Down-regulation of Myc as a Potential Target for Growth Arrest Induced by Human Polynucleotide Phosphorylase (hPNPase) in Human Melanoma Cells
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m302421200
ISSN1083-351X
AutoresDevanand Sarkar, Magdalena Leszczyniecka, Dong-chul Kang, Irina V. Lebedeva, Kristoffer Valerie, Sonu Dhar, Tej K. Pandita, Paul B. Fisher,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoTerminal 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 (hPNPaseold-35), a 3′,5′-exoribonuclease. We previously demonstrated that hPNPaseold-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 hPNPaseold-35 resulted in growth suppression in HO-1 human melanoma cells. The present study examined the molecular mechanism of the growth-arresting property of hPNPaseold-35. When overexpressed by means of a replication-incompetent adenoviral vector (Ad.hPNPaseold-35), hPNPaseold-35 inhibited cell growth in all cell lines tested. Analysis of cell cycle revealed that infection of HO-1 cells with Ad.hPNPaseold-35 resulted in arrest in the G1 phase and eventually apoptosis accompanied by marked reduction in the S phase. Infection with Ad.hPNPaseold-35 resulted in reduction in expression of the c-myc mRNA and Myc protein and modulated the expression of proteins regulating G1 checkpoint and apoptosis. In vitro mRNA degradation assays revealed that hPNPaseOLD-35 degraded c-myc mRNA. Overexpression of Myc partially but significantly protected HO-1 cells from Ad.hPNPaseold-35-induced growth arrest, indicating that Myc down-regulation might directly mediate the growth-inhibitory properties of Ad.hPNPaseold-35. Inhibition of hPNPaseold-35 by an antisense approach provided partial but significant protection against interferon-β-mediated growth inhibition, thus demonstrating the biological significance of hPNPaseold-35 in interferon action. 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 (hPNPaseold-35), a 3′,5′-exoribonuclease. We previously demonstrated that hPNPaseold-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 hPNPaseold-35 resulted in growth suppression in HO-1 human melanoma cells. The present study examined the molecular mechanism of the growth-arresting property of hPNPaseold-35. When overexpressed by means of a replication-incompetent adenoviral vector (Ad.hPNPaseold-35), hPNPaseold-35 inhibited cell growth in all cell lines tested. Analysis of cell cycle revealed that infection of HO-1 cells with Ad.hPNPaseold-35 resulted in arrest in the G1 phase and eventually apoptosis accompanied by marked reduction in the S phase. Infection with Ad.hPNPaseold-35 resulted in reduction in expression of the c-myc mRNA and Myc protein and modulated the expression of proteins regulating G1 checkpoint and apoptosis. In vitro mRNA degradation assays revealed that hPNPaseOLD-35 degraded c-myc mRNA. Overexpression of Myc partially but significantly protected HO-1 cells from Ad.hPNPaseold-35-induced growth arrest, indicating that Myc down-regulation might directly mediate the growth-inhibitory properties of Ad.hPNPaseold-35. Inhibition of hPNPaseold-35 by an antisense approach provided partial but significant protection against interferon-β-mediated growth inhibition, thus demonstrating the biological significance of hPNPaseold-35 in interferon action. 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-β) 1The abbreviations used are: IFN, interferon; MEZ, mezerein; m.o.i., multiplicity of infection; pfu, plaque-forming unit(s); AS, antisense; HA, hemagglutinin; CDK, cyclin-dependent kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Rb, retinoblastoma; GAPDH, glyceradehyde-3-phosphate dehydrogenase. 