Phosphoinositide 3-kinase signaling overrides a G2 phase arrest checkpoint and promotes aberrant cell cycling and death of hematopoietic cells after DNA damage

DNA damage activates arrest checkpoints to halt cell cycle progression in G1 and G2 phases. These checkpoints can be overridden in hematopoietic cells by cytokines, such as erythropoietin, through the activation of a phosphoinositide 3-kinase (PI3K) signaling pathway. Here, we show that PI3K activity specifically overrides delayed mechanisms effecting permanent G1 and G2 phase arrests, but does not affect transient checkpoints arresting cells up to 10 hours after gamma-irradiation. Assessing the status of cell cycle regulators in hematopoietic cells arrested after gamma-irradiation, we show that Cdk2 activity is completely inhibited in both G1 and G2 arrested cells. Despite the absence of Cdk2 activity, cells arrested in G2 phase did retain detectable levels of Cdk1 activity in the absence of PI3K signaling. However, reactivation of PI3K promoted robust increases in both Cdk1 and Cdk2 activity in G2-arrested cells. Reactivation of Cdks was accompanied by a resumption of cell cycling, but with strikingly different effectiveness in G1 and G2 phase arrested cells. Specifically, G1-arrested cells resumed normal cell cycle progression with little loss in viability when PI3K was activated after gamma-irradiation. Conversely, PI3K activation in G2-arrested cells promoted endoreduplication and death of the entire population. These observations show that cytokine-induced PI3K signaling pathways promote Cdk activation and override permanent cell cycle arrest checkpoints in hematopoietic cells. While this activity can rescue irradiated cells from permanent G1 phase arrest, it results in aberrant cell cycling and death when activated in hematopoietic cells arrested at the G2 phase DNA damage checkpoint.     

Plasma and platelet associated factors act in G1 to maintain proliferation and to stabilize arrested cells in a viable quiescent state : A temporal map of control points in the G1 phase

In medium supplemented with defibrinogenated, platelet-poor human plasma and a low molecular weight growth factor derived from human platelets (PDGF), Swiss 3T3 cells proliferate exponentially with the same cell cycle kinetics as cells cultured in medium supplemented with commercial calf serum. Removal of PDGF from the culture medium arrests proliferating cells in a stable, reversible G0/G1 quiescent state. This arrested state is similar to the known quiescent state induced by deprivation of calf serum in cell exit kinetics and cytoplasmic proteins synthesized. Cells are sensitive to PDGF deprivation only at the beginning of G1. Reduction of the plasma concentration in the culture medium also arrests cells in G1. The resulting arrested population is unstable and exhibits progressive cell death. Reduced levels of plasma block cellular transit through the cell cycle at a median time of approx. 2.1 h following mitosis, approx. 3.3 h prior to S phase initiation. In addition to being required by cycling cells, plasma associated factors are required to maintain G1 cells blocked by PDGF deprivation in a stable quiescent state. Establishment of a stable, viable G0/G1 growth-arrested state, therefore, apparently involves two distinct processes: arrest of cellular proliferation in G1 and stabilization of the arrested cells in a viable quiescent state. Together with previously reported findings on serum and isoleucine starvation, these results provide a temporal map of growth control points in the G1 phase.     

Modulation of growth-related gene expression and cell cycle synchronization by a sialoglycopeptide inhibitor

When an 18-kDa cell surface sialoglycopeptide (SGP), isolated from intact bovine cerebral cortex cells, was incubated with exponentially growing Swiss 3T3 cells, cell proliferation was efficiently arrested. The inhibition was totally reversible since after removal of the SGP the arrested cells resumed their progress in the cell cycle in a synchronized manner for at least two divisions. Readdition of the GSP 4 h after reversal of the inhibition did not, however, affect the commitment of the cells to advance through metaphase, although progress through the cell cycle was once again inhibited after the cells reentered the G1 phase. The efficient nature of the SGP-mediated cell cycle arrest in G1 provided us with a basis to examine potential changes in the expression of several competence genes, and genes associated with mid and late G1, that have been implicated in cell cycle progression. Upon serum stimulation of quiescent Swiss 3T3 cells, the induction of c-myc and c-fos expression was not influenced by the SGP at concentrations highly inhibitory to cell cycling. Expression of JE was induced by serum, and the presence of the SGP had little effect on the expression of this growth-related gene. KC expression was not appreciably stimulated by serum although, surprisingly, the addition of the SGP resulted in a significant increase in expression. In addition, we learned that the SGP did not alter expression of ornithine decarboxylase, c-ras, or thymidine kinase, which are induced later than the genes associated with the initial stages of competence.     

