Heterozygosity of p21WAF1/CIP1 enhances tumor cell proliferation and cyclin D1-associated kinase activity in a murine mammary cancer model.
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The p21(WAF1/cIP1) cyclin-dependent kinase (cdk) inhibitor is a regulator of the G(1)-S cell cycle checkpoint. Despite the importance of p21 in cell cycle inhibition, its role as a tumor suppressor is uncertain. p21 mutations are infrequent in human tumors, and p21 null mice exhibit no increased tumor incidence. To ascertain whether p21 could influence tumor formation or progression in the context of other oncogenic stimuli, we crossed p21-deficient mice with mammary tumor susceptible Wnt-1 transgenic mice. The p21+/+, p21+/-, and p21-/- Wnt-1 transgenic female offspring were monitored for mammary tumor incidence and growth rates. p21 status had no effect on the age at which mammary tumors formed. However, p21+/- mammary tumors grew significantly faster than p21+/+ and p21-/- mammary tumors. The increased growth rates were confirmed by mitotic index counts and by BrdUrd labelling assays, indicating that a significantly higher percentage of p21+/- tumor cells were in S phase and mitosis than their p21+/+ and p21-/- counterparts. Moreover, cyclin D1-associated phosphorylation of retinoblastoma protein was significantly increased in p21+/- tumor lysates compared with p21+/+ and p21-/- lysates. These results are consistent with data indicating that reduced levels of p21 can facilitate cyclin/cdk complex formation while enhancing cdk activity. Thus, a reduction of p21 dosage may promote tumor progression in the presence of other oncogenic initiators. The dependence of p21 on prior oncogenic stimuli for its tumor-promoting activities suggests that it may behave as a tumor modifier gene rather than as a tumor suppressor gene. (+info)
Overexpression of c-Myc inhibits p21WAF1/CIP1 expression and induces S-phase entry in 12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive human cancer cells.
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The c-Myc oncoprotein is a transcription factor involved in cellular transformation. We previously found (M. V. Blagosklonny, et al., Cancer Res., 57: 320-325, 1997) that exposure of human SkBr3 breast cancer and LNCaP prostate cancer cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a growth arrest associated with the up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/cIP1) and the inhibition of c-Myc expression. We show here that exogenous c-Myc inhibits p21 expression in SkBr3 and LNCaP cells induced to enter into S-phase. p27 expression was not increased from basal levels in TPA-treated growth-arrested cells. A time course after infection of TPA-arrested cells using a c-Myc-expressing adenovirus revealed that the inhibition of p21 expression preceded entry into S-phase. In contrast, after infection by E2F-1-expressing adenovirus, p21 expression was reduced after the cells entered S-phase. Overexpression of c-Myc reduced the levels of endogenous p21 mRNA, and transfection of c-Myc repressed p21-promoter luciferase-reporter gene expression. The results suggest that the down-regulation of p21 expression may contribute to c-Myc-dependent entry into S-phase, possibly in situations in which growth arrest is associated with increased p21 expression. (+info)
Activation and repression of the 2-5A synthetase and p21 gene promoters by IRF-1 and IRF-2.
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The Interferon Regulatory Factors-1 and -2 (IRF-1 and IRF-2) were originally identified as transcriptional regulators of the interferon (IFN) and IFN-stimulated genes. These factors also modulate immune response and play a role in cell growth regulation. In this study we analysed the effect of the ectopic expression of IRF-1 and IRF-2 on the regulation of two potential IRF target genes involved in cell growth regulation, 2-5A synthetase and p21 (WAF/CP1), both of which contain consensus binding sites for IRF family members within their promoters. Following ectopic expression, IRF-1 transactivated 2-5A synthetase and p21 genes, an effect that was counterbalanced by concomitant ectopic expression of IRF-2. These effects were mediated by direct binding of IRF to the gene promoters. A construct expressing an IRF-2 antisense (FRI-2) was able to revert the inhibitory effect of IRF-2 on the IRF-1 transactivation. IRF-1 also induced expression of its homologous repressor IRF-2 as indicated by EMSA analysis using an IRF-E probe from the IRF-2 promoter; and by cotransfection of IRF-1 together with an IRF-2 promoter CAT construct. Therefore, the induction of IRF-1 by IFNs or other stimuli acts as a transactivator of genes involved in cell growth regulation, as well as of its own repressor IRF-2, thus providing autoinhibitory regulation of IRF-1 activated genes. (+info)
Mechanisms of apoptosis induced by the synthetic retinoid CD437 in human non-small cell lung carcinoma cells.
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The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been shown to induce apoptosis in various tumor cell lines including human non-small cell lung carcinoma (NSCLC) cells, which are resistant to the natural all-trans retinoic acid and to many synthetic receptor-selective retinoids. Although the mechanism of this effect was not elucidated, it was found to be independent of nuclear retinoid receptors. In the present study, we analysed the mechanisms by which CD437 induces apoptosis in two human NSCLC cell lines: H460 with wild-type p53 and H1792 with mutant p53. Both cell lines underwent apoptosis after exposure to CD437, although the cell line with wild-type p53 (H460) was more sensitive to the induction of apoptosis. CD437 increased the activity of caspase in both cell lines, however, the effect was much more pronounced in the H460 cells. The caspase inhibitors (Z-DEVD-FMK and Z-VAD-FMK) suppressed CD437-induced CPP32-like caspase activation and apoptosis in both cell lines. CD437 induced the expression of the p53 gene and its target genes, p21, Bax, and Killer/DR5, only in the H460 cells. These results suggest that CD437-induced apoptosis is more extensive in NSCLC cells that express wild-type p53, possibly due to the involvement of the p53 regulated genes Killer/DR5, and Bax although CD437 can also induce apoptosis by means of a p53-independent mechanism. Both pathways of CD437-induced apoptosis appear to involve activation of CPP32-like caspase. (+info)
Effects of chromosomal integration site upon p53 interactions with DNA consensus sequence homologies.
