Altered regulation of cyclin G in human breast cancer and its specific localization at replication foci in response to DNA damage in p53+/+ cells. (65/5371)

Cyclin G, a recent addition to the cyclin family, was initially identified in screens for new src kinase family members and soon thereafter by differential screening for transcriptional targets of the tumor suppressor gene, p53. We have identified cyclin G as being overexpressed in breast and prostate cancer cells using differential display polymerase chain reaction screening. We demonstrate here that cyclin G is overexpressed in human breast and prostate cancer cells and in cancer cells in situ from tumor specimens. Cyclin G expression was tightly regulated throughout the cell cycle in normal breast cells, peaking at the S and G2/M phases of the cell cycle with lower levels in G1. The cell cycle-dependent expression was absent in breast cancer cells. Following DNA damage in normal p53+/+ cells, cyclin G is triggered to cluster in discrete nuclear DNA replication foci that contain replication-associated proteins such as proliferating cell nuclear antigen (PCNA). While p53-/- cells displayed a faint cyclin G nuclear staining pattern, there was no increased expression and no change in distribution of the staining pattern after DNA damage. The specific subcellular localization of cyclin G at DNA replication foci provides an additional link between p53-mediated growth arrest and cell cycle regulation and suggests that cyclin G may act as an effector of p53-mediated events by functional association with replication foci protein(s).  (+info)

Binding of CtIP to the BRCT repeats of BRCA1 involved in the transcription regulation of p21 is disrupted upon DNA damage. (66/5371)

Mutations in BRCA1 are responsible for nearly all of the hereditary ovarian and breast cancers, and about half of those in breast cancer-only kindreds. The ability of BRCA1 to transactivate the p21 promoter can be inactivated by mutation of the conserved BRCA1 C-terminal (BRCT) repeats. To explore the mechanisms of this BRCA1 function, the BRCT repeats were used as bait in a yeast two-hybrid screen. A known protein, CtIP, a co-repressor with CtBP, was found. CtIP interacts specifically with the BRCT repeats of BRCA1, both in vitro and in vivo, and tumor-derived mutations in this region abolished these interactions. The association of BRCA1 with CtIP was also abrogated in cells treated with DNA-damaging agents including UV, gamma-irradiation, and adriamycin, a response correlated with BRCA1 phosphorylation. The transactivation of the p21 promoter by BRCA1 was diminished by expression of exogenous CtIP and CtBP. These results suggest that the binding of the BRCT repeats of BRCA1 to CtIP/CtBP is critical in mediating transcriptional regulation of p21 in response to DNA damage.  (+info)

Activation of caspases and p53 by bovine herpesvirus 1 infection results in programmed cell death and efficient virus release. (67/5371)

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. Bovine herpesvirus 1 (BHV-1) induces PCD in peripheral blood mononuclear cells at the G0/G1 phase of the cell cycle (E. Hanon, S. Hoornaert, F. Dequiedt, A. Vanderplasschen, J. Lyaku, L. Willems, and P.-P. Pastoret, Virology 232:351-358, 1997). However, penetration of virus particles is not required for PCD (E. Hanon, G. Meyer, A. Vanderplasschen, C. Dessy-Doize, E. Thiry, and P. P. Pastoret, J. Virol. 72:7638-7641, 1998). The mechanism by which BHV-1 induces PCD in peripheral blood mononuclear cells is not understood, nor is it clear whether nonlymphoid cells undergo PCD following infection. This study demonstrates that infection of bovine kidney (MDBK) cells with BHV-1 leads to PCD, as judged by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, DNA laddering, and chromatin condensation. p53 appears to be important in this process, because p53 levels and promoter activity increased after infection. Expression of proteins that are stimulated by p53 (p21(Waf1) and Bax) is also activated after infection. Cleavage of Bcl-xL, a protein that inhibits PCD, occurred after infection, suggesting that caspases (interleukin-1beta-converting enzyme-like proteases) were activated. Other caspase substrates [poly(ADP-ribose) polymerase and actin] are also cleaved during the late stages of infection. Inhibition of caspase activity delayed cytotoxic activity and virus release but increased the overall virus yield. Taken together, these results indicate that nonlymphoid cells undergo PCD near the end of productive infection and further suggest that caspases enhance virus release.  (+info)

