Identification and cloning of human polynucleotide phosphorylase, hPNPase ^old-35 , in the context of terminal differentiation and cellular senescence

Abstract

Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. overlapping-pathway screen ͉ terminal cell differentiation ͉ senescent phenotype ͉ interferon-inducible gene ͉ evolutionary conserved gene P lasticity of the transformed phenotype is suggested by the ability of differentiation-inducing agents to revert the cancerous properties of specific tumors (1-3). This attribute of tumor cells provides the basis for a potentially less toxic form of therapy, ''differentiation therapy.'' In metastatic human melanoma, a combination of IFN-␤ and the protein kinase C activator mezerein (MEZ) produces irreversible growth arrest, a loss of tumorigenic competence, and terminal differentiation (1, 4). To define gene-expression changes associated with induction of terminal differentiation, a subtracted cDNA library enriched for genes associated with terminal differentiation was constructed (5). This construction was accomplished by subtracting control HO-1 human melanoma mRNAs from IFN-␤ ϩ MEZ-treated HO-1 mRNAs, which were temporally collected over a 24-h period (5). This subtracted cDNA library then was screened by random isolation of phage colonies and Northern blotting, high-density cDNA microarray analysis, and reverse Northern screening followed by Northern blotting (5-7). These approaches have identified both unknown and known genes associated with tumor and normal growth control, cell-cycle regulation, IFN response, differentiation, and apoptosis (5-12). Four classes of melanoma differentiation-associated (mda) genes have been identified (5, 10).Terminal cell differentiation and cellular senescence are characterized by changes in cell morphology, lack of responsiveness to mitogenic stimulation, and irreversible growth arrest (1, 4, 13-18).Normal cells cultured in vitro lose their proliferative potential after a finite number of doublings in a process described as cellular senescence (13). Experiments in human diploid fibroblasts and additional cell types document an inverse correlation between replicative senescence and donor age and a direct relationship between replicative senescence and donor-species life span (13,19,20). In agreement with this relationship, cells from patients with premature aging syndromes such as Werner's syndrome and progeria achieve a quiescent state more rapidly than normal human fibroblasts (21). Although senescence is a time-dependent process, terminal differentiation can be induced in a variety of cell types by appropriate treatme...

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

“…7,8 In total, 75 ESTs, termed old 1-75, were identified by OPS and old-35, which was later recognized to be the human version of PNPase, was found to be consistently overexpressed during the terminal differentiation process in human melanoma cells and senescent human fibroblasts. 3 While its expression was virtually undetectable in human melanoma cells under basal condition, hPNPase old- 35 was markedly induced within 6 h following treatment with IFN-b, highlighting a potentially prominent role in mediating IFN-b-induced molecular events. 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.…”

“…[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][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. 5 The human homolog was recently identified by our laboratory using a unique cloning strategy termed 'overlapping pathway screening' (OPS), in which a terminally differentiated human melanoma cDNA library was screened with cDNAs derived from senescent fibroblasts.…”

“…5 The human homolog was recently identified by our laboratory using a unique cloning strategy termed 'overlapping pathway screening' (OPS), in which a terminally differentiated human melanoma cDNA library was screened with cDNAs derived from senescent fibroblasts. 3 Terminal differentiation of human melanoma cells, induced by combined treatment with IFN-b and the protein kinase C activator mezerein (MEZ), and cellular senescence share several common end points, one of which is irreversible growth arrest, and the premise underlying the OPS approach was to identify genes that are critical mediators of two significant physiological processes, namely terminal differentiation and senescence. 7,8 In total, 75 ESTs, termed old 1-75, were identified by OPS and old-35, which was later recognized to be the human version of PNPase, was found to be consistently overexpressed during the terminal differentiation process in human melanoma cells and senescent human fibroblasts.…”

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].…”