(1/1162) The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis.
The first appearance of G1 during Drosophila embryogenesis, at cell cycle 17, is accompanied by the down-regulation of E2F-dependent transcription. Mutant alleles of rbf were generated and analyzed to determine the role of RBF in this process. Embryos lacking both maternal and zygotic RBF products show constitutive expression of PCNA and RNR2, two E2F-regulated genes, indicating that RBF is required for their transcriptional repression. Despite the ubiquitous expression of E2F target genes, most epidermal cells enter G1 normally. Rather than pausing in G1 until the appropriate time for cell cycle progression, many of these cells enter an ectopic S-phase. These results indicate that the repression of E2F target genes by RBF is necessary for the maintenance but not the initiation of a G1 phase. The phenotype of RBF-deficient embryos suggests that rbf has a function that is complementary to the roles of dacapo and fizzy-related in the introduction of G1 during Drosophila embryogenesis. (+info)
(2/1162) Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts.
The irreversible G1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G1 cyclin-cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21(Sdi1,Cip1,Waf1), which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16(Ink4a), suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by =50% compared to young cells. We also provide new evidence that p21 may play a role in inactivation of the DNA replication factor proliferating cell nuclear antigen during early senescence. Finally, because p16 accumulates in parallel with the increases in senescence-associated beta-Gal activity and cell volume that characterize the senescent phenotype, we suggest that p16 upregulation may be part of a differentiation program that is turned on in senescent cells. Since p21 decreases after senescence is achieved, this upregulation of p16 may be essential for maintenance of the senescent-cell-cycle arrest. (+info)
(3/1162) Progesterone inhibits estrogen-induced cyclin D1 and cdk4 nuclear translocation, cyclin E- and cyclin A-cdk2 kinase activation, and cell proliferation in uterine epithelial cells in mice.
The response of the uterine epithelium to female sex steroid hormones provides an excellent model to study cell proliferation in vivo since both stimulation and inhibition of cell proliferation can be studied. Thus, when administered to ovariectomized adult mice 17beta-estradiol (E2) stimulates a synchronized wave of DNA synthesis and cell division in the epithelial cells, while pretreatment with progesterone (P4) completely inhibits this E2-induced cell proliferation. Using a simple method to isolate the uterine epithelium with high purity, we have shown that E2 treatment induces a relocalization of cyclin D1 and, to a lesser extent, cdk4 from the cytoplasm into the nucleus and results in the orderly activation of cyclin E- and cyclin A-cdk2 kinases and hyperphosphorylation of pRb and p107. P4 pretreatment did not alter overall levels of cyclin D1, cdk4, or cdk6 nor their associated kinase activities but instead inhibited the E2-induced nuclear localization of cyclin D1 to below the control level and, to a lesser extent, nuclear cdk4 levels, with a consequent inhibition of pRb and p107 phosphorylation. In addition, it abrogated E2-induced cyclin E-cdk2 activation by dephosphorylation of cdk2, followed by inhibition of cyclin A expression and consequently of cyclin A-cdk2 kinase activity and further inhibition of phosphorylation of pRb and p107. P4 is used therapeutically to oppose the effect of E2 during hormone replacement therapy and in the treatment of uterine adenocarcinoma. This study showing a novel mechanism of cell cycle inhibition by P4 may provide the basis for the development of new antiestrogens. (+info)
(4/1162) Growth-inhibitory effect of cyclic GMP- and cyclic AMP-dependent vasodilators on rat vascular smooth muscle cells: effect on cell cycle and cyclin expression.
1. The possibility that the antiproliferative effect of cyclic GMP- and cyclic AMP-dependent vasodilators involves an impaired progression of vascular smooth muscle cells (VSMC) through the cell cycle and expression of cyclins, which in association with the cyclin-dependent kinases control the transition between the distinct phases of the cell cycle, was examined. 2. FCS (10%) stimulated the transition of quiescent VSMC from the G0/G1 to the S phase (maximum within 18-24 h and then to the G2/M phase (maximum within 22-28 h). Sodium nitroprusside and 8-Br-cyclic GMP, as well as forskolin and 8-Br-cyclic AMP markedly reduced the percentage of cells in the S phase after FCS stimulation. 3. FCS stimulated the low basal protein expression of cyclin D1 (maximum within 8-24 h) and E (maximum within 8-38 h) and of cyclin A (maximum within 14-30 h). The stimulatory effect of FCS on cyclin D1 and A expression was inhibited, but that of cyclin E was only minimally affected by the vasodilators. 4. FCS increased the low basal level of cyclin D1 mRNA after a lag phase of 2 h and that of cyclin A after 12 h. The vasodilators significantly reduced the FCS-stimulated expression of cyclin D1 and A mRNA. 5. These findings indicate that cyclic GMP- and cyclic AMP-dependent vasodilators inhibit the proliferation of VSMC by preventing the progression of the cell cycle from the G0/G1 into the S phase, an effect which can be attributed to the impaired expression of cyclin D1 and A. (+info)
(5/1162) Heparin inhibits proliferation of myometrial and leiomyomal smooth muscle cells through the induction of alpha-smooth muscle actin, calponin h1 and p27.
