The no-observed-adverse-effect level of phthalates promotes proliferation and cell cycle progression in normal human breast cells.

OBJECTIVE: The data on the association between phthalates and breast cancer risk remains inconsistent. This study aimed to explore the possible mechanism of low-dose exposures of phthalates, including Butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(20ethylhexyl) phthalate (DEHP), on breast tumorigenesis. METHODS AND METHODS: MCF-10A normal breast cells were treated with phthalates (10 and 100 nM) and 17ß-estradiol (E2, 10 nM), which were co-cultured with fibroblasts from normal mammary tissue. Cell viability, cycle, and apoptosis were detected by MTT assay, flow cytometry, and TUNEL assay respectively. The expression levels of related proteins were determined by Western blot. RESULTS: Like E2, both 10 nM and 100 nM phthalates exerted significantly higher cell viability, lower apoptosis, and increased cell numbers in the S and G2/M phases with up-regulation of cyclin D/CDK4, cyclin E/CDK2, cyclin A/CDK2, cyclin A/CDK1, and cyclin B/CDK1, compared with the control group. Significant increase in PDK1, P13K, p-AKT, p-mTOR, and BCL-2 expression and a decrease in Bax protein, cytochrome C, caspase 8, and caspase 3 levels were noted in cells treated with 10 nM and 100 nM phthalates and E2, compared with the control group and MCF-10A cells co-cultured with fibroblasts. The effects of the three phthalates were noted to be dose-dependent. CONCLUSIONS: The results indicate that phthalates at a level below its no-observed-adverse-effect concentration, as defined by the current standards, still induce cell cycle progression and proliferation as well as inhibit apoptosis of normal breast cells. Thus, the possibility of breast tumorigenesis through chronic phthalate exposure should be considered.

Daidzein and Chicory Extract Arrest the Cell Cycle via Inhibition of Cyclin D/CDK4 and Cyclin A/CDK2 Gene Expression in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers, associated with a high rate of mortality. A disturbance between cell proliferation and cell death is one of the cancer hallmarks including HCC. Cell proliferation is mainly controlled by the cell cycle. The arrest of the cell cycle is one of the important targets of anticancer agents. OBJECTIVES: The present study tries to clarify the exact role of some natural products such as daidzein (DAZ) and alcoholic chicory leaf extract (CE), as possible regulators of cell cycle and apoptosis. METHODS: HCC in rats was induced using diethylnitrosamine (DENA). Ninety rats were allocated and divided equally into nine groups, treated with CE, DAZ, a combination of both, and sorafenib with non-treated control groups. RESULTS: Treatment with CE, DAZ, and their combination significantly downregulated hepatic tissue expression of cyclin D1/CDK4 axis as well as cyclin A/CDK2 axis. The suggested therapeutic protocol inhibited the proliferation and dampened Bcl-2 expression. Furthermore, the efficiency of combining CE and DAZ demonstrated a potency comparable to sorafenib in terms of cyclin D/CDK4 axis expression, as well as; this combination protocol was more potent in revealing a potentiated inhibitory effect on cyclin A and Ki-67 expression. CONCLUSION: Treatment with DAZ or CE alone, or in combination, could possess an inhibitory effect on hepatocarcinogenesis via cell cycle arrest, inhibition of proliferation through suppression of Ki-67 expression, and apoptosis induction, mediated by downregulation of Bcl-2.

High Glucose Induced Upregulation of Cyclin a Associating with a Short Survival of Patients with Cholangiocarcinoma: A Potential Target for Treatment of Patients with Diabetes Mellitus.

