PPARgamma1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansion

Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I), dexamethasone (D), and methylisobutylxanthine (M), administered with a fresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPARgamma1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, l-mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPARgamma1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPARgamma1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPARgamma was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPARgamma1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. Importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPARgamma synthesis and subsequent adipocyte differentiation.     

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Differential Effect of Jasmonic Acid and Abscisic Acid on Cell Cycle Progression in Tobacco BY-2 Cells

Environmental stress affects plant growth and development. Several plant hormones, such as salicylic acid, abscisic acid (ABA), jasmonic acid (JA), and ethylene play a crucial role in altering plant morphology in response to stress. Developmental regulation often has the cell cycle machinery among its targets. We analyzed the effect of JA and ABA on cell cycle progression in synchronized tobacco (Nicotiana tabacum) BY-2 cells. Both compounds were found to prevent DNA replication, keeping the cells in the G1 stage, when applied just before the G1/S transition. However, ABA did not have any effect on subsequent phases of the cell cycle when applied at a later stage, whereas JA effectively prevented mitosis on application during DNA synthesis. This demonstrates that JA treatment can freeze synchronized BY-2 cells in both the G1 and G2 stages of the cell cycle. Jasmonate administered after the S-phase was less effective in decreasing the mitotic index, suggesting that cell sensitivity toward JA is dependent on the cell cycle phase. In cultures detained in the G2-phase, we observed a reduced histone H1 kinase activity of kinases associated with the p13(suc1) protein.     

Autoradiographic evaluation of vitamin A effects on Triturus alpestris blastema cells

Autoradiographic studies on Triturus alpestris regenerates treated with vitamin A palmitate for 4, 7, 11 and 14 days after amputation, reveal a considerable diminution in the percentage of cells duplicating DNA, as judged by measurements of labelling indices. Based on previous observations, that vitamin A promotes DNA-synthesis, mitosis and growth of regenerates, we conclude that the lower labelling indices calculated are due to an acceleration of S-phase processes, resulting in a concomitant reduction in its duration.     

Cell cycle analysis of synchronized chinese hamster cells using bromodeoxyuridine labeling and flow cytometry

Summary Chinese hamster ovary cells were synchronized into purified populations of viable G1-, S-, G2-, and M-phase cells by a combination of methods, including growth arrest, aphidicolin block, cell cycle progression, mitotic shake-off, and centrifugal elutriation. The DNA content and bromodeoxyuridine (BrdUrd) labeling index were measured in each purified fraction by dual-parameter flow cytometry. The cell cycle distributions determined from the DNA measurements alone (single parameter) were compared with those calculated from both DNA and BrdUrd data (dual parameter). The results show that highly purified cells can be obtained using these methods, but the assessed purity depends on the method of cell cycle analysis. Using the single versus dual parameter measurement to determine cell cycle distributions gave similar results for most phases of the cell cycle, except for cells near the transition from G1- to S-phase and S- to G2-phase. There the BrdUrd labeling index determined by flow cytometry was more sensitive for detecting small amounts of DNA synthesis. As an alternative to flow cytometry, a simple method of measuring BrdUrd labeling index on cell smears was used and gave the same result as flow cytometry. Measuring both DNA content and DNA synthesis improves characterization of synchronized cell populations, especially at the transitions in and out of S-phase, when cells are undergoing dramatic shifts in biochemical activity.     

The effect of bleomycin on rapidly proliferating epidermis. A comparative investigation using micro-flow fluorometry, H3Tdr incorporation and a stathmokinetic method (colcemid).