Mast cells are widely distributed in human tissues, including the human uterus. However, the function of mast cells in uterine smooth muscle has not been clearly established. Mast cells possess secretory granules containing such substances as heparin, serotonin, histamine and many cytokines. To help establish the role of mast cells in the human myometrium, the action of heparin was investigated using smooth muscle cells (SMC) from normal myometrium and from leiomyoma. The proliferation of cultured myometrial and leiomyomal SMC was inhibited by heparin treatment. Flow cytometric analysis showed that the population in the G1 phase of the cell cycle increased under heparin treatment. Western blotting analysis showed that markers of SMC differentiation such as alpha-smooth muscle actin (alpha-SMA), calponin h1 and cyclin-dependent kinase inhibitor p27 were induced by heparin, whereas cell-cycle-related gene products from the G1 phase of the cell cycle, such as cyclin E and cdk2, were not changed. Taken together, these results indicate that heparin inhibits the proliferation of myometrial and leiomyomal SMC through the induction of alpha-SMA, calponin h1 and p27. We suggest that heparin from mast cells may induce differentiation in uterine SMC and may influence tissue remodelling and reconstruction during physiological and pathophysiological events. (+info)
(6/1162) Impact of 9-(2-phosphonylmethoxyethyl)adenine on (deoxy)ribonucleotide metabolism and nucleic acid synthesis in tumor cells.
Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases alpha, delta and epsilon), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)-adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5-4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radio-labelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2-4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3-4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16-40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 microM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle. (+info)
(7/1162) Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27(Kip1).
Proliferation and apoptosis are increased in many types of inflammatory diseases. A role for the cyclin kinase inhibitor p27(Kip1) (p27) in limiting proliferation has been shown. In this study, we show that p27(-/-) mesangial cells and fibroblasts have strikingly elevated rates of apoptosis, not proliferation, when deprived of growth factors. Apoptosis was rescued by restoration of p27 expression. Cyclin A-cyclin-dependent kinase 2 (CDK2) activity, but not cyclin E-CDK2 activity, was increased in serum-starved p27(-/-) cells, and decreasing CDK2 activity, either pharmacologically (Roscovitine) or by a dominant-negative mutant, inhibited apoptosis. Our results show that a new biological function for the CDK inhibitor p27 is protection of cells from apoptosis by constraining CDK2 activity. These results suggest that CDK inhibitors are necessary for coordinating the cell cycle and cell-death programs so that cell viability is maintained during exit from the cell cycle. (+info)
(8/1162) Retardation of cell proliferation after expression of p202 accompanies an increase in p21(WAF1/CIP1).
p202 is an IFN-inducible, primarily nuclear, phosphoprotein (52-kDa) whose constitutive overexpression in transfected cells inhibits colony formation. To investigate the molecular mechanism(s) by which expression of p202 protein impairs colony formation, we established stable cell lines that inducibly express p202. Using this cell model, we demonstrate that the induced expression of p202 in asynchronous cultures of these cells was accompanied by: (a) an increase in steady-state levels of p21(WAF1/CIP1/SDI1) (p21) mRNA and protein; (b) a decrease in Cdk2 protein kinase activity; and (c) an increase in the functional form of retinoblastoma protein (pRb). Transient transfection of a p202-encoding plasmid in Saos-2 cells, which do not harbor a wild-type p53 protein, resulted in an increase in p21 protein, which indicated that p202 could regulate expression of p21 protein independent of p53 protein. Moreover, we demonstrate that expression of p202 in these cells increased cell doubling time without accumulation of cells in a particular phase of the cell cycle. Taken together, these results are consistent with the possibility that p202 protein contributes to the cell growth retardation activity of the IFNs, at least in part, by modulating p21 protein levels. (+info)