(1/4298) The yeast ser/thr phosphatases sit4 and ppz1 play opposite roles in regulation of the cell cycle.
Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition. (+info)
(2/4298) p27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry.
In this study the direct involvement of cadherins in adhesion-mediated growth inhibition was investigated. It is shown here that overexpression of N-cadherin in CHO cells significantly suppresses their growth rate. Interaction of these cells and two additional fibroblastic lines with synthetic beads coated with N-cadherin ligands (recombinant N-cadherin ectodomain or specific antibodies) leads to growth arrest at the G1 phase of the cell cycle. The cadherin-reactive beads inhibit the entry into S phase and the reduction in the levels of cyclin-dependent kinase (cdk) inhibitors p21 and p27, following serum-stimulation of starved cells. In exponentially growing cells these beads induce G1 arrest accompanied by elevation in p27 only. We propose that cadherin-mediated signaling is involved in contact inhibition of growth by inducing cell cycle arrest at the G1 phase and elevation of p27 levels. (+info)
(3/4298) Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A.
The PLZF gene was identified by its fusion with the RARalpha locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RARalpha-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RARalpha-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL. (+info)
(4/4298) Interleukin-6 dependent induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 is lost during progression of human malignant melanoma.
Human melanoma cell lines derived from early stage primary tumors are particularly sensitive to growth arrest induced by interleukin-6 (IL-6). This response is lost in cell lines derived from advanced lesions, a phenomenon which may contribute to tumor aggressiveness. We sought to determine whether resistance to growth inhibition by IL-6 can be explained by oncogenic alterations in cell cycle regulators or relevant components of intracellular signaling. Our results show that IL-6 treatment of early stage melanoma cell lines caused G1 arrest, which could not be explained by changes in levels of G1 cyclins (D1, E), cdks (cdk4, cdk2) or by loss of cyclin/cdk complex formation. Instead, IL-6 caused a marked induction of the cdk inhibitor p21WAF1/CIP1 in three different IL-6 sensitive cell lines, two of which also showed a marked accumulation of the cdk inhibitor p27Kip1. In contrast, IL-6 failed to induce p21WAF1/CIP1 transcript and did not increase p21WAF1/CIP1 or p27kip1 proteins in any of the resistant lines. In fact, of five IL-6 resistant cell lines, only two expressed detectable levels of p21WAF1/CIP1 mRNA and protein, while in three other lines, p21WAF1/CIP1 was undetectable. IL-6 dependent upregulation of p21WAF1/CIP1 was associated with binding of both STAT3 and STAT1 to the p21WAF1/CIP1 promoter. Surprisingly, however, IL-6 stimulated STAT binding to this promoter in both sensitive and resistant cell lines (with one exception), suggesting that gross deregulation of this event is not the unifying cause of the defect in p21WAF1/CIP1 induction in IL-6 resistant cells. In somatic cell hybrids of IL-6 sensitive and resistant cell lines, the resistant phenotype was dominant and IL-6 failed to induce p21WAF1/CIP1. Thus, our results suggest that in early stage human melanoma cells, IL-6 induced growth inhibition involves induction of p21WAF1/CIP1 which is lost in the course of tumor progression presumably as a result of a dominant oncogenic event. (+info)
(5/4298) Caffeine can override the S-M checkpoint in fission yeast.
The replication checkpoint (or 'S-M checkpoint') control prevents progression into mitosis when DNA replication is incomplete. Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells. We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. By contrast, no comparable effects of caffeine on the S. pombe DNA damage checkpoint were seen. S. pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2. Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions. This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest. The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest. In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S. pombe genes isolated in a cDNA library screen. These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control. (+info)
(6/4298) Regulation of the start of DNA replication in Schizosaccharomyces pombe.
Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S. We suggest that cell mass at the G1/S transition point is variable and determined by a set of molecular parameters. In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate. (+info)
(7/4298) Immunologic proliferation marker Ki-S2 as prognostic indicator for lymph node-negative breast cancer.
BACKGROUND: Proper treatment of lymph node-negative breast cancer depends on an accurate prognosis. To improve prognostic models for this disease, we evaluated whether an immunohistochemical marker for proliferating cells, Ki-S2 (a monoclonal antibody that binds to a 100-kd nuclear protein expressed in S, G2, and M phases of the cell cycle), is an accurate indicator of prognosis. METHODS: We studied 371 Swedish women with lymph node-negative breast cancer; the median follow-up time was 95 months. The fraction of tumor cells in S phase was assessed by flow cytometry, and tumor cell proliferation was measured immunohistochemically with the monoclonal antibodies Ki-S2 and Ki-S5 (directed against the nuclear antigen Ki-67). A combined prognostic index was calculated on the basis of the S-phase fraction, progesterone receptor content, and tumor size. RESULTS: In multivariate analyses that did or did not (263 and 332 observations, respectively) include the S-phase fraction and the combined prognostic index, the Ki-S2 labeling index (percentage of antibody-stained tumor cell nuclei) emerged as the most statistically significant predictor of overall survival, disease-specific survival, and disease-free survival (all two-sided P<.0001). In the risk group defined by a Ki-S2 labeling index of 10% or less, life expectancy was not statistically significantly different from that of age-matched women without breast cancer, whereas the group with a high Ki-S2 labeling index had an increased risk of mortality of up to 20-fold. CONCLUSIONS: Cellular proliferation is a major determinant of the biologic behavior of breast cancer. Prognosis is apparently best indicated by the percentage of cells in S through M phases of the cell cycle. Measurement of the Ki-S2 labeling index of a tumor sample may improve a clinician's ability to make an accurate prognosis and to identify patients with a low risk of recurrence who may not need adjuvant therapy. (+info)
(8/4298) Fus3p and Kss1p control G1 arrest in Saccharomyces cerevisiae through a balance of distinct arrest and proliferative functions that operate in parallel with Far1p.
In Saccharomyces cerevisiae, mating pheromones activate two MAP kinases (MAPKs), Fus3p and Kss1p, to induce G1 arrest prior to mating. Fus3p is known to promote G1 arrest by activating Far1p, which inhibits three Clnp/Cdc28p kinases. To analyze the contribution of Fus3p and Kss1p to G1 arrest that is independent of Far1p, we constructed far1 CLN strains that undergo G1 arrest from increased activation of the mating MAP kinase pathway. We find that Fus3p and Kss1p both control G1 arrest through multiple functions that operate in parallel with Far1p. Fus3p and Kss1p together promote G1 arrest by repressing transcription of G1/S cyclin genes (CLN1, CLN2, CLB5) by a mechanism that blocks their activation by Cln3p/Cdc28p kinase. In addition, Fus3p and Kss1p counteract G1 arrest through overlapping and distinct functions. Fus3p and Kss1p together increase the expression of CLN3 and PCL2 genes that promote budding, and Kss1p inhibits the MAP kinase cascade. Strikingly, Fus3p promotes proliferation by a novel function that is not linked to reduced Ste12p activity or increased levels of Cln2p/Cdc28p kinase. Genetic analysis suggests that Fus3p promotes proliferation through activation of Mcm1p transcription factor that upregulates numerous genes in G1 phase. Thus, Fus3p and Kss1p control G1 arrest through a balance of arrest functions that inhibit the Cdc28p machinery and proliferative functions that bypass this inhibition. (+info)