Antiproliferative effect of bcl-2 gene does not concern the control of mitotic events

E1A + c-Ha-ras-transformants overexpressing bcl-2 oncogene are able to be arrested at the G1/S boundary of the cell cycle after DNA damage and upon serum starvation, this cell cycle blockage being accompanied by a decrease in the activity of cyclin E--Cdk2 complexes. Roscovitine-induced inhibition of cyclin-dependent kinases (Cdks) activity does not result in the G1/S arrest of E1A + c-Ha-ras + bcl-2-transformants. Roscovitine treatment causes an accumulation of G2/M cells, mainly at the expense of mitotic cells. However, the expression of Bcl-2 oncoproducts does not re-establish the regulation of mitotic events broken by introduction of E1A and c-Ha-ras oncogenes in normal cells, as revealed by the treatment of E1A + c-Ha-ras + bcl-2-transformants with nocodazole inducing mitotic arrest in normal cells. In spite of the elevated expression of antiapoptotic bcl-2 gene in transformants, nocodazole treatment results in mass apoptotic death preceded by polyploidy. Roscovitine also induces apoptosis with no polyploid cell accumulation being observed. Inhibition of Cdks activity with Roscovitine, as well as violation of microtubule depolymerization with nocodazole result in the apoptotic death in the tested cell lines sensitive (E1A + c-Ha-ras) and resistant (E1A + c-Ha-ras + bcl-2) to damaging agents. Thus, the application of Roscovitine, a specific inhibitor of Cdks, suggests that the decrease in Cdks activity in E1A + c-Ha-ras + bcl-2-transformants is not likely to be responsible for G1/S cell cycle arrest realization after damaging influences. Moreover, an antiproliferative effect of Bcl-2 in E1A + c-Ha-ras-transformants is restricted by restoration of cell cycle events at G1/S and G2/M boundaries, and does not concern the program of mitotic events regulation.     

Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of 7-hydroxystaurosporine and the kinetics of Cdc25C activation

DNA damage causes cell cycle arrest in G(1), S, or G(2) to prevent replication on damaged DNA or to prevent aberrant mitosis. The G(1) arrest requires the p53 tumor suppressor, yet the topoisomerase I inhibitor SN38 induces p53 after the G(1) checkpoint such that the cells only arrest in S or G(2). Hence, SN38 facilitates comparison of p53 wild-type and mutant cells with regard to the efficacy of drugs such as 7-hydroxystaurosporine (UCN-01) that abrogate S and G(2) arrest. UCN-01 abrogated S and G(2) arrest in the p53 mutant breast tumor cell line MDA-MB-231 but not in the p53 wild-type breast line, MCF10a. This resistance to UCN-01 in the p53 wild-type cells correlated with suppression of cyclins A and B. In the p53 mutant cells, low concentrations of UCN-01 caused S phase cells to progress to G(2) before undergoing mitosis and death, whereas high concentrations caused rapid premature mitosis and death of S phase cells. UCN-01 inhibits Chk1/2, which should activate the mitosis-inducing phosphatase Cdc25C, yet this phosphatase remained inactive during S phase progression induced by low concentrations of UCN-01, probably because Cdc25C is also inhibited by the constitutive kinase, C-TAK1. High concentrations of UCN-01 caused rapid activation of Cdc25C, which is attributed to inhibition of C-TAK1, as well as Chk1/2. Hence, UCN-01 has multiple effects depending on concentration and cell phenotype that must be considered when investigating mechanisms of checkpoint regulation.     

Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21(WAF1/Cip1) resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3sigma, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3sigma (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.     