A new compound which reversibly arrests T lymphocyte cell cycle near the G1/S boundary

A novel cell cycle blocking agent profoundly suppressed the proliferation of mitogen-stimulated T lymphocytes. The carboxythiazole derivative arrested cells in the G1 phase of the cell cycle but did not inhibit the induction of cell surface receptors for either interleukin-2 or transferrin. The uncoupling of transferrin receptor expression from DNA synthesis indicated that a previously undefined restriction point in the cell cycle has been identified which occurs after transferrin receptor expression in late G1 and just prior to the initiation of DNA replication in S phase. T cells incubated in an inhibitory dose of the carboxythiazole derivative resumed cell cycle progression subsequent to its removal, indicating that the compound reversibly arrests cells at the late G1 restriction point. In contrast to other techniques which have been inefficient in achieving T cell synchronization, T cells released from the block mediated by the carboxythiazole compound progress through S phase with a considerable degree of synchrony.     

Ca~(2+)在粟酒裂殖酵母细胞周期时相中的作用

以粟酒裂殖酵母(Schizosaccharomyces pombe)为研究材料,研究了Ca~(2+)在细胞周期时相中的作用。当外源Ca~(2+)浓度在0.5-20 mmol/L范围内,随Ca~(2+)浓度增加,细胞增殖速度加快,延滞期逐渐缩短。但SD-Ca（CaCl2省略）并不能终止Sch. pombe的细胞周期。采用缺氮对群体细胞进行同步化,并以EGTA 螯合培养介质中低浓度的Ca~(2+),Sch. pombe 细胞增殖被完全抑制,细胞流式法测定结果表明：细胞周期被终止在G1期。分析认为Ca~(2+) 对Sch. pombe 细胞增殖是必不可少的,外源Ca~(2+)在G1期向S期转化过程中起着关键性的作用。     

Cell cycle regulation by light in Prochlorococcus strains

The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 micromol of quanta x m(-2) x s(-1), the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the "light on" signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G1 phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G2 phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.     

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.     

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.     

High resolution analysis of cell cycle-correlated vimentin expression in asynchronously grown,TPA-treated MPC-11 cells by the novel flow cytometric multiparameter BrdU-hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC-11 mouse plasmacytoma cell cultures treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA-treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time-dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication. © 1994 Wiley-Liss, Inc.     

THE DETERMINATION OF THE FAR-RED EFFECT IN MARINE PHYTOPLANKTON1,2

A far-red effect exists in 4 marine phytoplankton species: the diatom Ditylum brightwelli, the coccolithophorid Coccolithus huxleyi, the green flagellate Dunaliella tertiolecta, and the dinoflagellate Pyrocystis lunula. The effect is reversible and is manifested through a change in cell division rate. Cultures of algae which received 30-min far-red (FR) light (750 nm) prior to the dark period were compared to controls which received, no FR. Reversal of the FR effect was studied by exposing experimental cultures to 30 min FR followed by 5-min red (R) light (650 nm) prior to the dark period. Controls received only FR. Cultures were exposed to light at 6 different enumerated wavelengths between 460 and 750 nm. A decrease in division rate runs evident only with light at 750 nm. These results give evidence for the presence of the phytochrome system in these phytoplankton species.     

Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.     

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.     

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of cell cycle on GFPuv gene expression in the baculovirus expression system.

The effects of cell cycle on recombinant protein production and infection yield in the baculovirus-insect cell expression system (BES) were investigated. When, at any cell cycle phase, the host cell was infected by baculovirus, the cell cycle was finally arrested at the S or G(2)/M phase with 4n DNA. In the case of G(1) or S phase-infection, cell cycle of virus-infected cells began to be arrested at S phase from 8 h post-infection or at G(2)/M phase from 4 h post-infection, respectively; while, in the case of M phase-infection, cell cycle was arrested at S phase after 12 h post-infection. When the host cell was infected at the G(1) phase, average intracellular GFPuv fluorescence intensity was 1.3-fold higher than that at G(2)/M phase at 24 h post-infection. The GFPuv expression corresponded to the profile of the G(1) cell cycle in the BES. Infection yield was measured by detection of intracellular DNA binding protein using immunohistochemical method within 7 h post-infection. The infection yield at G(1) or S phase-infection was 1.5-1.8-fold higher than that at G(2)/M phase-infection.     

Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae.

We showed that the heat killing curve for exponentially growing Saccharomyces cerevisiae was biphasic. This suggests two populations of cells with different thermal killing characteristics. When exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were heat shocked, the fractions enriched in small unbudded cells showed greater resistance to heat killing than did other cell cycle fractions. Cells arrested as unbudded cells fell into two groups on the basis of thermotolerance. Sulfur-starved cells and the temperature-sensitive mutants cdc25, cdc33, and cdc35 arrested as unbudded cells were in a thermotolerant state. Alpha-factor-treated cells arrested in a thermosensitive state, as did the temperature-sensitive mutant cdc36 when grown at the restrictive temperature. cdc7, which arrested at the G1-S boundary, arrested in a thermosensitive state. Our results suggest that there is a subpopulation of unbudded cells in exponentially growing cultures that is in G0 and not in G1 and that some but not all methods which cause arrest as unbudded cells lead to arrest in G0 as opposed to G1. It has been shown previously that yeast cells acquire thermotolerance to a subsequent challenge at an otherwise lethal temperature during a preincubation at 36 degrees C. We showed that this acquisition of thermotolerance was corrected temporally with a transient increase in the percentage of unbudded cells during the preincubation at 36 degrees C. The results suggest a relationship between the heat shock phenomenon and the cell cycle in S. cerevisiae and relate thermotolerance to transient as well as to more prolonged residence in the G0 state.     