Diabetes mellitus (DM) is associated with an increased risk and progression of cholangiocarcinoma (CCA). High glucose underlying the association between DM and CCA by modulating the intracellular signaling has been demonstrated. However, the effects of DM and hyperglycemia on cell cycle machineries and progression of CCA remain elucidated. CCA cells, KKU-213A and KKU-213B were cultured in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) resembling euglycemia and hyperglycemia. Western blotting was used to determine expressions of cell cycle machineries in CCA cells. The expression of cyclin A in CCA tissues from patients with or without hyperglycemia was determined by immunohistochemistry. Pan-cyclin dependent kinases (CDKs) inhibitor and silencing of cyclin A expression were investigated as a possible modality targeting CCA treatment in patients with DM. High glucose induced expression of cell cycle machinery proteins in both CCA cells. Among these, cyclin A was consistently and significantly upregulated. Nuclear cyclin A was significantly increased in tumor tissues from CCA patients with hyperglycemia and was significantly associated with post-operative survival of shorter than 5 mo. Silencing cyclin A expression sensitized CCA cells to pan-CDKs inhibitor, suggesting the combined treatment as an alternative approach for treatment of CCA patients with DM.

Falcarindiol and dichloromethane fraction are bioactive components in Oplopanax elatus: Colorectal cancer chemoprevention via induction of apoptosis and G2/M cell cycle arrest mediated by cyclin A upregulation.

Oplopanax elatus (Nakai) Nakai has a long history of use as an ethnomedicine by the people living in eastern Asia. However, its bioactive constituents and cancer chemopreventive mechanisms are largely unknown. The aim of this study was to prepare O. elatus extracts, fractions, and single compounds and to investigate the herb's antiproliferative effects on colon cancer cells and the involved mechanisms of action. Two polyyne compounds were isolated from O. elatus, falcarindiol and oplopandiol. Based on our HPLC analysis, falcarindiol and oplopandiol are major constituents in the dichloromethane (CH2Cl2) fraction. For the HCT-116 cell line, the dichloromethane fraction showed significant effects. Furthermore, the IC50 for falcarindiol and oplopandiol was 1.7 µM and 15.5 µM, respectively. In the mechanistic study, after treatment with 5 µg/ml for 48 h, dichloromethane fraction induced cancer cell apoptosis by 36.5% (p < 0.01% vs. control of 3.9%). Under the same treatment condition, dichloromethane fraction caused cell cycle arrest at the G2/M phase by 32.6% (p < 0.01% vs. control of 23.4%), supported by upregulation of key cell cycle regulator cyclin A to 21.6% (p < 0.01% vs. control of 8.6%). Similar trends were observed by using cell line HT-29. Data from this study filled the gap between phytochemical components and the cancer chemoprevention of O. elatus. The dichloromethane fraction is a bioactive fraction, and falcarindiol is identified as an active constituent. The mechanisms involved in cancer chemoprevention by O. elatus were apoptosis induction and G2/M cell cycle arrest mediated by a key cell cycle regulator cyclin A.

NFATc1 targets cyclin A in the regulation of vascular smooth muscle cell multiplication during restenosis.

Platelet-derived growth factor BB (PDGF-BB) induced cyclin A expression and CDK2 activity in vascular smooth muscle cells (VSMC). Inhibition of nuclear factors of activated T cell (NFAT) activation by cyclosporin A (CsA) and VIVIT suppressed PDGF-BB-induced cyclin A expression and CDK2 activity, resulting in blockade of VSMC in the G(1) phase. In addition, CsA- and VIVIT-mediated inhibition of NFATs and small interfering RNA-targeted down-regulation of cyclin A levels suppressed PDGF-BB-induced VSMC DNA synthesis. PDGF-BB also induced cyclin A mRNA levels in VSMC in an NFAT-dependent manner. Cloning and bioinformatic analysis of rat cyclin A promoter revealed the presence of NFAT-binding elements, and PDGF-BB induced the binding of NFATs to these regulatory sequences in a CsA- and VIVIT-sensitive manner. Chromatin immunoprecipitation analysis showed that NFATc1 binds to the cyclin A promoter in response to PDGF-BB in a VIVIT-sensitive manner. Furthermore, PDGF-BB induced cyclin A promoter-luciferase reporter gene activity in VSMC, and it was inhibited by both CsA and VIVIT. Balloon injury induced cyclin A expression and CDK2 activity in rat carotid arteries, and these responses were also blocked by VIVIT. In addition, VIVIT attenuated balloon injury-induced SMC proliferation, resulting in reduced restenosis. Down-regulation of NFATc1 by its small interfering RNA inhibited PDGF-BB-induced cyclin A expression and DNA synthesis both in rat and human VSMC. Together, these findings demonstrate that the cyclin A-CDK2 complex may be a potential effector of NFATs, specifically NFATc1, in mediating SMC multiplication leading to neointima formation. Therefore, NFATs may be used as target molecules for the development of therapeutic agents against vascular diseases such as restenosis.