The nucleoside analog sangivamycin induces apoptotic cell death in breast carcinoma MCF7/adriamycin-resistant cells via protein kinase Cdelta and JNK activation

Sangivamycin has shown a potent antiproliferative activity against a variety of human cancers. However, little is known about the mechanism of action underlying its antitumor activity. Here we demonstrate that sangivamycin has differential antitumor effects in drug-sensitive MCF7/wild type (WT) cells, causing growth arrest, and in multidrug-resistant MCF7/adriamycin-resistant (ADR) human breast carcinoma cells, causing massive apoptotic cell death. Comparisons between the effects of sangivamycin on these two cell lines allowed us to identify the mechanism underlying the apoptotic antitumor effect. Fluorescence-activated cell sorter analysis indicated that sangivamycin induced cell cycle arrest in the G(2)/M phase in MCF7/ADR cells. A marked induction of c-Jun expression as well as phosphorylation of c-Jun and JNK was observed after sangivamycin treatment of MCF7/ADR cells but not MCF7/WT cells. Sangivamycin also induced cleavage of lamin A and poly(ADP-ribose) polymerase (PARP) in MCF7/ADR cells, probably via activation of caspase-6, -7, and -9. Pretreatment with a caspase-9-specific inhibitor or pan-caspase inhibitor abolished sangivamycin-induced cleavage of lamin A and PARP but not sangivamycin induction of c-Jun expression and phosphorylation. Pretreatment of MCF7/ADR cells with SP600125, a specific inhibitor of JNK, or with rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta), significantly reduced the sangivamycin-induced apoptosis and almost completely abolished sangivamycin-induced phosphorylation of c-Jun and cleavage of lamin A and PARP. Transfection of MCF7/ADR cells with PKCdelta small interfering RNAs or PKCdelta antibody or rottlerin pretreatment significantly suppressed the phosphorylation of JNK. Taken together, our data suggest that sangivamycin induces mitochondria-mediated apoptotic cell death of MCF7/ADR cells via activation of JNK in a protein kinase Cdelta-dependent manner.     

Acetonic extract of Buxus sempervirens induces cell cycle arrest, apoptosis and autophagy in breast cancer cells

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC(50) ranging from 7.74 μg/ml to 12.5 μg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC(50) of 19.24 μg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC(50) did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer.     

Effects of 4-hydroperoxycyclophosphamide (4-OOH-CP) and 4-hydroperoxydechlorocyclophosphamide (4-OOH-deCICP) on the cell cycle of post implantation rat embryos.

In this study, we used preactivated forms of cyclophosphamide (CP) and dechlorocyclophosphamide (deClCP) to examine the effects of phosphoramide mustard (PM) and acrolein, respectively, on the cell cycle of postimplantation rat embryos. The percentage distribution of cells in the G1/G0, S, and G2/M phases of the cell cycle was determined by flow-cytometric analysis. At embryotoxic concentrations, 4-OOH-CP (PM) induced major cell cycle perturbations whereas 4-OOH-deClCP (acrolein) caused no major perturbation of the cell cycle. These data support the hypothesis that the mechanism of the embryotoxic action of PM involves alkylation of DNA, whereas the mechanism of action of acrolein does not. The primary effect of PM on the cell cycle was an initial delay in the S phase followed by a G2/M arrest. At low embryotoxic concentrations of 4-OOH-CP, there was apparent reversal of the G2/M arrest; at higher embryotoxic concentrations there was little recovery from the G2/M arrest. The high level of cell death found at higher drug concentrations suggests that prolonged G2/M arrest leads to cell death. Using radiolabeled CP and cell sorting, it was determined that PM predominantly alkylated DNA in the S phase of the cell cycle. Overall, the data from this study support the hypothesis that DNA cross-links, induced by the alkylation of DNA by PM, induce cell cycle perturbations. Furthermore, these cell cycle alterations may be one of the early steps in the mechanism leading to the embryotoxicity of PM.     