Cell cycle analysis of p53-induced cell death in murine erythroleukemia cells.

A temperature-sensitive mutant of murine p53 (p53Val-135) was transfected by electroporation into murine erythroleukemia cells (DP16-1) lacking endogenous expression of p53. While the transfected cells grew normally in the presence of mutant p53 (37.5 degrees C), wild-type p53 (32.5 degrees C) was associated with a rapid loss of cell viability. Genomic DNA extracted at 32.5 degrees C was seen to be fragmented into a characteristic ladder consistent with cell death due to apoptosis. Following synchronization by density arrest, transfected cells released into G1 at 32.5 degrees C were found to lose viability more rapidly than did randomly growing cultures. Following release into G1, cells became irreversibly committed to cell death after 4 h at 32.5 degrees C. Commitment to cell death correlated with the first appearance of fragmented DNA. Synchronized cells allowed to pass out of G1 prior to being placed at 32.5 degrees C continued to cycle until subsequently arrested in G1; loss of viability occurred following G1 arrest. In contrast to cells in G1, cells cultured at 32.5 degrees C for prolonged periods during S phase and G2/M, and then returned to 37.5 degrees C, did not become committed to cell death. G1 arrest at 37.5 degrees C, utilizing either mimosine or isoleucine deprivation, does not lead to rapid cell death. Upon transfer to 32.5 degrees C, these G1 synchronized cell populations quickly lost viability. Cells that were kept density arrested at 32.5 degrees C (G0) lost viability at a much slower rate than did cells released into G1. Taken together, these results indicate that wild-type p53 induces cell death in murine erythroleukemia cells and that this effect occurs predominantly in the G1 phase of actively cycling cells.     

A model of cell cycle control: effects of thymidine on synchronous cell cultures.

Further evidence is presented in support of a model for growth control in which commitment for cell division is determined by an event in the preceding cell cycle. A study was made of conditions affecting synchronous growth following treatment of murine mastocytoma cells with excess thymidine at different phases of the cell cycle. Cells were synchronized by a physical procedure involving velocity sedimentation in a zonal rotor. Pulse treatment of such cultures with thymidine at times corresponding to the S, G2, and M periods had no effect on further growth. However, addition at G1, although having no immediate effect, arrested cell growth in the next cell cycle. This temporal effect may account for the decay of synchrony observed during double thymidine blockade or thymidine-FUdR blockade. When the time interval between two such blocks was 7 hr or less, P815Y cells were arrested after one synchronous division. At this critical time a majority of cells were at, or near, G1. It is suggested that thymidine exerts a hitherto unrecognized effect at the G1 interval.     

Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1

Chemical agents for cell cycle synchronization have greatly facilitated the study of biochemical events driving cell cycle progression. G1, S and M phase inhibitors have been developed and used widely in cell cycle research. However, currently there are no effective G2 phase inhibitors and synchronization of cultured cells in G2 phase has been challenging. Recently, a selective CDK1 inhibitor, RO-3306, has been identified that reversibly arrests proliferating human cells at the G2/M phase border and provides a novel means for cell cycle synchronization. A single-step protocol using RO-3306 permits the synchronization of >95% of cycling cancer cells in G2 phase. RO-3306 arrested cells enter mitosis rapidly after release from the G2 block thus allowing for isolation of mitotic cells without microtubule poisons. RO-3306 represents a new molecular tool for studying CDK1 function in human cells.     

Study of the cytolethal distending toxin-induced cell cycle arrest in HeLa cells: involvement of the CDC25 phosphatase

HeLa cells exposed to Escherichia coli cytolethal distending toxins (CDT) arrest their cell cycle at the G2/M transition. We have shown previously that in these cells the CDK1/cyclin B complex is inactive and can be reactivated in vitro using recombinant CDC25 phosphatase. Here we have investigated in vivo the effects of CDC25 on this cell cycle checkpoint. We report that overexpression of CDC25B or CDC25C overrides an established CDT-induced G2 cell cycle arrest and leads the cells to accumulate in an abnormal mitotic stage with condensed chromatin and high CDK1 activity. This effect can be counteracted by coexpression of the WEE1 kinase. In contrast, overexpression of CDC25B or C prior to CDT treatment prevents G2 arrest and allows most of the cells to progress through mitosis with only a low percentage of cells arrested in abnormal mitosis. The implications of these results on the biochemical nature of the CDT-induced cell cycle arrest are discussed.     

Indomethacin-induced G1/S phase arrest of the plant cell cycle

In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.