Induction of senescence-like growth arrest in melanocytes and malignant melanoma cells by human polynucleotide phosphorylase (hPNPaseold-35). Free

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Terminal differentiation and senescence are two physiological processes that show overlapping properties including irreversible growth arrest and changes in gene expression profiles. To identify genes that lie at the core of these two phenomena we employed an overlapping pathway screening strategy that involved probing a library from melanoma cells, that are induced to differentiate with interferon-β and the protein kinase C activator mezerein, with cDNAs from senescent progeroid cells. This led to the identification of human polynucleotide phosphorylase (hPNPaseold-35), an early interferon-responsive gene having 3’-5’ exoribonuclease properties, that is upregulated during differentiation and senescence. Overexpression of hPNPaseold-35 in melanoma cells via a replication-incompetent adenovirus (Ad.hPNPaseold-35) inhibited growth and colony formation indicating the involvement of hPNPaseold-35 in differentiation and senescence-associated growth arrest. We presently investigated the molecular mechanism of growth inhibition by hPNPaseold-35. Infection of melanoma cells with Ad.hPNPaseold-35 resulted in cell cycle arrest in G1 phase, followed by apoptotic death, with concomitant reduction in S phase indicating inhibition of DNA synthesis. Ad.hPNPaseold-35 infection in melanocytes and melanoma cells resulted in a significant increase in senescence-associated β-galatosidase positive cells, a classical marker of senescence. At the molecular level, Ad.hPNPaseold-35 infection modulated the expression of genes that control G1 check-point, such as cyclin-dependent kinase inhibitors, Rb, E2F1 and c-myc. In melanoma cells, infection with Ad.hPNPaseold-35 reduced c-myc, both mRNA and protein, and hPNPaseOLD-35 protein could degrade c-myc mRNA in vitro indicating that c-myc might be a direct target of hPNPaseold-35. This observation was supported by the finding that overexpression of c-myc could rescue melanoma cells from hPNPaseold-35-mediated growth arrest. Additionally, Ad.hPNPaseold-35 infection significantly inhibited telomerase activity, which is also a feature of senescence. These findings indicate that hPNPaseold-35 might be a novel component of the molecular machinery governing senescence and inhibition of hPNPaseold-35 expression might serve as a potential approach for prolonging longevity.