Both ERK1 and ERK2 kinases promote G2/M arrest in etoposide-treated MCF7 cells by facilitating ATM activation

The MEK–ERK pathway plays a role in DNA damage response (DDR). This has been thoroughly studied by modulating MEK activation. However, much less has been done to directly examine the contributions of ERK1 and ERK2 kinases to DDR. Etoposide induces G2/M arrest in a variety of cell lines, including MCF7 cells. DNA damage-induced G2/M arrest depends on the activation of the protein kinase ataxia-telangiectasia mutated (ATM). ATM subsequently activates CHK2 by phosphorylating CHK2 threonine 68 (T68) and CHK2 inactivates CDC25C via phosphorylation of its serine 216 (S216), resulting in G2/M arrest. To determine the contribution of ERK1 and ERK2 to etoposide-induced G2/M arrest, we individually knocked-down ERK1 and ERK2 in MCF7 cells using specific small interfering RNA (siRNA). Knockdown of either kinases significantly reduced ATM activation in response to etoposide treatment, and thereby attenuated phosphorylation of the ATM substrates, including the S139 of H2AX (γH2AX), p53 S15, and CHK2 T68. Consistent with these observations, knockdown of either ERK1 or ERK2 reduced etoposide-induced CDC25C S216 phosphorylation and significantly compromised etoposide-induced G2/M arrest in MCF7 cells. Taken together, we demonstrated that both ERK1 and ERK2 kinases play a role in etoposide-induced G2/M arrest by facilitating activation of the ATM pathway. These observations suggest that a cellular threshold level of ERK kinase activity is required for the proper checkpoint activation in MCF7 cells.     

Cell expression of a four extra octarepeat mutated PrPC modifies cell structure and cell cycle regulation

RK13 cell lines generated to express bovine PrP(C) with a four extra octarepeat insertional mutation (Bo-10ORPrP(C)) show partially insoluble PrP(C) and lower rates of cell growth when compared to either the same cells expressing wild type Bo-6ORPrP(C) or the original RK13 cell line. The expression of Bo-10ORPrP(C) in cell cultures was also associated with changes in cell size and reorganization of the actin cytoskeleton. This last process was reversed by Clostridium difficile toxin-B, a specific inhibitor of small GTPase proteins. Further, in clones expressing Bo-10ORPrP(C), increased proportions of cells at cell cycle stage G2/M were observed. Proteasome inhibitors caused a further expansion of G2/M-stage cells that was more marked in cell lines expressing Bo-10ORPrP(C) than those expressing Bo-6ORPrP(C), while this effect was minimal or null in the original RK13 cell line. Hence, the presence of Bo-10ORPrP(C) in RK13 cells promotes cell cycle arrest at G2/M, and the effect is amplified by proteasome inhibition. These findings suggest a role for PrP(C) in cell morphology and cell cycle regulation, and open new avenues for understanding the mechanisms underlying PrP mutation-associated diseases.     

SHP-2 phosphatase regulates DNA damage-induced apoptosis and G2/M arrest in catalytically dependent and independent manners, respectively

SHP-2, a tyrosine phosphatase implicated in diverse signaling pathways induced by growth factors and cytokines, is also involved in DNA damage-triggered signaling and cellular responses. We previously demonstrated that SHP-2 played an important role in DNA damage-induced apoptosis and G2/M cell cycle checkpoint. In the present studies, we have provided evidence that SHP-2 functions in DNA damage apoptosis and G2/M arrest in catalytically dependent and independent manners, respectively. Mutant embryonic fibroblasts with the Exon 3 deletion mutation in SHP-2 showed decreased apoptosis and diminished G2/M arrest in response to cisplatin treatment. Wild type (WT), but not catalytically inactive mutant SHP-2 (SHP-2 C459S), rescued the apoptotic response of the mutant cells. Interestingly, both WT and SHP-2 C459S efficiently restored the G2/M arrest response. Furthermore, inhibition of the catalytic activity of endogenous SHP-2 in WT cells by overexpression of SHP-2 C459S greatly decreased cell death but not G2/M arrest induced by cisplatin. Biochemical analyses revealed that activation of c-Abl kinase was decreased in SHP-2 C459S-overexpressing cells. However, DNA damage-induced translocation of Cdc25C from the nucleus to the cytoplasm was fully restored in both WT and SHP-2 C459S "rescued" cells. Additionally, we demonstrated that the role of SHP-2 in DNA damage-induced cellular responses was independent of the tumor suppressor p53. Embryonic stem cells with the SHP-2 deletion mutation showed markedly decreased sensitivity to cisplatin-induced apoptosis, attributed to impaired induction of p73 but not p53. In agreement with these results, DNA damage-induced apoptosis and G2/M arrest were also decreased in SHP-2/p53 double mutant embryonic fibroblasts. Collectively, these studies have further defined the mechanisms by which SHP-2 phosphatase regulates DNA damage responses.     

