p16INK4a and p21WAF1, two major cyclin-dependent kinase inhibitors, are the products of two tumor suppressor genes that play important roles in various cellular metabolic pathways. p21WAF1 is up-regulated in response to different DNA damaging agents. While the activation of p21WAF1 is p53-dependent following γ-rays, the effect of ultraviolet (UV) light on p21WAF1 protein level is still unclear. In the present report, we show that the level of the p21WAF1 protein augments in response to low UVC fluences in different mammalian cells. This up-regulation is mediated through the stabilization of p21WAF1 mRNA in a p16INK4a-dependent manner in both human and mouse cells. Furthermore, using p16-siRNA treated human skin fibroblast; we have shown that p16 controls the UV-dependent cytoplasmic accumulation of the mRNA binding HuR protein. In addition, HuR immunoprecipitations showed that UV-dependent binding of HuR to p21 mRNA is p16-related. This suggests that p16 induces p21 by enabling the relocalization of HuR from the nucleus to the cytoplasm. Accordingly, we have also shown that p16 is necessary for efficient UV-dependent p53 up-regulation, which also requires HuR. These results indicate that, in addition to its role in cell proliferation, p16INK4a is also an important regulator of the cellular response to UV damage.

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BackgroundThe cyclin-D/CDK4,6/p16INK4a/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined.Methodology/Principal FindingsWe have shown here that the tumor suppressor p16INK4a protein positively controls the expression of cyclin D1 and E2F1 in both human and mouse cells. p16INK4a stabilizes the mRNAs of the corresponding genes through negative regulation of the mRNA decay-promoting AUF1 protein. Immunoprecipitation of AUF1-associated RNAs followed by RT-PCR indicated that endogenous AUF1 binds to the cyclin D1 and E2F1 mRNAs. Furthermore, AUF1 down-regulation increased the expression levels of these genes, while concurrent silencing of AUF1 and p16INK4a, using specific siRNAs, restored normal expression of both cyclinD1 and E2F1. Besides, we have shown the presence of functional AU-rich elements in the E2F1 3′UTR, which contributed to p16/AUF1-mediated regulation of E2F1 post-transcriptional events in vivo. Importantly, genome-wide gene expression microarray analysis revealed the presence of a large number of genes differentially expressed in a p16INK4a -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis.Conclusion/SignificanceThese findings show that the cyclin-dependent kinase inhibitor p16 INK4a is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1.

BackgroundBRCA1 promoter methylation has been detected in DNA from peripheral blood cells of both breast cancer patients and cancer-free females. However, the pathological significance of this epigenetic change in white blood cells (WBC) remains an open question. In this study, we hypothesized that if constitutional BRCA1 methylation reflects an elevated risk for developing breast cancer (BC), WBC that harbor methylated BRCA1 in both cancer-free females and BC patients should exhibit similar molecular changes.MethodsBRCA1 promoter methylation was examined by methylation-specific PCR in WBC from 155 breast cancer patients and 143 cancer-free females. The Human Breast Cancer EpiTect Methyl II Signature PCR Array and The Human Breast Cancer RT2 Profiler™ PCR Array were used to study the methylation status and the expression profile of several breast cancer-related genes, respectively. In addition, we used label-free MS-based technique to study protein expression in plasma.ResultsWe have shown that 14.2% of BC patients and 9.1% of cancer-free females (carriers) harbored methylated BRCA1 promoter in their WBC. Interestingly, 66.7% of patients harbored methylated BRCA1 promoter in both WBC and tumors. Importantly, we have shown the presence of epigenetic changes in 9 other BC-related genes in WBC of both patients and carriers. Additionally, BRCA1 and 15 other important cancer –related genes were found to be differentially expressed in WBC from patients and carriers as compared to controls. Furthermore, we have shown that the carriers exhibited a unique plasma protein pattern different from those of BC patients and controls, with 10 proteins similarly differentially expressed in patients and carriers as compared to controls.ConclusionsThe present results suggest the presence of a strong link between aberrant methylation of the BRCA1 promoter in WBC and breast cancer –related molecular changes, which indicate the potential predisposition of the carriers for developing breast cancer. This informs the potential use of the aberrant methylation of BRCA1 promoter in WBC as a powerful non-invasive molecular marker for detecting predisposed individuals at a very early age.

BackgroundConstitutive methylation of tumor suppressor genes are associated with increased cancer risk. However, to date, the question of epimutational transmission of these genes remains unresolved. Here, we studied the potential transmission of BRCA1 and MGMT promoter methylations in mother-newborn pairs.MethodsA total of 1014 female subjects (cancer-free women, n = 268; delivering women, n = 295; newborn females, n = 302; breast cancer patients, n = 67; ovarian cancer patients, n = 82) were screened for methylation status in white blood cells (WBC) using methylation-specific PCR and bisulfite pyrosequencing assays. In addition, BRCA1 gene expression levels were analyzed by quantitative real-time PCR.ResultsWe found similar methylation frequencies in newborn and adults for both BRCA1 (9.9 and 9.3%) and MGMT (12.3 and 13.1%). Of the 290 mother-newborn pairs analyzed for promoter methylation, 20 mothers were found to be positive for BRCA1 and 29 for MGMT. Four mother-newborn pairs were positive for methylated BRCA1 (20%) and nine pairs were positive for methylated MGMT (31%). Intriguingly, the delivering women had 26% lower BRCA1 and MGMT methylation frequencies than those of the cancer-free female subjects. BRCA1 was downregulated in both cancer-free woman carriers and breast cancer patients but not in newborn carriers. There was a statistically significant association between the MGMT promoter methylation and late-onset breast cancers.ConclusionsOur study demonstrates that BRCA1and MGMT epimutations are present from the early life of the carriers. We show the transmission of BRCA1 and MGMT epimutations from mother to daughter. Our data also point at the possible demethylation of BRCA1and MGMT during pregnancy.