The proto-oncogene c-myc is a direct target gene of Epstein-Barr virus nuclear antigen 2. (68/5371)

Epstein-Barr virus (EBV) infects and transforms primary B lymphocytes in vitro. Viral infection initiates the cell cycle entry of the resting B lymphocytes. The maintenance of proliferation in the infected cells is strictly dependent on functional EBNA2. We have recently developed a conditional immortalization system for EBV by rendering the function of EBNA2, and thus proliferation of the immortalized cells, dependent on estrogen. This cellular system was used to identify early events preceding induction of proliferation. We show that LMP1 and c-myc are directly activated by EBNA2, indicating that all cellular factors essential for induction of these genes by EBNA2 are present in the resting cells. In contrast, induction of the cell cycle regulators cyclin D2 and cdk4 are secondary events, which require de novo protein synthesis.  (+info)

Expression of catalytically active telomerase does not prevent premature senescence caused by overexpression of oncogenic Ha-Ras in normal human fibroblasts. (69/5371)

All normal cells in culture display a limited capacity to divide and eventually undergo an irreversible growth arrest known as replicative cellular senescence. The development of cellular immortality has been implicated as an important factor in the progression of human cancers. Expression of telomerase has been shown to elicit a bypass of senescence and the acquirement of an extended life span. Although oncogenic Ras induces malignant transformation in most immortal cells, it has been shown to cause a senescence-like cell cycle arrest in presenescent human fibroblasts. To test the relationship between the senescence-inducing effect of Ras and the senescence-bypassing activity of telomerase, we used retroviral vector infection to introduce the catalytic subunit of human telomerase into normal human lung fibroblasts. Cell clones displaying in vitro telomerase catalytic activity and life span extension were obtained. However, these cells still became senescent after infection with a retrovirus vector expressing oncogenic Ha-Ras. No differences in premature senescence phenotypes between normal and telomerase-expressing cells were observed. A small number of colonies were recovered after the introduction of Ha-Ras into either normal or telomerase-expressing cells, but in all cases, these clones failed to express the exogenously introduced Ras. We propose that even in the presence of active telomerase, the cellular senescence machinery remains intact and can be activated by appropriate signals. Consequently, interventions aimed at the activation of the latent senescence program may be a fruitful approach in cancer therapy.  (+info)

Initiation of growth arrest and apoptosis of MCF-7 mammary carcinoma cells by tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate, is associated with mitochondrial activity. (70/5371)

We investigated the effects of tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate and an approved food additive, establishing induction of growth arrest and apoptosis of MCF-7 human mammary carcinoma cells. Transient increased mitochondria-associated bax, dissipation of the mitochondrial membrane potential (delta(psi)m), and caspase-3-independent cleavage of poly(ADP-ribose) polymerase are evident as early as 4 h after treatment of cells with tributyrin. These events are followed by the transient accumulation of mitochondrial cytochrome c in the cytosol and, finally, the generation and accumulation of cells with subdiploid DNA content. During the period in which mitochondria-associated bax levels are elevated, the delta(psi)m is disrupted, and cytochrome c is detected in the cytosol, we show induction of p21WAF1/Cip1 in the absence of increased p53 and arrest of cells in G2-M. Thus, early mitochondria-associated events may play a key role in initiating and/or coordinating tributyrin-mediated growth arrest and apoptosis of wild-type p53 MCF-7 cells. Because effective chemoprevention has been associated with agents that restore or maintain the balance between proliferation and apoptosis, dietary tributyrin, particularly during the critical period of mammary gland development, may be a promising chemopreventive agent.  (+info)

Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists. (71/5371)

Recent studies identified a short peptide motif that serves as a docking site for cyclin/cyclin-dependent kinase (cdk) 2 complexes. Peptides containing this motif block the phosphorylation of substrates by cyclin A/cdk2 or cyclin E/cdk2. Here we report that cell membrane-permeable forms of such peptides preferentially induced transformed cells to undergo apoptosis relative to nontransformed cells. Deregulation of E2F family transcription factors is a common event during transformation and was sufficient to sensitize cells to the cyclin/cdk2 inhibitory peptides. These results suggest that deregulation of E2F and inhibition of cdk2 are synthetically lethal and provide a rationale for the development of cdk2 antagonists as antineoplastic agents.  (+info)

A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. (72/5371)

Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21(WAF1/CIP1) tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.  (+info)