Pin1 stabilizes Emi1 during G2 phase by preventing its association with SCF(betatrcp).

The anaphase-promoting complex (APC) early mitotic inhibitor 1 (Emi1) is required to induce S- and M-phase entries by stimulating the accumulation of cyclin A and cyclin B through APC(Cdh1/cdc20) inhibition. In this report, we show that Emi1 proteolysis can be induced by cyclin A/cdk (cdk for cyclin-dependent kinase). Paradoxically, Emi1 is stable during G2 phase, when cyclin A/cdk, Plx1 and SCF(betatrcp) (SCF for Skp1-Cul1-Fbox protein)--which play a role in its degradation--are active. Here, we identify Pin1 as a new regulator of Emi1 that induces Emi1 stabilization by preventing its association with SCF(betatrcp). We show that Pin1 binds to Emi1 and prevents its association with betatrcp in an isomerization-dependent pathway. We also show that Emi1-Pin1 binding is present in vivo in XL2 cells during G2 phase and that this association protects Emi1 from being degraded during this phase of the cell cycle. We propose that S- and M-phase entries are mediated by the accumulation of cyclin A and cyclin B through a Pin1-dependent stabilization of Emi1 during G2.

Reactivity of Cdc25 phosphatase at low pH and with thiophosphorylated protein substrate.

Cdc25s, dual-specificity phosphatases that dephosphorylate and activate cyclin-dependent kinases, are important regulators of the eukaryotic cell cycle. Herein, we probe the protonation state of the phosphate on the protein substrate of Cdc25 by pH-dependent studies and thiosubstitution. We have extended the useable range of pH for this enzyme substrate pair by using high concentrations of glycerol under acidic conditions. Using the protein substrate, we find a slope of 2 for the acidic side of the bell-shaped pH-rate profile, as found with other protein tyrosine phosphatases. Using thiophosphorylated protein substrate, we find no change in the basic side of the pH-rate profile, despite a large reduction in activity as measured by kcat/Km (0.18%) or kcat (0. 11%). In contrast, the acidic side of the profile changes shows a slope of 1, consistent with the 1.5 pH unit shift associated with thiosubstitution. Thus, Cdc25, like other protein phosphatases, uses a dianionic phosphorylated substrate.

Characterization of a novel ATR-dependent, Chk1-independent, intra-S-phase checkpoint that suppresses initiation of replication in Xenopus.

In most eukaryotes, replication origins fire asynchronously throughout S-phase according to a precise timing programme. When replication fork progression is inhibited, an intra-S-phase checkpoint is activated that blocks further origin firing and stabilizes existing replication forks to prevent them undergoing irreversible collapse. We show that chromatin incubated in Xenopus egg extracts displays a replication-timing programme in which firing of new replication origins during S phase depends on the continued activity of S-phase-inducing cyclin-dependent kinases. We also show that low concentrations of the DNA-polymerase inhibitor aphidicolin, which only slightly slows replication-fork progression, strongly suppress further initiation events. This intra-S-phase checkpoint can be overcome by caffeine, an inhibitor of the ATM/ATR checkpoint kinases, or by neutralizing antibodies to ATR. However, depletion or inhibition of Chk1 did not abolish the checkpoint. We could detect no significant effect on fork stability when this intra-S-phase checkpoint was inhibited. Interestingly, although caffeine could prevent the checkpoint from being activated, it could not rescue replication if added after the timing programme would normally have been executed. This suggests that special mechanisms might be necessary to reverse the effects of the intra-S-phase checkpoint once it has acted on particular origins.