Overexpression of the signaling adapter FRS2 reconstitutes the cell cycle deficit of a nerve growth factor non-responsive TrkA receptor mutant

We have characterized the cell cycle deficit of a novel TrkA receptor mutant (TrkAS3) that fails to support nerve growth factor (NGF)-dependent cell cycle arrest and neurite outgrowth. TrkAS3 receptors fail to support an NGF-dependent increase in the expression of cyclin D1 and the cell cycle inhibitor, p21(Waf1/Cip1), two important regulators of G(1) /S transition, and do not down-regulate expression of the G(2) /M phase marker, cdc2/cdk1, or the S phase marker, proliferating cell nuclear antigen. Moreover, NGF-activated TrkAS3 receptors do not down-regulate cyclin-dependent kinase 4 phosphorylation of the retinoblastoma protein, essential for G(1) arrest, in comparison to NGF-activated wild-type TrkA. Collectively these data indicate that TrkAS3 receptors fail to support NGF-dependent G(1) arrest. Interestingly, ectopic expression of regulators of G(1) /S arrest, such as cyclin D1 or inhibitors of cell cycle (p21(Waf1/Cip1), p16(INK4A) ), or the fibroblast growth factor (FGF) receptor substrate-2 (FRS2) in cells expressing TrkAS3 reconstitutes NGF-dependent neurite outgrowth. Collectively, these data suggest a model in which NGF-stimulated TrkA-dependent activation of FRS2 supports neurite outgrowth through a mechanism that likely involves the induction of p21(Waf1/Cip1) expression and the arrest of cells at G(1) /S.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Both low and high concentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and cdk2 expression

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.     

Influence of G2 arrest on the cytotoxicity of DNA topoisomerase inhibitors toward human carcinoma cells with different p53 status

We here report the influence of the cell cycle abrogator UCN-01 on RKO human colon carcinoma cells differing in p53 status following exposure to two DNA damaging agents, the topoisomerase inhibitors etoposide and camptothecin. Cells were treated with the two drugs at the IC90 concentration for 24 h followed by post-incubation in drug-free medium. RKO cells expressing wild-type, functional p53 arrested the cell cycle progression in both the G1 and G2 phases of the cell cycle whereas the RKO/E6 cells, which lack functional p53, only arrested in the G2 phase. Growth-arrested cells did not resume proliferation even after prolonged incubation in drug-free medium (up to 96 h). To evaluate the importance of the cell cycle arrest on cellular survival, a non-toxic dose of UCN-01 (100 nM) was added to the growth-arrested cells. The addition of UCN-01 was accompanied by mitotic entry as revealed by the appearance of condensed chromatin and the MPM-2 phosphoepitope, which is characteristic for mitotic cells. G2 exit and mitotic transit was accompanied by a rapid activation of caspase-3 and apoptotic cell death. The influence of UCN-01 on the long-term cytotoxic effects of the two drugs was also determined. Unexpectedly, abrogation of the G2 arrest had no influence on the overall cytotoxicity of either drug. In contrast, addition of UCN-01 to cisplatin-treated RKO and RKO/E6 cells greatly increased the cytotoxic effects of the alkylating agent. These results strongly suggest that even prolonged cell cycle arrest in the G2 phase of the cell cycle is not necessarily coupled to efficient DNA repair and enhanced cellular survival as generally believed.     

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.     

cAMP-mediated growth inhibition of a B-lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1

This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell-cycle progression in the B-lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for approximately 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S-phase cells were selectively stained by pulsing asynchronously growing cells with bromo-deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/m, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.