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 (1Fisher P.B. Grant S. Pharmacol. Ther. 1985; 27: 143-166Google Scholar, 2Fisher P.B. Prignoli D.R. Hermo Jr., H. Weinstein I.B. Pestka S. J. Interferon Res. 1985; 5: 11-22Google Scholar, 3Graham G.M. Guarini L. Moulton T.A. Datta S. Ferrone S. Giacomini P. Kerbel R.S. Fisher P.B. Cancer Immunol. Immunother. 1991; 32: 382-390Google Scholar, 4Guarini L. Graham G.M. Jiang H. Ferrone S. Zucker S. Fisher P.B. Pigment Cell Res. Suppl. 1992; 2: 123-131Google Scholar, 5Jiang H. Su Z.Z. Boyd J. Fisher P.B. Mol. Cell Differ. 1993; 1: 41-66Google Scholar). 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 (6Hayflick L. N. Engl. J. Med. 1976; 295: 1302-1308Google Scholar). 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. 7Serrano M. Blasco M.A. Curr. Opin. Cell Biol. 2001; 13: 748-753Google Scholar). 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 (8Leszczyniecka M. Kang D.-C. Sarkar D. Su Z.Z. Holmes M. Valerie K. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16636-16641Google Scholar). 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 (hPNPaseold-35), a 3′,5′ exoribonuclease involved in RNA degradation (8Leszczyniecka M. Kang D.-C. Sarkar D. Su Z.Z. Holmes M. Valerie K. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16636-16641Google Scholar). hPNPaseold-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 hPNPaseold-35 is involved in RNA degradation. Analysis of the expression profile of hPNPaseold-35 revealed that it is predominantly a type I interferon-inducible gene, and its expression is also induced in senescent fibroblasts in comparison with young fibroblasts. These findings indicate that hPNPaseold-35 might play an essential role in interferon- and senescence-induced growth arrest. Indeed, when hPNPaseold-35 is transfected by plasmid or transduced via a replication-incompetent adenovirus (Ad.hPNPaseold-35), there is a marked reduction in the colony-forming ability of HO-1 cells. The objective of the present study was to elucidate the molecular mechanism of the growth-suppressing property of Ad.hPNPaseold-35. The protooncogene c-myc is involved in a wide range of cellular processes including proliferation, differentiation, and tumorigenesis (for a recent review, see Ref. 9Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar). Myc belongs to the Max network, a group of transcription factors containing basic helix-loop-helix zipper motifs (10Ferre-D'Amare A.R. Pognonec P. Roeder R.G. Burley S.K. EMBO J. 1994; 13: 180-189Google Scholar, 11Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschka S.D. Lassar A.B. et al.Cell. 1989; 58: 537-544Google Scholar). Myc heterodimerizes with Max and binds to the E-box sequence (CACGTG), thereby activating transcription (12Blackwood E.M. Eisenman R.N. Science. 1991; 251: 1211-1217Google Scholar). Max is constitutively expressed throughout the cell cycle (13Blackwood E.M. Luscher B. Eisenman R.N. Genes Dev. 1992; 6: 71-80Google Scholar), and it also heterodimerizes with the Mad family of transcription factors: Mad1, Mxi1, Mad3, and Mad4 (14Ayer D.E. Eisenman R.N. Genes Dev. 1993; 7: 2110-2119Google Scholar, 15Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 223-232Google Scholar, 16Hurlin P.J. Queva C. Koskinen P.J. Steingrimsson E. Ayer D.E. Copeland N.G. Jenkins N.A. Eisenman R.N. EMBO J. 1995; 14: 5646-5659Google Scholar); however, in contrast to Myc-Max, the Mad-Max heterodimers act as transcriptional repressors at the same binding sites. The most important function of Myc is its essential role in controlling cell proliferation. Expression of exogenous Myc in cultured fibroblasts promotes S phase entry and shortens G1 phase of the cell cycle, whereas activation of a conditional Myc is sufficient to drive quiescent cells into the cell cycle (17Eilers M. Picard D. Yamamoto K.R. Bishop J.M. Nature. 1989; 340: 66-68Google Scholar, 18Karn J. Watson J.V. Lowe A.D. Green S.M. Vedeckis W. Oncogene. 