At different time intervals after injection of Bleomycin (BLM) th effect on several kinetic parameters of the hairless mouse epidermis stimulated to proliferate by previous adhesive tape stripping was measured. Micro-flow fluorometry was used to determine the relative number of cells in the various phases of the cell cycle (G1, S and G2). Tritiated thymidine was used to determine labelling indices and grain counts. Colcemid was used to observe the mitotic rate. An initial decrease followed by a subsequent significant increase compared to the non-BLM-treated controls was observed in all parameters studied except the mitotic rate, which remained lower than in the control animals during all 48 hours. The transit time of the cells through the S-phase was initially slightly prolonged, but thereafter it seemed to be shorter than that of the controls. BLM seems to provoke a partial blocking of cells in the G1 phase. When the block is released, a greater number of cells pass through the S phase in partial synchrony at a higher than normal speed. BLM induced a low mitotic rate which remained below the level of that of the normal animals after stripping, even though there obviously was a considerably higher influx of cells from the S phase to the G2 phase. This resulted in a subsequent accumulation of cells in the G2-phase. Thus, BLM has also a blocking effect on the G2-M boundary of the cell cycle. This inhibitory effect of BLM on the mitotic rate was shown to be independent of the effect of BLM on the DNA synthesis. BLM therefore seems to have complex influence on epidermal cell kinetics in vivo. Cells in G1-phase are partially and transiently blocked, but this block is soon released. These cells thereafter pass through the S-phase and pile up in the G2-phase, because BLM also blocks the passage of cells from the G2-phase to mitosis. The overall reduction in cell proliferation seen after BLM in vivo seems mainly to be due to the effect on the G2-M boundray of the cell cycle.     

Lymphoblasts already in the DNA synthesis phase of the cell cycle can be reversibly arrested at the R/G transition

Early and late S-phase of the cell cycle are separated by the R-band/G-band (R/G) transition. This corresponds to the time at which R-band synthesis has been completed while G-band synthesis has yet to begin. The aim of this work was to study cell cycle kinetics during S-phase using different blocking agents: mimosine, methotrexate, 5-fluorouracil, 5-fluoro-2'-deoxyuridine and an excess of thymidine. The stage at which these blocking agents arrest the cell cycle and their efficiency at blocking Epstein-Barr virus transformed lymphoblasts at the R/G transition were evaluated using flow cytometric techniques. Mimosine blocked 90% of the cells near the G1/S-phase boundary. Methotrexate, 5-fluoro-2'-deoxyuridine and 5-fluorouracil, and particularly thymidine, let a significant proportion of cells enter S-phase. The cells were released from the arrest state and their progression through early S-phase was monitored by flow cytometry. Before the cells reached the R/G transition, a second agent was added to inhibit cell cycle progression. For example, the use of mimosine followed by thymidine allowed up to 60% of the cells to be blocked at the R/G transition. The arrest of DNA replication at the R/G transition was confirmed by a marked decrease of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation, revealed by using bivariate flow cytometric analysis. The blocking agent was then removed and the cell cohort was released in the presence of BrdUrd so that replication banding analysis could be performed on the harvested mitotic cells. This yielded a mitotic index of approximately 10% and chromosomes showing replication bands. Flow cytometric analysis combined with cytogenetic banding analysis suggested that the R/G transition is an arrest point within the S-phase of the cell cycle and allowed us to conclude that only cells that have already initiated S-phase are blocked at this point. It corresponds to a susceptible site where S-phase can be arrested easily. The R/G transition could also be a regulatory checkpoint within S-phase, a checkpoint that could respond to imbalance in deoxyribonucleotide pools.     

Cell cycle-dependent effects of wortmannin on radiation survival and mutation

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.     

The blocking of the proliferation of the human endothelial cells in culture by beta-irradiation from internal and external sources of the radiation. S-block is induced by the 3H2O beta-particles

Recently we shown that low doses (0.12-0.46 Gy) of (methyl-3H)-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. The temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the induction of the G2-block estimated by flow cytometry analysis of DNA content and in the induction of the chromosome aberrations (bridges and fragments in anaphase). The aim of this study was the comparative investigation of efficiency of beta-rays emitted 3H from 3H-thymidine and 3H2O by several of the cellular parameters. Here we shown that at the equal conditions of the incubation of the cells in medium with 3H2O induced the accumulation cells in S-phase without decreasing of the mitotic activity and without increasing of the chromosome aberrations level. Unlike from 3H2O the incubation of the cells with 3H-thymidine induced the accumulation cells in G2-phase with decrease of the mitotic activity and with increase of the chromosome aberrations level. Concurrent treatment cells with 3H-thymidine and thymidine abrogate these cellular effects of the 3H-thymidine. Inhibitor ATM-kinase caffeine abrogate as G2-block as S-phase block. These results suggest that the low-dose beta-radiation activates S-phase and G2-phase checkpoints requiring ATM-mediated signal transduction pathway. The factors, which impact on the efficiency of the internal and of the external sources of the irradiation, depend on theirs disposition in relation to radiosensitive target--DNA was discussed.     

Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.     

Centrosome duplication proceeds during mimosine-induced G1 cell cycle arrest

Centrosome duplication must remain coordinated with cell cycle progression to ensure the formation of a strictly bipolar mitotic spindle, but the mechanisms that regulate this coordination are poorly understood. Previous work has shown that prolonged S-phase is permissive for centrosome duplication, but prolonging either G2 or M-phase cannot support duplication. To examine whether G1 is permissive for centrosome duplication, we release serum-starved G0 cells into mimosine, which delays the cell cycle in G1. We find that in mimosine, centrosome duplication does occur, albeit slowly compared with cells that progress into S-phase; centrosome duplication in mimosine-treated cells also proceeds in the absence of a rise in Cdk2 kinase activity normally associated with the G1/S transition. CHO cells arrested with mimosine can also assemble more than four centrioles (termed "centrosome amplification"), but the extent of centrosome amplification during prolonged G1 is decreased compared to cells that enter S-phase and activate the Cdk2-cyclin complex. Together, our results suggest a model, which predicts that entry into S-phase and the rise in Cdk2 activity associated with this transition are not absolutely required to initiate centrosome duplication, but rather, serve to entrain the centrosome reproduction cycle with cell cycle progression.     

Inhibitors of Histone Deacetylases Alter Kinetochore Assembly by Disrupting Pericentromeric Heterochromatin

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-? on pericentromeric heterochromatin in S-phase and G2, decreased pericentromeric targeting of Aurora B kinase, resulting in decreased pre-mitotic phosphorylation of pericentromeric histone H3(S10) in G2, followed by assembly of deficient kinetochores in M-phase. HP1-?, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.     

Cytostatic effect of cyclophosphane on esophageal tumors and epithelium of mice

Investigation of cytostatic activity of cyclophosphamide in sarcoma 37 and esophagus epithelium in albino mice with respect to the diurnal rhythm of mitotic activity and the number of labeled nuclei was performed. Apparently the tumour cells in the G1-phase and at the beginning of the S-phase of the mitotic cycle were the most sensitive to the inhibitory effect of this drug. During the completion of the DNA-synthesis period the cell resistance to the action of the cytostatic increased. Cells at the G1-phase of the mitotic cycle were sensitive to the inhibitory action of cyclophosphane in the esophageal epithelium.     

Heat production of mammalian cells at different cell-cycle phases

1. 1.|Heat production of Reuber H35 rat hepatoma cells and murine C1300 neuroblastoma cells at different stages of the cell cycle were measured microcalorimetrically.

2. 2.|Reuber H35 monolayer cultures of G1-phase cells and cells in S-phase were trypsinized, reincubated in suspension culture and immediately used for microcalorimetric measurements. There was a remrkable difference in the heat evolution of H35-cells in suspension derived from a monolayer culture of G1-phase cells and that of cells in S-phase of the cell cycle. Heat production of G1-cells was relatively continuous during the experiment, in contrast to S-phase cells that showed a decrease in heat production after an initial maximum.

3. 3.|Neuroblastoma cells synchronized by mitotic shake-off and cultured in suspension progressed through their cell cycle. They showed maximal heat production shortly before and durign mitosis.