Cyclin A correlates with the sensitivity of human cancer cells to cytotoxic effects of 5-FU.

We investigated the role of cyclin A in the cytotoxic effect of 5-fluorouracil (5-FU) on cancer cell lines. Experiments were performed using gastric cancer chemosensitive NUGC3, and chemoresistant NUGC3/5FU/L established by repeated exposure to 5-FU. 5-FU inhibited cell growth of NUGC3 in a dose-dependent manner. Low concentrations of 5-FU did not inhibit cell growth of NUGC3/5FU/L, while high concentrations slightly inhibited cell growth. Examination of the cell cycle pattern of NUGC3 cells showed accumulation at S-phase at 10 micro M and at G1-S-phase at 100 micro M of 5-FU. Cell cycle pattern of NUGC3/5FU/L cells did not change 5-FU concentrations. 5-FU increased cyclin A mRNA level in NUGC3 cells but not NUGC3/5FU/L cells. In the presence of 100 micro M 5-FU, cyclin A protein level increased 2.6-fold in NUGC3 and 1.47-fold in NUGC3/5FU/L. 5-FU dose-dependently increased the percentage of cyclin A-positive NUGC3 cells, but not NUGC3/5FU/L cells. The percentage of cyclin A-positive cells in other 5-FU sensitive esophageal, colon and gastric cancer cell lines (T.Tn, LOVO, DLD-1, MKN-7), increased in the presence of 1 and 10 micro M 5-FU, while cyclin A-positive cells in 5-FU resitant hepatocellular and colon carcinoma cell lines (HCC50 and C-1), did not increase with the same treatment. Our results indicate that the cytotoxic effects of 5-FU in human cancer cell lines correlate with cyclin A and it may be used as a predictive factor for chemotherapy response.

Tumor necrosis factor alpha inhibits cyclin A expression and retinoblastoma hyperphosphorylation triggered by insulin-like growth factor-I induction of new E2F-1 synthesis.

Cyclin A is required for cell cycle S phase entry, and its overexpression contributes to tumorigenesis. Release of pre-existing E2Fs from inactive complexes of E2F and hypophosphorylated retinoblastoma (RB) is the prevailing dogma for E2F transcriptional activation of target genes such as cyclin A. Here we explored the hypothesis that new synthesis of E2F-1 is required for insulin-like growth factor-I (IGF-I) to induce cyclin A accumulation and RB hyperphosphorylation, events that are targeted by tumor necrosis factor alpha (TNFalpha) to arrest cell cycle progression. We first established that IGF-I increases expression of cyclin A, causes hyperphosphorylation of RB, and augments the mass of E2F-1 in a time-dependent manner. As expected, E2F-1 small interfering RNA blocks the ability of IGF-I to increase synthesis of E2F-1. Most important, this E2F-1 small interfering RNA also blocks the ability of IGF-I to increase cyclin A accumulation and to hyperphosphorylate RB. We next established that TNFalpha dose-dependently inhibits IGF-I-induced phosphorylation of both RB and histone H1 by cyclin A-dependent cyclin-dependent kinases. Cyclin-dependent kinase 2 (Cdk2) mediates this suppression because co-immunoprecipitation experiments revealed that TNFalpha reduces the amount of IGF-I-induced cyclin A that binds Cdk2, leading to a reduction in Cdk2 enzymatic activity. TNFalpha antagonizes the ability of IGF-I to increase mass of both E2F-1 and cyclin A but not cyclin E or D1. The cytostatic property of TNFalpha is also shown by its ability to block IGF-I-stimulated luciferase activity of a cyclin A promoter reporter. Deletion of an E2F recognition site from this reporter eliminates the regulatory effects of both IGF-I and TNFalpha on cyclin A transcription, indicating the essential role of E2F-1 in mediating their cross-talk. Collectively, these results establish that TNFalpha targets IGF-I-induced E2F-1 synthesis, leading to inhibition of the subsequent accumulation in cyclin A, formation of cyclin A-Cdk2 complexes, hyperphosphorylation of RB, and cell cycle arrest.

