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Abstract: Background: Together with p53, the NAD-dependent lysine deacetylase SIRT1 and the microRNA miR-34a form a feedback loop which self-regulates SIRT1 expression and modulates p53-dependent responses. In addition to its well-described role in mediating transcriptional responses to genotoxic stress, p53 may also regulate microRNA processing and maturation. Objective: This study explored the functional relationship among p53, SIRT1 and miR-34a, and the influence of p53 and SIRT1 on microRNA biogenesis and maturation in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. Methods: RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors were used to modulate activity and expression of SIRT1 and p53. Changes in microRNA and mRNA were analysed by qRT-PCR and protein expression was determined by immunoblotting. Results: Mature miR-34a decreased in p53-suppressed NHEK cells, whereas ablation of SIRT1 reduced the primary transcript (pri-miR-34a). When either SIRT1 expression or activity was inhibited in combination with p53 ablation, pri-miR-34a levels increased and mature miR-34a levels decreased. Under these same conditions, additional p53-regulated microRNAs (miRs 16-1/15, 145 and 107) also failed to mature. In HaCaT cells, primary microRNA transcripts for miR-16-1/15, miR-145 miR200c/141 and miRNA-107, but not miR-34a, were approximately 8-fold higher than in NHEK cells. However, the levels of mature microRNA sequences in HaCaT cells were only 1.5–2 fold higher (miR-16-1, miR-145), unchanged (miR-107) or decreased (miR-200c/141, miR-34a) compared to NHEK cells. Conclusions: Our results suggest that p53 mutations interfere with efficient microRNA biogenesis in keratinocytes, and that SIRT1 functions in combination with p53 in this process. [Copyright &y& Elsevier]

Abstract: Arsenic (As) is both a human carcinogen and an effective anticancer drug. These aspects of arsenic toxicity develop as a consequence of arsenic-induced oxidative stress and modifications to signal pathway activity which alter gene expression. Resveratrol (RVL) a food antioxidant found in grapes and other fruits, exhibits anti-carcinogenic properties by reducing oxidative stress and restoring signal pathway control. This study investigated the impact of RVL on arsenite [As(III)]-induced cell signalling in HaCaT keratinocytes by assaying phosphorylation status of epidermal growth factor receptor (EGFR) signalling intermediates and measuring changes in expression of Phase II and DNA repair biomarkers. As(III) exposure produced dose-dependent toxicity which was associated with increased activation of EGFR pathway intermediates, cSrc, Rac1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Arsenic-mediated ERK1/2 activation negatively regulated DNA polymerase beta expression and up regulated heme-oxygenase-1 at toxic concentrations. RVL treatment modulated As(III)-mediated ERK1/2 activation by shifting the balance of cSrc regulatory domain phosphorylation. These effects significantly altered the response of the EGFR pathway to growth factor-induced stimulation. Our research provides evidence that treatment with pharmacologically relevant doses of RVL influences cellular responses to As(III), largely due to RVL-mediated changes to Src and ERK1/2 activation. [Copyright &y& Elsevier]