1989; 4: 773-787Google Scholar). The progression of cell cycle beyond the G1 phase is also augmented by the activities of the cyclin-dependent kinase (CDK) complexes cyclin D-CDK4 and cyclin E-CDK2, and the activities of these complexes are inhibited by CDK inhibitors, the Cip/Kip (CDK-interacting protein/kinase-inhibitory protein) family, including p27KIP1 and p21CIP1/WAF-1/MDA-6 and the INK (inhibitors of CDK4) family including p16INK4A and p15INK4B (19Bartek J. Lukas J. Curr. Opin. Cell Biol. 2001; 13: 738-747Google Scholar). Cyclins D and E are essential for G1-S progression in higher eukaryotic cells and when overexpressed are able to shorten the G1 interval (20Baldin V. Lukas J. Marcote M.J. Pagano M. Draetta G. Genes Dev. 1993; 7: 812-821Google Scholar, 21Resnitzky D. Gossen M. Bujard H. Reed S.I. Mol. Cell. Biol. 1994; 14: 1669-1679Google Scholar). The major pathway by which Myc induces cell cycle progression is by activating cyclin D2 and CDK4 (22Bouchard C. Thieke K. Maier A. Saffrich R. Hanley-Hyde J. Ansorge W. Reed S. Sicinski P. Bartek J. Eilers M. EMBO J. 1999; 18: 5321-5333Google Scholar, 23Perez-Roger I. Solomon D.L. Sewing A. Land H. Oncogene. 1997; 14: 2373-2381Google Scholar, 24Hermeking H. Rago C. Schuhmacher M. Li Q. Barrett J.F. Obaya A.J. O'Connell B.C. Mateyak M.K. Tam W. Kohlhuber F. Dang C.V. Sedivy J.M. Eick D. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2229-2234Google Scholar, 25Coller H.A. Grandori C. Tamayo P. Colbert T. Lander E.S. Eisenman R.N. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3260-3265Google Scholar). An important consequence of the induction by Myc of cyclin D2 is its sequestration of p27KIP1 CDK inhibitor, permitting unfettered and prolonged activity of the cyclin E-CDK2 complex (26Pusch O. Bernaschek G. Eilers M. Hengstschlager M. Oncogene. 1997; 15: 649-656Google Scholar). Increased cyclin E-CDK2 activity shortens G1, whereas increased CDK2 and CDK4 activities result in hyperphosphorylation of the retinoblastoma (Rb) protein. This leads to release of E2F, a family of transcription factors that regulate a battery of genes necessary for cell cycle progression, from complexes with Rb and together with the direct induction of E2F2 by Myc, may further contribute to cell cycle progression (27Sears R. Ohtani K. Nevins J.R. Mol. Cell. Biol. 1997; 17: 5227-5235Google Scholar). In addition Myc can directly repress p27KIP1 and p21CIP1/WAF-1/MDA-6 transcription (28Yang W. Shen J. Wu M. Arsura M. FitzGerald M. Suldan Z. Kim D.W. Hofmann C.S. Pianetti S. Romieu-Mourez R. Freedman L.P. Sonenshein G.E. Oncogene. 2001; 20: 1688-1702Google Scholar, 29Claassen G.F. Hann S.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9498-9503Google Scholar). In the present study, we show that infection of HO-1 melanoma cells with Ad.hPNPaseold-35resulted in cell cycle arrest in the G1 phase and eventually apoptosis with marked reduction in DNA synthesis. Ad.hPNPaseold-35infection caused reduction in expression of the c-myc mRNA and Myc protein that was accompanied by induction of Mad1 protein. Overexpression of Myc protected HO-1 cells against Ad.hPNPaseold-35-mediated cell death. These findings argue that Myc might play a pivotal role in mediating the growth-inhibitory properties of Ad.hPNPaseold-35. We also show that inhibition of hPNPaseold-35 by an antisense strategy partially but significantly rescues HO-1 cells from interferon-β-mediated growth inhibition, thereby documenting a potential role of hPNPaseold-35 in mediating interferon action. Cell Lines and Cell Viability Assays—Normal immortal human melanocyte (FM516-SV; FM516), WM35 early radial growth phase, and WM278 vertical growth phase primary human melanomas, and HO-1, FO-1, and MeWo metastatic melanoma cell lines and HEK-293 cells were cultured as previously described (30Lebedeva I.V. Su Z.Z. Chang Y. Kitada S. Reed J.C. Fisher P.B. Oncogene. 