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Paul B Fisher - One of the best experts on this subject based on the ideXlab platform.

  • defining the domains of human polynucleotide phosphorylase hpnpaseold 35 mediating cellular senescence
    Molecular and Cellular Biology, 2005
    Co-Authors: Devanand Sarkar, Kristoffer Valerie, Eunsook Park, Luni Emdad, Aaron Randolph, Paul B Fisher
    Abstract:

    RNases are enzymes that are master regulators of stability and decay of RNA (7-9). Depending upon their degradative characteristics, RNases are divided into two functional classes, endo- and exoribonucleases (8). In Escherichia coli, there are eight distinct exoribonucleases, of which two, RNase PH (RPH) and polynucleotide phosphorylase (PNPase), catalyze RNA degradation only in a phosphate-dependent manner (2, 8). In vivo, PNPase catalyzes mRNA decay in the 3′-5′ direction, while RPH is primarily involved in 3′ processing of tRNA precursors (15, 22, 29, 30). RPHs are proteins of ∼250 amino acid (aa) residues, whereas PNPase is a large molecule that contains two RPH domains separated by an α-helix (PNPase domain) and two COOH-terminal RNA binding domains, KH and S1 (29, 37, 38, 42). The structure of PNPase containing the five motifs is conspicuously preserved through evolution extending from prokaryotes and plants to mammals (23). In E. coli, PNPase to a small degree functions as a constituent of the degradosome, a multiprotein complex also consisting of RNase E, RNA helicase, enolase, and possibly other molecules (4). However, PNPase in the chloroplast forms a homotrimeric complex (2), and X-ray crystallographic analysis of the PNPase from the bacterium Streptomyces antibioticus also reveals a homotrimeric complex forming a “doughnut” shape surrounding a central channel capable of accommodating a single-stranded RNA molecule (37, 38). In addition to the characteristic five motifs, plant PNPase contains an N-terminal target peptide allowing translocation to chloroplasts and the mammalian PNPase contains an N-terminal mitochondrial localization signal facilitating its subcellular localization in mitochondria (28, 31, 40). Cloning of human polynucleotide phosphorylase was first achieved using an overlapping pathway screening (OPS) strategy designed to identify genes involved in the processes of cellular differentiation and senescence (24). Human melanoma HO-1 cells undergo terminal cell differentiation resembling melanocytes when treated with fibroblast beta interferon (IFN-β) and the protein kinase C activator Mezerein (MEZ) (11-13, 16, 21). Senescence is a state of irreversible growth arrest induced spontaneously in primary cells after a finite number of population doublings (replicative senescence) or induced by endogenous and exogenous acute and chronic stress signals (stress- or aberrant-signaling-induced senescence) (17, 34). Although terminal cell differentiation in HO-1 cells by IFN-β plus MEZ and cellular senescence represent two discrete phenomena, there are several overlapping characteristics of these processes. Both are distinguished by irreversible growth arrest associated with marked inhibition of DNA synthesis, inhibition of telomerase activity, and modulation of gene expression, especially up-regulation of cyclin-dependent kinase inhibitors (CDKI) (3, 12, 17, 21, 32). Screening of a temporal cDNA library generated from terminally differentiated HO-1 cells with cDNAs from senescent progeroid fibroblasts identified 75 genes, termed old-1 to -75, that were upregulated during terminal differentiation and senescence (24). Sequence analysis of one particular clone, old-35, confirmed identity to the PNPase gene, resulting in the gene being renamed hPNPaseold-35 (24). hPNPaseold-35 is an early type I interferon-inducible gene, and its expression is augmented in senescent progeroid fibroblasts in comparison to actively proliferating young counterparts (24, 25). In vitro assays confirmed the phosphate-dependent RNA degradation properties of hPNPaseOLD-35, thus confirming the structural and functional conservation of this molecule through evolution (24). Interestingly, overexpression of hPNPaseold-35 via an adenoviral vector in HO-1 cells promotes a senescence-like phenotype characterized by growth arrest in the G1 phase, inhibition of DNA synthesis and telomerase activity, and modulation of gene expression, most notably downregulation of c-myc and upregulation of CDKI p27KIP1 (32). In vitro assays documented that c-myc mRNA is specifically degraded by hPNPaseOLD-35, thus revealing a specific mRNA substrate for hPNPaseOLD-35 (32). The present study examines the different motifs of hPNPaseOLD-35 and their roles in mediating its senescence-inducing properties. We observed that either of the RPH domains is capable of inducing the morphological, biochemical, and gene expression changes leading to the senescence phenotype. This is the first demonstration of functional activity and specificity of discrete domains of hPNPaseOLD-35 molecule, which will now pave the way for an in-depth understanding of this intriguing molecule as well as permitting a comparative functional analysis of the domains of PNPase molecules in different species.

