Down-regulation of Myc as a Potential Target for Growth Arrest Induced by Human Polynucleotide Phosphorylase (hPNPase) in Human Melanoma Cells

Abstract

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

Defining the mechanism by which IFN-β dowregulates c-myc expression in human melanoma cells: pivotal role for human polynucleotide phosphorylase (hPNPaseold-35)

“…6 Moreover, hPNPase old-35 induces gene expression changes that are consistent with G 1 cell cycle arrest, such as downregulation of c-myc, upregulation of p27 KIP-1 and hypophosphorylation of Rb. 6 We previously found that downregulation of c-myc plays a critical part in hPNPase old-35 -induced growth inhibition, since overexpression of c-myc partially but significantly ablated this growth-inhibitory effect. 6,9 In vitro mRNA degradation assays revealed that, as an exoribonuclease, hPNPase old-35 directly and specifically degraded c-myc mRNA.…”

“…[3][4][5][6] Biochemically, hPNPase old-35 is a 3 0 ,5 0 exoribonuclease catalyzing mRNA decay in a 3 0 -5 0 direction. 3 Polynucleotide phosphorylase (PNPase) is an evolutionary conserved gene and the bacterial and plant enzymes have been cloned and extensively studied for decades.…”

“…3 Of direct relevance to the present study, overexpression of hPNPase old-35 via an adenoviral vector (Ad.hPNPase old-35 ) induces profound morphological, biochemical and gene expression changes that mimic the critical molecular and biochemical signatures of senescence as well as differentiation. 6 hPNPase old-35 induces cell cycle arrest in the G 1 phase with inhibition of DNA synthesis and telomerase activity, and activates the senescence-associated b-galactosidase enzyme. 6 Moreover, hPNPase old-35 induces gene expression changes that are consistent with G 1 cell cycle arrest, such as downregulation of c-myc, upregulation of p27 KIP-1 and hypophosphorylation of Rb.…”

“…6 hPNPase old-35 induces cell cycle arrest in the G 1 phase with inhibition of DNA synthesis and telomerase activity, and activates the senescence-associated b-galactosidase enzyme. 6 Moreover, hPNPase old-35 induces gene expression changes that are consistent with G 1 cell cycle arrest, such as downregulation of c-myc, upregulation of p27 KIP-1 and hypophosphorylation of Rb. 6 We previously found that downregulation of c-myc plays a critical part in hPNPase old-35 -induced growth inhibition, since overexpression of c-myc partially but significantly ablated this growth-inhibitory effect.…”

The PNPase, exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

“…Part of the PNPase population in E. coli is associated with the endoribonuclease RNase E, an RNA helicase, enolase, and possibly other proteins in a highmolecular weight complex called the degradosome [10][11][12][13][14]. Human PNPase was identified in an overlapping pathway screen to discover genes displaying coordinated expression as a consequence of terminal differentiation and senescence of melanoma cells [15][16][17]. In human cells PNPase is mostly located in the mitochondrial intermembrane space and may not play the major role in the processing and degradation of RNA that it plays in bacteria, chloroplasts and plant mitochondria [18,19].…”