determinedusing flow cytometry. For both types of growth rate limitation,the increases in generation time caused by increasing degreesof limitation were due solely to expansion of the G₁ phase ofthe cell cycle. The durations of the S and G₂ + M phases wereindependent of growth rate. Furthermore, when cells were deprivedcompletely of light and nitrogen, they arrested in the G₁ phaseof the cell cycle. The results indicate that light- and nitrogen-dependentprocesses are heavily concentrated in the early part of thecell cycle, while DNA replication and cell division, once initiated,are independent of light or nitrogen supply.     

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.