  • induction of senescence like growth arrest in melanocytes and malignant melanoma cells by human polynucleotide phosphorylase hpnpaseold 35
    Cancer Research, 2004
    Co-Authors: Devanand Sarkar, Dongchul Kang, Magdalena Leszczyniecka, Kristoffer Valerie, Tej K Pandita, Paul B Fisher
    Abstract:

    2413 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.

  • identification and cloning of human polynucleotide phosphorylase hpnpaseold 35 in the context of terminal differentiation and cellular senescence
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Magdalena Leszczyniecka, Dongchul Kang, Zaozhong Su, Devanand Sarkar, Matthew Holmes, Kristoffer Valerie, Paul B Fisher
    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. This strategy identified old-35, which encodes an evolutionary conserved gene, human polynucleotide phosphorylase (hPNPaseold-35), that is regulated predominantly by type I IFNs. The hPNPaseOLD-35 protein localizes in the cytoplasm of human cells and induces RNA degradation in vitro, as does its purified bacterial protein homologue. Ectopic expression of hPNPaseold-35 in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPaseold-35, an IFN-inducible 3′-5′ RNA exonuclease, provides additional support for a relationship between IFN action and RNA processing and suggests an important role for this gene in growth control associated with terminal differentiation and cellular senescence.

  • mda 5 an interferon inducible putative rna helicase with double stranded rna dependent atpase activity and melanoma growth suppressive properties
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Dongchul Kang, Paul B Fisher, Rahul V Gopalkrishnan, Qingping Wu, Eckhard Jankowsky, Anna Marie Pyle
    Abstract:

    Human melanoma cells can be reprogrammed to terminally differentiate and irreversibly lose proliferative capacity by appropriate pharmacological manipulation. Subtraction hybridization identified melanoma differentiation-associated gene-5 (mda-5) as a gene induced during differentiation, cancer reversion, and programmed cell death (apoptosis). This gene contains both a caspase recruitment domain and putative DExH group RNA helicase domains. Atypical helicase motifs of MDA-5 deviate from consensus sequences but are well conserved in a potentially new group of cloned and hypothetical proteins. mda-5 is an early response gene inducible by IFN and tumor necrosis factor-α, responding predominantly to IFN-β. Protein kinase C activation by Mezerein further augments mda-5 expression induced by IFN-β. Expression of mda-5 is controlled transcriptionally by IFN-β, and the MDA-5 protein localizes in the cytoplasm. mda-5 displays RNA-dependent ATPase activity, and ectopic expression of mda-5 in human melanoma cells inhibits colony formation. In these contexts, mda-5 may function as a mediator of IFN-induced growth inhibition and/or apoptosis. MDA-5 is a double-stranded RNA-dependent ATPase that contains both a caspase recruitment domain and RNA helicase motifs, with a confirmed association with growth and differentiation in human melanoma cells.

  • cell cycle gene expression and e2f transcription factor complexes in human melanoma cells induced to terminally differentiate
    Oncogene, 1995
    Co-Authors: Hongping Jiang, Jian Lin, Shumin Young, Neil I Goldstein, S Waxman, V Davila, S P Chellappan, Paul B Fisher
    Abstract:

    Defects in cellular differentiation are a common occurrence in human cancers. The combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound Mezerein (MEZ) results in an irreversible loss of proliferative capacity and terminal cell differentiation in H0-1 human melanoma cells. In contrast, either agent alone induces reversible growth arrest and/or specific components of the differentiation process without inducing terminal differentiation. The current study investigates changes in cell cycle, cell cycle gene expression and E2F transcription factor complex formation during the processes of reversible and irreversible (terminal) differentiation. Induction of both terminal differentiation and reversible differentiation (MEZ treatment) results in a temporal decrease in DNA synthesis and the percentage of cells in S phase and a decrease in the expression of cell cycle and growth regulated genes, including cdc2, cyclin A, cyclin B, histone H1, histone H4, nm23-H1, p53 and c-myc. Persistent gene expression changes occur in terminally differentiated cells, but not in reversibly differentiated cells. H0-1 cells contain several E2F binding activities, including uncomplexed E2F, an E2F-p107-cyclin A-cdk2 kinase complex and an Rb-E2F complex. Induction of growth arrest by MEZ results in a slow migrating gelshift band that contains E2F associated with the pRb2/p130 protein. There is also a loss of the Rb-E2F complex. Induction of terminal differentiation after treatment with IFN-beta + MEZ generates a second pRb2/p130-E2F complex that migrates considerably faster than the pRb2/p130-E2F complex resulting from growth arrest. The slower migrating complex may contribute to growth arrest, whereas the faster migrating complex may play a role in terminal differentiation. Our results demonstrate that terminal cell differentiation involves a co-ordinate and continuous suppression of a number of cell cycle and growth related genes and results in the development of a novel E2F transcription factor complex not apparent in growth arrested and reversibly differentiated human melanoma cells.

Hongping Jiang - One of the best experts on this subject based on the ideXlab platform.

  • genomic structure chromosomal localization and expression profile of a novel melanoma differentiation associated mda 7 gene with cancer specific growth suppressing and apoptosis inducing properties
    Oncogene, 2001
    Co-Authors: Eric Y Huang, Dongchul Kang, Rahul V Gopalkrishnan, Hongping Jiang, Magdalena Leszczyniecka, Malavi T Madireddi, Irina V Lebedeva, Jiao Jiao Lin, Deborah Alexandre
    Abstract:

    Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-β) and the protein kinase C activator Mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-β plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-β+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-β+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

  • cell cycle gene expression and e2f transcription factor complexes in human melanoma cells induced to terminally differentiate
    Oncogene, 1995
    Co-Authors: Hongping Jiang, Jian Lin, Shumin Young, Neil I Goldstein, S Waxman, V Davila, S P Chellappan, Paul B Fisher
    Abstract:

    Defects in cellular differentiation are a common occurrence in human cancers. The combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound Mezerein (MEZ) results in an irreversible loss of proliferative capacity and terminal cell differentiation in H0-1 human melanoma cells. In contrast, either agent alone induces reversible growth arrest and/or specific components of the differentiation process without inducing terminal differentiation. The current study investigates changes in cell cycle, cell cycle gene expression and E2F transcription factor complex formation during the processes of reversible and irreversible (terminal) differentiation. Induction of both terminal differentiation and reversible differentiation (MEZ treatment) results in a temporal decrease in DNA synthesis and the percentage of cells in S phase and a decrease in the expression of cell cycle and growth regulated genes, including cdc2, cyclin A, cyclin B, histone H1, histone H4, nm23-H1, p53 and c-myc. Persistent gene expression changes occur in terminally differentiated cells, but not in reversibly differentiated cells. H0-1 cells contain several E2F binding activities, including uncomplexed E2F, an E2F-p107-cyclin A-cdk2 kinase complex and an Rb-E2F complex. Induction of growth arrest by MEZ results in a slow migrating gelshift band that contains E2F associated with the pRb2/p130 protein. There is also a loss of the Rb-E2F complex. Induction of terminal differentiation after treatment with IFN-beta + MEZ generates a second pRb2/p130-E2F complex that migrates considerably faster than the pRb2/p130-E2F complex resulting from growth arrest. The slower migrating complex may contribute to growth arrest, whereas the faster migrating complex may play a role in terminal differentiation. Our results demonstrate that terminal cell differentiation involves a co-ordinate and continuous suppression of a number of cell cycle and growth related genes and results in the development of a novel E2F transcription factor complex not apparent in growth arrested and reversibly differentiated human melanoma cells.

