HLA class I-mediated induction of cell proliferation involves cyclin E-mediated inactivation of Rb function and induction of E2F activity.
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Chronic rejection of transplanted organs is manifested as atherosclerosis of the blood vessels of the allograft. HLA class I Ags have been implicated to play a major role in this process, since signaling via HLA class I molecules can induce the proliferation of aortic endothelial as well as smooth muscle cells. In this study, we show that HLA class I-mediated induction of cell proliferation correlates with inactivation of the Rb protein in the T cell line Jurkat as well as human aortic endothelial cells. HLA class I-mediated inactivation of Rb can be inhibited specifically by neutralizing Abs to basic fibroblast growth factor (bFGF), suggesting a role for FGF receptors in the signaling process. Signaling through HLA class I molecules induced cyclin E-associated kinase activity within 4 h in quiescent endothelial cells, and appeared to mediate the inactivation of Rb. A cdk2 inhibitor, Olomoucine, as well as a dominant-negative cdk2 construct prevented HLA class I-mediated inactivation of Rb; in contrast, dominant-negative cdk4 and cdk6 constructs had no effect. Furthermore, there was no increase in cyclin D-associated kinase activity upon HLA class I ligation, suggesting that cyclin E-dependent kinase activity mediates Rb inactivation, leading to E2F activation and cell proliferation. (+info)
Bcl-2-induced changes in E2F regulatory complexes reveal the potential for integrated cell cycle and cell death functions.
(34/1403)
Proliferation and cell death are tightly linked fates during cell and tissue differentiation. In the past few years, it has been shown that Bcl-2 exhibits a potent cell cycle inhibitory effect, in addition to its better known role in the antagonism of cell death. In the present study, we show that the cell cycle effects of Bcl-2 apparently occur at the level of E2F control of gene transcription. Under conditions of normal cell growth, or under conditions that lead to cell death in the absence of Bcl-2, bcl-2 expression results in a reduction of free (active) E2F isoforms and in an increase in the formation of higher-order (inactive) complexes. Bcl-2-induced changes in E2F complex formation are paralleled by an apparent increase in pRb regulatory activity, by the up-regulation of p130 protein expression, and by the formation of E2F/p130 complexes at the expense of those consisting of E2F/p107. Cells lacking bcl-2 expression respond to growth factor withdrawal in the opposite manner, by the liberation of E2F from inactivating complexes and by continued cell cycle leading to cell death. These analyses reveal a mechanism for cell cycle regulation by Bcl-2 that occurs at the level of E2F transcriptional activity. Further, since specific E2F activities are clearly linked to the induction of cell death, these findings may help to consolidate the cell survival and cell cycle effects of Bcl-2 through a common transcriptional mechanism. (+info)
E2F mediates developmental and cell cycle regulation of ORC1 in Drosophila.
(35/1403)
Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins. Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, we show that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, we find that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila. (+info)
RBP1 induces growth arrest by repression of E2F-dependent transcription.
(36/1403)
Growth arrest and cell cycle progression are regulated by the retinoblastoma tumour suppressor pRB and related proteins p130 and p107 that bind to and inhibit the E2F family of transcription factors. Although the precise mechanism of this inhibition remains to be established, previous studies indicated the presence of transcriptional repression activity in the 'pocket' of RB family members. We show here that RBP1, a known pRB pocket-binding protein, possesses transcriptional repression activity and associates with p130-E2F and pRB-E2F complexes specifically during growth arrest. Overexpression of RBP1 both inhibited E2F-dependent gene expression and suppressed cell growth. Thus repression of E2F-dependent transcription by RBP1 via RB family members may play a central role in inducing growth arrest. (+info)
Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F.
(37/1403)
Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle. Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary. In this study, we examined the transcriptional activities of isolated human MCM gene promoters. Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression. In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation. Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells. Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters. (+info)
Activation of the murine dihydrofolate reductase promoter by E2F1. A requirement for CBP recruitment.
(38/1403)
The E2F family of heterodimeric transcription factors plays an important role in the regulation of gene expression at the G1/S phase transition of the mammalian cell cycle. Previously, we have demonstrated that cell cycle regulation of murine dihydrofolate reductase (dhfr) expression requires E2F-mediated activation of the dhfr promoter in S phase. To investigate the mechanism by which E2F activates an authentic E2F-regulated promoter, we precisely replaced the E2F binding site in the dhfr promoter with a Gal4 binding site. Using Gal4-E2F1 derivatives, we found that E2F1 amino acids 409-437 contain a potent core transactivation domain. Functional analysis of the E2F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the dhfr promoter and protein-protein interactions with CBP, transcription factor (TF) IIH, and TATA-binding protein (TBP). However, additional amino acid substitutions for phenylalanine 429 demonstrated a strong correlation between activation of the dhfr promoter and binding of CBP, but not TFIIH or TBP. Finally, transactivator bypass experiments indicated that direct recruitment of CBP is sufficient for activation of the dhfr promoter. Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates the dhfr promoter. (+info)
Prognostic significance of transcription factor E2F-1 in bladder cancer: genotypic and phenotypic characterization.
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BACKGROUND: We sought to identify and characterize potential alterations in E2F-1, a transcription factor that binds to the retinoblastoma protein (pRB), in bladder neoplasms and to elucidate a possible role for E2F-1 as an oncogene or a tumor suppressor gene. METHODS: Tumor samples from 133 evaluable patients with bladder cancer were analyzed for E2F-1 gene mutations by use of polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, tumors were studied for E2F-1 and pRB protein expression by use of immunohistochemistry. Results from the above analyses were correlated with clinicopathologic parameters and outcome. All P values are two-sided. RESULTS: A polymorphism, consisting of a nucleotide change at amino acid codon 393 in exon 7 (GGC-->AGC [Gly-->Ser]), was identified in seven of 133 case patients, being present in both tumor and corresponding normal tissues. No bandshifts were identified in the nuclear-localization or DNA-binding domains on PCR-SSCP analysis. On immunohistochemical analysis, E2F-1 nuclear reactivity was observed in less than 5% of the cells from 53 tumors and in 5%-75% of the cells from the remaining 80 tumors. The pattern of E2F-1 protein expression was not altered in relation to the identified polymorphism. pRB nuclear reactivity greater than 20% (of tumor cells stained) was present in 66% of the samples. E2F-1 nuclear reactivity correlated inversely with the percentage of cells showing pRB reactivity (Kendall tau(b) = -0.18; P = .019). On multivariate analysis, patients with lower E2F-1 reactivity had statistically significantly increased risks of progression to metastases (P = .001) and death (P = .02). CONCLUSIONS: E2F-1 alterations occur at the phenotypic level, rather than at the genotypic level, in bladder cancer. The adverse outcome for patients whose tumors exhibit low E2F-1 nuclear expression suggests a possible tumor suppressor role for E2F-1 in bladder cancer. (+info)
Physical and functional interactions of neuronal growth suppressor necdin with p53.
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Necdin is expressed in virtually all postmitotic neurons, and ectopic expression of this protein suppresses cell proliferation. Necdin, like the retinoblastoma protein, interacts with cell cycle promoting proteins such as simian virus 40 large T antigen, adenovirus E1A, and the transcription factor E2F1. Here we demonstrate that necdin interacts with the tumor suppressor protein p53 as well. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region (amino acids 35-62) located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed p53-dependent activation of the p21/WAF promoter. Necdin and p53 inhibited cell growth in an additive manner as assessed by the colony formation of SAOS-2 cells, suggesting that necdin does not affect p53-mediated growth suppression. On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons. (+info)