The role of RBF in developmentally regulated cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth

During Drosophila eye development, cell proliferation is coordinated with differentiation. Immediately posterior to the morphogenetic furrow, cells enter a synchronous round of S phase called second mitotic wave. We have examined the role of RBF, the Drosophila RB family homolog, in cell cycle progression in the second mitotic wave. RBF-280, a mutant form of RBF that has four putative cdk phosphorylation sites mutated, can no longer be regulated by Cyclin D or Cyclin E. Expression of RBF-280 in the developing eye revealed that RBF-280 does not inhibit G1/S transition in the second mitotic wave, rather it delays the completion of S phase and leads to abnormal eye development. These observations suggest that RB/E2F control the rate of S-phase progression instead of G1/S transition in the second mitotic wave. Characterization of the role of RBF in Cyclin D/Cdk4-mediated cellular growth showed that RBF-280 blocks Cyclin D/Cdk4 induced cellular growth in the proliferating wing disc cells but not in the non-dividing eye disc cells. By contrast, RBF-280 does not block activated Ras-induced cellular growth. These results suggest that the ability of Cyclin D/Cdk4 to drive growth in the proliferating wing cells is distinct from that in the none-dividing eye cells or the ability of activated Ras to induce growth, and that RBF may have a role in regulating growth in the proliferating wing discs.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Control over the onset of DNA synthesis in fission yeast

The fission yeast Schizosaccharomyces pombe has been used to identify gene functions required for the cell to become committed to the mitotic cell cycle and to initiate the processes leading to chromosome replication in S-phase. Two gene functions cdc2 and cdc10 must be executed for the cell to traverse 'start' and proceed from G1 into S-phase. Before the completion of these two functions the cell is in an uncommitted state and can undergo alternative developmental fates such as conjugation. A third gene, suc1, has also been identified whose product may interact directly with that of cdc2 at 'start'. The molecular functions of the genes involved in the completion of 'start' have been investigated. The cdc2 gene has been shown to be a protein kinase, suggesting that phosphorylation may be involved in the control over the transition from G1 into S-phase. The biochemical functions of the cdc10 and suc1 gene products have not yet been elucidated. A control at 'start' has also been shown to exist in the budding yeast Saccharomyces cerevisiae. Traverse of 'start' requires the execution of the CDC28 gene function. The cdc2 and CDC28 gene products (lower-case letters represent genes of Schizosaccharomyces pombe, and capital letters genes of Saccharomyces cerevisiae) are functionally homologous, suggesting that the processes involved in traverse of 'start' are highly conserved. An analogous control may also exist in the G1 period of mammalian cells, suggesting that the 'start' control step, after which cells become committed to the mitotic cell cycle, may have been conserved through evolution.     

Reduced expression of GINS complex members induces hallmarks of pre-malignancy in primary untransformed human cells

In cancer cells ablation of the GINS complex member Psf2 elicits chromosome mis-segregation yet the precise role of Psf2 in mitosis is unknown. We investigated the putative mitotic role of the GINS complex using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Metaphase spreads from Psf1/Psf2-depleted HDF were normal and mitotic exit of Psf1/Psf2-depleted cells was only slightly delayed, suggesting no direct role for the GINS complex in mitosis of untransformed cells. Because the GINS complex is required for initiation and elongation events during DNA replication we hypothesized that the mitotic delay of Psf1/Psf2-deficient cells resulted indirectly from defective DNA synthesis during a prior S-phase. Therefore, we investigated the effects of Psf1/Psf2-depletion on DNA replication. Recruitment of Mcm7 to chromatin during G1 was unaffected by Psf1/Psf2-ablation, indicating that replication licensing does not require GINS. However, chromatin-binding of Cdc45 and PCNA, onset of DNA synthesis and accumulation of G2/M markers were delayed in Psf1/Psf2-ablated cells. The cell cycle delay of Psf1/Psf2-depleted HDF was associated with several hallmarks of pre-malignancy including γH2AX, Thr 68-phosphorylated Chk2, and increased numbers of aberrant fragmented nuclei. Ectopic expression of catalytically-inactive Chk2 promoted S-phase and G2/M progression in Psf1/Psf2-depleted cells, as evidenced by modestly-increased rates of DNA synthesis and increased dephosphorylation of Cdc2. Therefore, S-phase progression of untransformed cells containing sub-optimal levels of Psf1/2 is associated with replication stress and acquisition of DNA damage. The ensuing Chk2-mediated DNA damage signalling likely contributes to maintenance of chromosomal integrity.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.