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In the present study, we report that, despite the presence of one perfect p53 consensus sequence homology (designated SCL CS) and four half-sites within the 3'-untranslated region of the stem cell leukemia (SCL) gene, the native endogenous gene is not regulated by p53. We employ a tet-repressible system to show that, under conditions in which the WAF1 mRNA steady-state level is upregulated fourfold by p53, the SCL mRNA level is not altered. In a previous report, we demonstrated that p53 interactions with the SCL CS can upregulate downstream reporter gene activity 43-fold in transient reporter assays. This disparity prompted us to explore the differences between p53 regulation of SCL CS activity in organized (chromosomally integrated) and disorganized (non-replicating episomal plasmid) chromatin. We show that p53 can increase (between 3-80-fold), decrease (between 5-33-fold) or have no effect upon transactivation of an SCL CS/reporter fusion gene depending upon chromosomal integration site. Most studies used to characterize p53 binding sites employ transient transfection assays. Our results suggest that characterization of consensus sequence homologies by assay of transiently transfected cells may be inaccurate. (+info)
Overriding of cyclin-dependent kinase inhibitors by high and low risk human papillomavirus types: evidence for an in vivo role in cervical lesions.
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High risk types of human papillomavirus (HPV) are agents in the aetiology of cervical carcinoma. The products of two early genes, E6 and E7, appear to be the principal transforming proteins. Studies of various monolayer cell culture systems have shown that the E7 oncoprotein of human papillomavirus type 16 is able to neutralize or bypass the inhibitory effect of the cell cycle-dependent kinase (CDK) inhibitors (CKIs) p21WAF1/CIP1 and p27KIP1. To understand whether the p21WAF1/CIP1 or p27KIP1 neutralization also plays a role in vivo, we performed studies on clinical specimens. Forty-five cervical biopsies, including HPV-negative mucosa, HPV 16-positive preinvasive (low and high grade lesions) and invasive neoplasia as well as HPV 6-positive condyloma acuminatum were analysed by single and double immunohistology. We examined the positive cell cycle regulator cyclin A and the universal cell cycle marker Ki67 as well as the negative cell cycle regulators p21WAF1/CIP1 and p27KIP1. Here, we show that in a significant fraction of cells the G1 block can be overcome despite high levels of CKIs in HPV lesions. This phenomenon, which was more evident for p21WAF1/CIP1 than for p27KIP1 was most marked in low grade lesions and in condylomata acuminata, in which a high viral productivity is expected. These results indicate that the overriding of CKI inactivation by viral oncoproteins appears to be a conserved property between low and high risk HPV types. We conclude that the CKI neutralization by HPVs is likely to be required for viral DNA replication rather than for malignant transformation of the host cell. (+info)
NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells.
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The mechanisms of nitric oxide (NO)-mediated inhibition of vascular smooth muscle (VSM) cell proliferation are still obscure. Cyclins A and E in association with cyclin-dependent kinase 2 (cdk2) serve as positive regulators for mammalian cell cycle progression through the G1/S checkpoint of the cell cycle and subsequent cell proliferation. Therefore, we have tested the effect of adenovirus-mediated transfection of the endothelial nitric oxide synthase (eNOS) gene into guinea pig coronary VSM cells on platelet-derived growth factor (BB homodimer) (PDGF-BB)-stimulated cell proliferation and the expression of cell cycle regulatory molecules. Transfection of the eNOS gene (eNOS) into VSM cells significantly inhibited (P < 0.05) [3H]thymidine incorporation into the DNA in response to PDGF-BB stimulation compared with lacZ-transfected control cells. The eNOS transfer significantly inhibited (P < 0.05) PDGF-BB-induced proliferating cell nuclear antigen (PCNA) and cyclin A expression in VSM cells compared with cells transfected with the control vector. The time course of cyclin E expression in response to PDGF-BB stimulation was delayed in eNOS-transfected cells. Levels of cyclin-dependent kinase inhibitors p21 and p27 were not significantly affected by eNOS transfer. eNOS transfer did not decrease PDGF-beta receptor number, affinity, and autophosphorylation measured by radioreceptor assay and Western analysis. These results suggest that inhibition of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA is the target of NO action. These findings could explain, at least in part, NO-mediated inhibition of VSM cell proliferation. (+info)
Inverse relationship between apoptosis and Bcl-2 expression in syncytiotrophoblast and fibrin-type fibrinoid in early gestation.
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The purpose of this study was to assess the role of apoptosis and cell cycle arrest in the trophoblast during early gestation by determining the location of apoptotic cells and examining the expression of Bcl-2 and p21. Using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) method on human chorionic villi, a cluster of apoptotic nuclei was demonstrated in perivillous fibrin-type fibrinoid, but no apoptotic changes were identified in the syncytiotrophoblast or in other subtypes of the trophoblast. The syncytiotrophoblast was diffusely positive for Bcl-2, but fibrin-type fibrinoid was negative for Bcl-2. Hence, there was an inverse relationship between apoptosis and Bcl-2 expression in both fibrin-type fibrinoid and syncytiotrophoblast. Expression of p21 was present to some extent in the syncytiotrophoblast, but not in fibrin-type fibrinoid. These results suggest that Bcl-2 may play an important role in preventing apoptosis in the syncytiotrophoblast; this may be necessary to prevent any DNA degradation from being spread to other nuclei in a multinuclear cell like the syncytiotrophoblast. (+info)