  • induction of differentiation in human promyelocytic hl 60 leukemia cells activates p21 waf1 cip1 expression in the absence of p53
    Oncogene, 1994
    Co-Authors: Hongping Jiang, Zaozhong Su, F Collart, Eliezer Huberman, Paul B Fisher
    Abstract:

    The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound Mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed inmore » Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.« less

  • induction of differentiation in human promyelocytic hl 60 leukemia cells activates p21 waf1 cip1 expression in the absence of p53
    Oncogene, 1994
    Co-Authors: Hongping Jiang, Eliezer Huberman, Jian Lin, Frank R Collart, Paul B Fisher
    Abstract:

    The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound Mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed in Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.

Eliezer Huberman - One of the best experts on this subject based on the ideXlab platform.

  • induction of differentiation in human promyelocytic hl 60 leukemia cells activates p21 waf1 cip1 expression in the absence of p53
    Oncogene, 1994
    Co-Authors: Hongping Jiang, Zaozhong Su, F Collart, Eliezer Huberman, Paul B Fisher
    Abstract:

    The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound Mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed inmore » Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.« less

  • induction of differentiation in human promyelocytic hl 60 leukemia cells activates p21 waf1 cip1 expression in the absence of p53
    Oncogene, 1994
    Co-Authors: Hongping Jiang, Eliezer Huberman, Jian Lin, Frank R Collart, Paul B Fisher
    Abstract:

    The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound Mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed in Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.

Zaozhong Su - One of the best experts on this subject based on the ideXlab platform.

  • identification and cloning of human polynucleotide phosphorylase hpnpaseold 35 in the context of terminal differentiation and cellular senescence
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Magdalena Leszczyniecka, Dongchul Kang, Zaozhong Su, Devanand Sarkar, Matthew Holmes, Kristoffer Valerie, Paul B Fisher
    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. This strategy identified old-35, which encodes an evolutionary conserved gene, human polynucleotide phosphorylase (hPNPaseold-35), that is regulated predominantly by type I IFNs. The hPNPaseOLD-35 protein localizes in the cytoplasm of human cells and induces RNA degradation in vitro, as does its purified bacterial protein homologue. Ectopic expression of hPNPaseold-35 in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPaseold-35, an IFN-inducible 3′-5′ RNA exonuclease, provides additional support for a relationship between IFN action and RNA processing and suggests an important role for this gene in growth control associated with terminal differentiation and cellular senescence.

  • induction of differentiation in human promyelocytic hl 60 leukemia cells activates p21 waf1 cip1 expression in the absence of p53
    Oncogene, 1994
    Co-Authors: Hongping Jiang, Zaozhong Su, F Collart, Eliezer Huberman, Paul B Fisher
    Abstract:

    The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound Mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed inmore » Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.« less

Devanand Sarkar - One of the best experts on this subject based on the ideXlab platform.

  • defining the domains of human polynucleotide phosphorylase hpnpaseold 35 mediating cellular senescence
    Molecular and Cellular Biology, 2005
    Co-Authors: Devanand Sarkar, Kristoffer Valerie, Eunsook Park, Luni Emdad, Aaron Randolph, Paul B Fisher
    Abstract:

