[Show abstract][Hide abstract] ABSTRACT: The transcription factors c-Myc and E2F-1 have been shown to harbour both mitogenic and apoptotic properties. Both factors have been implicated in the regulation of the transition from the G1 phase to the S phase in the mammalian cell cycle. However, whether cell death triggered by these molecules is dependent on the cell's position in the ongoing cell cycle remained elusive. Using centrifugal elutriation we here show for the first time that c-Myc induces apoptosis in G1 and in G2 phase, whereas E2F-1-induced apoptosis specifically occurs in G1. S phase cells are resistant to cell death triggered by these factors. We demonstrate that this is not a general phenomenon, since S phase cells are susceptible to apoptosis induced by treatment with actinomycin D and to the anti-apoptotic activity of Bcl-2. Our data indicate that S phase cells harbour specific protective activities against c-Myc- and E2F-1-induced apoptosis. Our results demonstrate that these transcription factors, although probably sharing specific apoptotic pathways, also take distinct routes to induce cell death and that apoptosis can occur at different phases of the cell cycle depending on the apoptotic stimulus. In this report we present the usefulness of a new approach to determine the regulation of apoptosis in the ongoing unperturbated cell cycle. This approach has clear implications for the identification of target genes involved in the regulation of cell death.
[Show abstract][Hide abstract] ABSTRACT: The initiation of anaphase and exit from mitosis depend on the activation of the cyclosome/anaphase-promoting complex (APC) that ubiquitinates regulatory proteins such as anaphase inhibitors and mitotic cyclins [1-4]. Genetic experiments have demonstrated that two related WD40-repeat proteins--called Cdc20p and Hct1p/Cdh1p in budding yeast and Fizzy and Fizzy-related in Drosophila--are essential for APC--dependent proteolysis [5-11]. Human orthologs of these proteins--hCDC20/p55CDC  and hCDH1--have recently been found to associate with APC in a cell-cycle-dependent manner [13,14]. Here, we show that the amount of hCDC20 and hCDH1 bound to APC correlates with a high ubiquitination activity of APC and that binding of recombinant hCDC20 and hCDH1 can activate APC in vitro. Our results suggest that the association between hCDH1 and APC is regulated by post-translational mechanisms, whereas the amount of hCDC20 bound to APC may in addition be controlled by hCDC20 synthesis and destruction . The temporally distinct association of hCDC20 and hCDH1 with APC suggests that these proteins are, respectively, mitosis-specific and G1-specific activating subunits of APC.
[Show abstract][Hide abstract] ABSTRACT: The G1-S transition in mammalian cells has been demonstrated to require the cyclin-dependent kinases cdk2, cdk3 and cdk4/6. Here we show that a novel kinase activity associated with cdk3 fluctuates throughout the cell cycle differently from the expression of cyclin D1-, E- and A-associated kinase activities. Cdk3 kinase activity is neither affected by p16 (in contrast to cdk4/6) nor by E2F-1 (in contrast to cdk2), but is downregulated upon transient p27 expression. We found cdk3 to bind to p21 and p27. We provide evidence that p27 could be involved in the regulation of the cell cycle fluctuation of cdk3 activity: cdk3 protein does not fluctuate and interaction of cdk3 with p27, but not with p21, is lost when cdk3 kinase becomes active during the cell cycle. In Myc-overexpressing cells, but not in normal Ratl cells, constitutive ectopic expression of cdk3 induces specific upregulation of cdk3-associated kinase activity that is still cell cycle phase dependent. Ectopic cdk3, but not cdk2, enhances Myc-induced proliferation and anchorage-independent growth associated with Myc activation, without effects on cyclin D1, E and A protein expression or kinase activities. High levels of cdk3 in Myc-overexpressing cells trigger up- and deregulation of E2F-dependent transcription without inducing the E2F-DNA binding capacity. In contrast to all other studied positive G regulators, cdk3 is unable to cooperate with ras in fibroblast transformation suggesting a function of cdk3 in G1 progression that is different from cyclin D- or E-associated kinase activities. Our data provide first insights into the regulation of cdk3-associated kinase activity and suggest a model how cdk3 participates in the regulation of the G1-S transition.
[Show abstract][Hide abstract] ABSTRACT: Activation of high ectopic levels of c-Myc in serum-deprived Rat1-MycER cells by 4-hydroxytamoxifen induces both proliferation and apoptosis. To further elucidate the role of G1 cyclin-dependent kinases (CDKs) in the process of Myc-induced apoptosis, we generated Rat1-MycER cells stably overexpressing CDK2 or CDK3. Ectopic expression of these CDKs in Myc-overexpressing cells was accompanied by upregulation of the specific kinase activities. Whereas neither high ectopic CDK2 nor CDK3 alone induced apoptosis in serum-deprived Rat1 cells, both CDKs markedly elevated the incidence of Myc-induced apoptosis. It was shown earlier that in Rat1-MycER cells, which are resistant to tumor necrosis factor-alpha (TNF) when grown in high serum concentrations, the addition of TNF with the concomitant activation of Myc resulted in apoptotic cell death. Here, we show that neither CDK2 nor CDK3 induces susceptibility to the cytotoxic action of TNF in Rat1 cells. However, both molecules heavily elevated the incidence of apoptosis induced by TNF together with Myc. It has earlier been reported that Myc-induced apoptosis in serum-deprived Rat1 fibroblasts is inhibited by specific cytokines, such as platelet-derived growth factor (PDGF). Here, we demonstrate that PDGF-mediated protection from Myc-induced apoptosis is almost lost in Rat1 cells overexpressing CDK2 or CDK3. These apoptotic effects of CDK2 or CDK3 are not accompanied by alterations of proliferation parameters, such as DNA distribution, time the cells spend in each phase of the cell cycle, thymidine incorporation into DNA, or cell size analyzed during Myc-induced apoptosis. However, we found CDK3 to deregulate E2F-dependent transcription. In this report, we provide evidence for a not yet described property of CDK2 or CDK3 besides their activity in promoting proliferation: these G1-CDKs can promote apoptosis by interfering with the cell's response to survival factors.
No preview · Article · Oct 1998 · DNA and Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Tuberous sclerosis is an autosomal dominant disorder. Besides the development of benign growths (hamartomas) in different tissues, one hallmark of this disease is the presence of highly epileptogenic dysplastic lesions in the cerebral cortex (tubers) composed of abnormal shaped neurones. Patients often show evidence of severe mental retardation. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid putative tumour suppressor protein, tuberin, that functions as a GTPase-activating protein. Here we show that tuberin expression is upregulated upon induction of neuronal differentiation in the neuroblastoma cell lines SK-N-SH and LAN-1. This upregulation occurs at post-transcriptional level and is independent of the proliferation status. TSC2 expression is unaffected during differentiation of C2C12 myoblasts into myotubes and of F9 embryonal carcinoma cells into cells resembling parietal endoderm. Antisense inhibition of tuberin expression in SK-N-SH or LAN-1 cells inhibits neuronal differentiation, but does not affect the differentiation of F9 cells. Ectopic overexpression of TSC2 not only reverts the antisense-associated phenotype but furthermore accelerates the neuronal differentiation process. Our data show for the first time that tuberin plays a critical role in neuronal differentiation. Such role is consistent with the phenotype of tuberous sclerosis patients, who inherit one defective TSC2 allele, and frequently lose the remaining normal allele in many of the tubers/hamartomas which develop in the central nervous system of these patients.