A p53-dependent checkpoint pathway prevents rereplication.

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.

Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes.

Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B.Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification.

The binding of ORC2 to chromatin from terminally differentiated cells.

Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the prereplication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.

Cyclin A transcriptional suppression is the major mechanism mediating homocysteine-induced endothelial cell growth inhibition.

Previously, it was reported that homocysteine (Hcy) specifically inhibits the growth of endothelial cells (ECs), suppresses Ras/mitogen-activated protein (MAP) signaling, and arrests cell growth at the G(1)/S transition of the cell cycle. The present study investigated the molecular mechanisms underlying this cell-cycle effect. Results showed that clinically relevant concentrations (50 microM) of Hcy significantly inhibited the expression of cyclin A messenger RNA (mRNA) in ECs in a dose- and time-dependent manner. G(1)/S-associated molecules that might account for this block were not changed, because Hcy did not affect mRNA and protein expression of cyclin D1 and cyclin E. Cyclin D1- and E-associated kinase activities were unchanged. In contrast, cyclin A-associated kinase activity and CDK2 kinase activity were markedly suppressed. Nuclear run-on assay demonstrated that Hcy decreased the transcription rate of the cyclin A gene but had no effect on the half-life of cyclin A mRNA. In transient transfection experiments, Hcy significantly inhibited cyclin A promoter activity in endothelial cells, but not in vascular smooth muscle cells. Finally, adenovirus-transduced cyclin A expression restored EC growth inhibition and overcame the S phase block imposed by Hcy. Taken together, these findings indicate that cyclin A is a critical functional target of Hcy-mediated EC growth inhibition.

Alteration of cell growth and morphology by overexpression of transforming growth factor beta type II receptor in human lung adenocarcinoma cells.

TGF-beta is a potent inhibitory regulator of cell growth, which is transduced through interaction between type I (RI) and type II (RII) receptors that form heteromeric kinase complexes. Abnormal expression of these receptors has been identified in several human epithelial cancers and has been shown to be highly associated with resistance to TGF-beta. In this study, we investigated the expression of RI and RII in 13 human non-small cell lung cancer cell lines (NSCLCs) and demonstrated decreased or loss of RII expression in five lung cancer cell lines, but not of RI. Of these cell lines, the role of RII in NCI-H358 adenocarcinoma, which lacks RII and is insensitive to TGF-beta, was investigated by transducing this cell line with a recombinant retrovirus expressing full-length TGF-beta RII. Stably transfected cells showed significant increase in RII mRNA and protein expression. These cells responded to exogenous TGF-beta1 with suppressed proliferation in a dose-dependent manner and G1 arrest accompanied by morphological change distinct from control cells. We also investigated whether overexpression of dominant-negative RII (dnRII) in NCI-H441 adenocarcinoma, which is sensitive but expresses low levels of RII, could block signaling through the receptor complex. The overexpression of this kinase-domain-truncated RII by expressing the retroviral dnRII construct led to loss of the ability to respond to TGF-beta1 and an exhibition of uncontrolled growth. These results suggest a close association between the loss of the expression of wild-type TGF-beta RII and carcinogenesis in human lung cancer cells.

Transcription factors of the Sp1 family: interaction with E2F and regulation of the murine thymidine kinase promoter.