    RNases are enzymes that are master regulators of stability and decay of RNA (7-9). Depending upon their degradative characteristics, RNases are divided into two functional classes, endo- and exoribonucleases (8). In Escherichia coli, there are eight distinct exoribonucleases, of which two, RNase PH (RPH) and polynucleotide phosphorylase (PNPase), catalyze RNA degradation only in a phosphate-dependent manner (2, 8). In vivo, PNPase catalyzes mRNA decay in the 3′-5′ direction, while RPH is primarily involved in 3′ processing of tRNA precursors (15, 22, 29, 30). RPHs are proteins of ∼250 amino acid (aa) residues, whereas PNPase is a large molecule that contains two RPH domains separated by an α-helix (PNPase domain) and two COOH-terminal RNA binding domains, KH and S1 (29, 37, 38, 42). The structure of PNPase containing the five motifs is conspicuously preserved through evolution extending from prokaryotes and plants to mammals (23). In E. coli, PNPase to a small degree functions as a constituent of the degradosome, a multiprotein complex also consisting of RNase E, RNA helicase, enolase, and possibly other molecules (4). However, PNPase in the chloroplast forms a homotrimeric complex (2), and X-ray crystallographic analysis of the PNPase from the bacterium Streptomyces antibioticus also reveals a homotrimeric complex forming a “doughnut” shape surrounding a central channel capable of accommodating a single-stranded RNA molecule (37, 38). In addition to the characteristic five motifs, plant PNPase contains an N-terminal target peptide allowing translocation to chloroplasts and the mammalian PNPase contains an N-terminal mitochondrial localization signal facilitating its subcellular localization in mitochondria (28, 31, 40). Cloning of human polynucleotide phosphorylase was first achieved using an overlapping pathway screening (OPS) strategy designed to identify genes involved in the processes of cellular differentiation and senescence (24). Human melanoma HO-1 cells undergo terminal cell differentiation resembling melanocytes when treated with fibroblast beta interferon (IFN-β) and the protein kinase C activator Mezerein (MEZ) (11-13, 16, 21). Senescence is a state of irreversible growth arrest induced spontaneously in primary cells after a finite number of population doublings (replicative senescence) or induced by endogenous and exogenous acute and chronic stress signals (stress- or aberrant-signaling-induced senescence) (17, 34). Although terminal cell differentiation in HO-1 cells by IFN-β plus MEZ and cellular senescence represent two discrete phenomena, there are several overlapping characteristics of these processes. Both are distinguished by irreversible growth arrest associated with marked inhibition of DNA synthesis, inhibition of telomerase activity, and modulation of gene expression, especially up-regulation of cyclin-dependent kinase inhibitors (CDKI) (3, 12, 17, 21, 32). Screening of a temporal cDNA library generated from terminally differentiated HO-1 cells with cDNAs from senescent progeroid fibroblasts identified 75 genes, termed old-1 to -75, that were upregulated during terminal differentiation and senescence (24). Sequence analysis of one particular clone, old-35, confirmed identity to the PNPase gene, resulting in the gene being renamed hPNPaseold-35 (24). hPNPaseold-35 is an early type I interferon-inducible gene, and its expression is augmented in senescent progeroid fibroblasts in comparison to actively proliferating young counterparts (24, 25). In vitro assays confirmed the phosphate-dependent RNA degradation properties of hPNPaseOLD-35, thus confirming the structural and functional conservation of this molecule through evolution (24). Interestingly, overexpression of hPNPaseold-35 via an adenoviral vector in HO-1 cells promotes a senescence-like phenotype characterized by growth arrest in the G1 phase, inhibition of DNA synthesis and telomerase activity, and modulation of gene expression, most notably downregulation of c-myc and upregulation of CDKI p27KIP1 (32). In vitro assays documented that c-myc mRNA is specifically degraded by hPNPaseOLD-35, thus revealing a specific mRNA substrate for hPNPaseOLD-35 (32). The present study examines the different motifs of hPNPaseOLD-35 and their roles in mediating its senescence-inducing properties. We observed that either of the RPH domains is capable of inducing the morphological, biochemical, and gene expression changes leading to the senescence phenotype. This is the first demonstration of functional activity and specificity of discrete domains of hPNPaseOLD-35 molecule, which will now pave the way for an in-depth understanding of this intriguing molecule as well as permitting a comparative functional analysis of the domains of PNPase molecules in different species.

  • induction of senescence like growth arrest in melanocytes and malignant melanoma cells by human polynucleotide phosphorylase hpnpaseold 35
    Cancer Research, 2004
    Co-Authors: Devanand Sarkar, Dongchul Kang, Magdalena Leszczyniecka, Kristoffer Valerie, Tej K Pandita, Paul B Fisher
    Abstract:

    2413 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.

  • identification and cloning of human polynucleotide phosphorylase hpnpaseold 35 in the context of terminal differentiation and cellular senescence
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Magdalena Leszczyniecka, Dongchul Kang, Zaozhong Su, Devanand Sarkar, Matthew Holmes, Kristoffer Valerie, Paul B Fisher
    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. This strategy identified old-35, which encodes an evolutionary conserved gene, human polynucleotide phosphorylase (hPNPaseold-35), that is regulated predominantly by type I IFNs. The hPNPaseOLD-35 protein localizes in the cytoplasm of human cells and induces RNA degradation in vitro, as does its purified bacterial protein homologue. Ectopic expression of hPNPaseold-35 in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPaseold-35, an IFN-inducible 3′-5′ RNA exonuclease, provides additional support for a relationship between IFN action and RNA processing and suggests an important role for this gene in growth control associated with terminal differentiation and cellular senescence.