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Terminal differentiation and senescence share several common properties, including irreversible cessation of growth and changes in gene expression profiles. To identify molecules that converge in both processes, an overlapping pathway screening was employed that identified old-35, which is human polynucleotide phosphorylase (hPNPase old-35 ), a 3,5-exoribonuclease. We previously demonstrated that hPNPase old-35 is a type I interferon-inducible gene that is also induced in senescent fibroblasts. In vitro RNA degradation assays confirmed its exoribonuclease properties, and overexpression of hPNPase There are two contrasting endpoints in the life of a replicating cell. One involves the normal physiological processes of differentiation or senescence. The other is the pathological process of neoplastic transformation characterized by uncontrolled proliferation and de-differentiation. Treatment of HO-1 metastatic human melanoma cells with fibroblast interferon (IFN-␤) 1 and the protein kinase C activator mezerein (MEZ) induces irreversible growth arrest and terminal differentiation characterized by changes in cell morphology, increase in melanin synthesis, modifications in gene expression, and alterations in surface antigen expression (1-5). Replicative or cellular senescence, a process leading to irreversible arrest of cell division, was first described in cultures of human fibroblasts that lost the ability to divide upon continuous subcultures (6). Replicative senescence can result from telomere shortening linked with a DNA end-replication problem, overexpression of certain oncogenes, or tumor suppressor genes, or it can be stress-induced premature senescence after exposure to a variety of oxidative stresses or DNA damaging agents (for a review, see Ref. 7).Terminal differentiation and cellular senescence share several common traits including irreversible growth arrest and changes in gene expression profiles. To understand the molecular and biochemical basis of the complex physiological changes associated with these phenomena, an overlapping pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes (8). A temporally spaced terminally differentiated human melanoma subtracted cDNA library was screened with cDNAs derived from senescent progeroid fibroblast cells. This led to the identification of old-35, which is human polynucleotide phosphorylase (hPNPase old-35 ), a 3Ј,5Ј exoribonuclease involved in RNA degradation (8). hPNPase old-35 is a highly evolutionary conserved gene in plants, prokaryotes and eukaryotes having similar domain structure and functional properties in all species. In vitro assays confirmed that hPNPase old-35 is involved in RNA degradation. Analysis of the expression profile of hPNPase old-35 revealed that it is predominantly a type I interferoninducible gene, and its expression is also induced in senescent fibroblasts in comparison with young fibroblasts. These findings indicate that hPNPase old-35 might play an essential role in

Human melanoma cells growth-arrest irreversibly and terminally differentiate on treatment with a combination of fibroblast interferon and the protein kinase C activator mezerein. This experimental protocol also results in a loss of tumorigenic potential and profound changes in gene expression. Various cloning and cDNA microarray strategies are being used to determine the complete spectrum of gene expression changes underlying these alterations in human melanoma cells. An efficient approach, Rapid Subtraction Hybridization (RaSH), has been developed that is permitting the identification of genes of potential relevance to cancer growth control and terminal cell differentiation. RaSH cDNA libraries are prepared from double-stranded cDNAs that are enzymatically digested into small fragments, ligated to adapters, and PCR amplified followed by incubation of tester and driver PCR fragments. This subtraction hybridization scheme is technically simple and results in the identification of a high proportion of differentially expressed sequences, including known genes and those not described in current DNA databases. The RaSH approach represents an efficient methodology for identifying and cloning genes displaying differential expression that associate with and potentially regulate complex biological processes.cDNA cloning ͉ reverse Northern blotting ͉ melanoma differentiation associated genes ͉ Northern blotting

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