Promoters of growth and cell cycle regulated genes frequently carry binding sites for transcription factors of the E2F and Sp1 families. We have demonstrated recently that direct interaction between Sp1 and a subgroup of the E2F factors is essential for the regulation of certain promoters. We show here that the amino acids necessary for this interaction in both cases are located within the DNA binding domain. This is in line with the assumption, that the interaction between E2F and Sp-factors contributes to promoter-specificity. Cyclin A, which binds to E2F-1 in close vicinity to Sp1 does not interfere with this interaction. Moreover we have investigated the ability of other members of the Sp1 family to interact with E2F-1 and to regulate the activity of the E2F and Sp1 dependent murine thymidine kinase promoter. All four factors of the Sp1 family are able to bind E2F-1 in co-immunoprecipitation and GST-pull down experiments. Mobility shift assays with oligonucleotides comprising the Sp1, or both the Sp1 and the E2F binding site suggest that Sp1 and Sp3 supply most if not all activity binding to the GC-box of the thymidine kinase promoter in murine fibroblasts. Reporter gene assays in Drosophila melanogaster SL2 cells and murine fibroblast 3T6 cells demonstrate that the thymidine kinase promoter is activated strongly by Sp1 and Sp3, weakly by Sp4, and not at all by Sp2. Co-expression of E2F-1 results in synergistic activation in 3T6 but not in SL2 cells.

NF-Y-mediated trans-activation of the human thymidine kinase promoter is closely linked to activation of cyclin-dependent kinase.

Transcriptional activation is important for the elevated expression of human thymidine kinase (hTK) in tumor cells. Here, we used TK(-133/+33)-luciferase reporter gene construct and bandshift assay to study the cis-elements involved in transcriptional activation of the hTK promoter. We found that two CCAAT boxes at -71/-67 and -40/-36 and Sp1 binding site located at -118/-113 were critical for maximal expression of the hTK promoter activity. As Sp1-mediated activation of the hTK promoter was not detectable for the promoter construct with double mutations at two CCAAT boxes, we proposed that NF-Y binding to the hTK promoter sequence is a requisite step for the functional interaction with Sp1. Here, we further showed that the hTK promoter activity was reduced in HeLa cells transfected with p16 or p21, both of which are inhibitors of cyclin-dependent kinases (CDKs). Inhibition of the hTK promoter activity by p16 could be abrogated by overexpression of cyclin A, indicating that the cyclin A activating event is more directly involved in transcriptional activation of the hTK promoter. We thus proposed that NF-Y-mediated activation of the hTK promoter is closely linked to the activation of CDK2/cyclin A pathway.

Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression.

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.

The cell cycle program in germ cells of the Drosophila embryo.

The germ cells of metazoans follow a program of proliferation that is distinct from proliferation programs of somatic cells. Despite their developmental importance, the cell proliferation program in the metazoan primordial germ cells is not well characterized and the regulatory controls are not understood. In Drosophila melanogaster, germ cell precursors (called pole cells) proliferate early in embryogenesis and then enter a prolonged quiescence. We found that polar nuclear divisions are asynchronous and lag behind somatic nuclear divisions during syncytial cycles 9 and 10. Thus, the polar division program deviates from the somatic division program when pole nuclei and somatic nuclei still share a common cytoplasm, earlier than previously thought. The lag in polar nuclear divisions is independent of grapes, which is required for lengthening somatic cell cycles 10-13. Mapping of the last S phase in pole cells and measurement of their DNA content indicate that pole cells become quiescent in G2 phase of the cell cycle. We were able to drive quiescent pole cells into mitosis by induction of either an activator of Cdc2 (Cdc25string phosphatase) or a mutant form of Cdc2 that cannot be inhibited by phosphorylation. In contrast, induction of wild-type Cdc2 with a mitotic cyclin did not induce mitosis in pole cells. We propose that inhibition of Cdc2 by phosphorylation contributes to G2 arrest in pole cells during embryogenesis. Furthermore, pole cells enter G1 following induced mitoses, indicating that entry into both mitosis and S phase is blocked in quiescent pole cells. These studies represent the first molecular characterization of proliferation in embryonic germ cells of Drosophila.