2002; 21: 708-718Google Scholar). HO-1-pREP4 and HO-1-hPNPaseold-35AS cell lines were generated by stable transfection of HO-1 cells with pREP4 (HO-1-pREP4) or antisense hPNPaseold-35 expressing pREP4 (HO-1-hPNPaseold-35AS), respectively, and selection with hygromycin. HO-1-Bcl-2 and HO-1-Bcl-xL cell lines were produced by stable transfection of HO-1 cells with Bcl-2 and Bcl-xL expression plasmids (kindly provided by Dr. John C. Reed) and selection with G418. Cell growth and viable cell numbers were monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) staining as described (30Lebedeva I.V. Su Z.Z. Chang Y. Kitada S. Reed J.C. Fisher P.B. Oncogene. 2002; 21: 708-718Google Scholar). Cultures were incubated with interferon-β (2000 units/ml) and mezerein (10 ng/ml) for 5 days prior to assaying for cell viability. Virus Construction and Infection Protocol—The construction and purification of hPNPaseold-35 expressing replication-defective adenovirus Ad.hPNPaseold-35were described previously (8Leszczyniecka M. Kang D.-C. Sarkar D. Su Z.Z. Holmes M. Valerie K. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16636-16641Google Scholar, 31Valerie K. Wu-Pong S. Rojanasakul Y. Biopharmaceutical Drug Design and Development. Humana Press, Totowa, NJ1999: 69-142Google Scholar). A similar method was employed to generate an antisense hPNPaseold-35 expressing replication-defective adenovirus (Ad.hPNPaseold-35AS). The empty adenoviral vector (Ad.vec) was used as a control. Viral infections were performed as previously described (30Lebedeva I.V. Su Z.Z. Chang Y. Kitada S. Reed J.C. Fisher P.B. Oncogene. 2002; 21: 708-718Google Scholar). Plasmid Construction, Transfection, and Colony Formation Assays— 3′-HA-tagged hPNPaseold-35 was created by PCR using the primers GCT AGC ATG GCG GCC TGC AGG TAC (sense) and GGA TCC TCA AGC GTA ATC TGG AAC ATC GTA TGG GTA CTG AGA ATT AGA TGA TGA (antisense). The authenticity of the amplified product was verified by sequencing, and it was cloned into the NheI/BamHI sites of pcDNA3.1 (Invitrogen) to generate hPNPaseold-35-HA. hPNPaseold-35AS was generated by ligating hPNPaseold-35 in an antisense orientation into BamHI/NotI sites of pREP4 (Invitrogen). The c-myc expression plasmid p290-myc (2Fisher P.B. Prignoli D.R. Hermo Jr., H. Weinstein I.B. Pestka S. J. Interferon Res. 1985; 5: 11-22Google Scholar, 3Graham G.M. Guarini L. Moulton T.A. Datta S. Ferrone S. Giacomini P. Kerbel R.S. Fisher P.B. Cancer Immunol. Immunother. 1991; 32: 382-390Google Scholar) was provided by Dr. Riccardo Dalla-Favera. HO-1 cells were plated at a density of 3 × 105 cells/6-cm dish and 24 h later were transfected with 5 μg of either empty vector or p290-myc (2Fisher P.B. Prignoli D.R. Hermo Jr., H. Weinstein I.B. Pestka S. J. Interferon Res. 1985; 5: 11-22Google Scholar, 3Graham G.M. Guarini L. Moulton T.A. Datta S. Ferrone S. Giacomini P. Kerbel R.S. Fisher P.B. Cancer Immunol. Immunother. 1991; 32: 382-390Google Scholar) using Superfect® (Qiagen, Hilden, Germany) transfection reagent according to the protocol from the manufacturer. After 36 h, the cells were infected with Ad.hPNPaseold-35 at a multiplicity of infection (m.o.i.) of 50 or 100 pfu/cell; 6 h later, the cells were trypsinized and counted and 103 cells were plated in 6-cm dishes. Colonies were counted after 3 weeks. Colony formation assays using hPNPaseold-35-HA in HO-1 cells were performed as described (32Kang D.C. Gopalkrishnan R.V. Wu Q. Jankowsky E. Pyle A.M. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 637-642Google Scholar). RNA Isolation and Northern Blot Analysis—Total RNA was extracted from the cells using Qiagen RNeasy mini kit (Qiagen) according to the protocol from the manufacturer, and Northern blotting was performed as described (33Sarkar D. Su Z.Z. Lebedeva I.V. Sauane M. Gopalkrishnan R.V. Valerie K. Dent P. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10054-10059Google Scholar). The cDNA probes used were a 400-bp fragment from human c-myc, a 500-bp fragment from hPNPaseold-35, a 500-bp fragment from human GADD34, full-length human c-jun, and full-length human GAPDH. In Vitro Translation and in Vitro mRNA Degradation Assays—In vitro translation was performed using the TNT-coupled Reticulocyte Lysate Systems (Promega, Madison, WI) using the plasmids pcDNA3.1 as a control, GADD153 expression plasmid, and hPNPaseold-35-HA according to the protocol from the manufacturer. Five μg of total RNA from HO-1 cells were incubated with 5 μl of each in vitro translated protein at 37 °C from 0.5 to 3 h. The RNA was repurified using the Qiagen RNeasy mini kit, and Northern blotting was performed (33Sarkar D. Su Z.Z. Lebedeva I.V. Sauane M. Gopalkrishnan R.V. Valerie K. Dent P. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10054-10059Google Scholar). Western Blot Analysis—Western blotting was performed as previously described (33Sarkar D. Su Z.Z. Lebedeva I.V. Sauane M. Gopalkrishnan R.V. Valerie K. Dent P. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10054-10059Google Scholar). Briefly, cells were harvested in radioimmune precipitation assay buffer containing protease inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany), 1 mm Na3VO4, and 50 mm NaF and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was used as total cell lysate. Thirty μg of total cell lysate were used for SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibodies included: Myc (1:200; mouse monoclonal), Max (1: 200; rabbit polyclonal), Mad1 (1:200; rabbit polyclonal), p16 (1:200; rabbit polyclonal), p21 (1:200; rabbit polyclonal), p27 (1:200; rabbit polyclonal), p53 (1:200; mouse monoclonal), cyclin E (1:200, rabbit polyclonal), and E2F1 (1:200, rabbit polyclonal) (all from Santa Cruz Biotechnology, Santa Cruz, CA); Rb (1:500, mouse monoclonal) and cyclin A (1:500, mouse monoclonal) (from BD Biosciences); Bcl-2, Bcl-xL, and Bax (1:1000; rabbit polyclonal; kindly provided by Dr. John C. Reed); anti-HA (1:3000; mouse monoclonal; Covance Research Products, Inc., Berkeley, CA); and EF1α (1:1000; mouse monoclonal; Upstate Biotechnology, Inc., Waltham, MA). [3H]Thymidine Incorporation Assay—HO-1 cells were plated at a density of 5 × 104 cells in each well of a 12-well plate. The next day the cells were infected with Ad.hPNPaseold-35 at an m.o.i. of 25 or 50 pfu/cell. After 4 days the cells were incubated with 10 μCi/ml [3H]thymidine for 12 h. The cells were washed with phosphate-buffered saline and incubated with 2 ml of ice-cold 10% trichloroacetic acid at 4 °C for 30 min. Trichloroacetic acid-precipitated materials were collected by centrifugation and solubilized with 1 ml of 2% SDS, and 100-μl aliquots were counted in a liquid scintillation counter. Cell Cycle Analysis— Cells were harvested, washed in phosphate-buffered saline, and fixed overnight at –20 °C in 70% ethanol. The cells were treated with RNase A (1 mg/ml) at 37 °C for 30 min and then with propidium iodide (50 μg/ml). Cell cycle was analyzed using a FACScan flow cytometer, and data were analyzed using CellQuest software (Becton Dickinson, San Jose, CA). Telomerase Assay—HO-1 cells were infected with either Ad.vec or Ad.hPNPaseold-35 for 1–4 days or untreated or treated with fibroblast IFN-β (2000 units/ml) plus MEZ (10 ng/ml) for 1–4 days, and telomerase assays were performed as described previously (34Wood L.D. Halvorsen T.L. Dhar S. Baur J.A. Pandita R.K. Wright W.E. Hande M.P. Calaf G. Hei T.K. Levine F. Shay J.W. Wang J.J. Pandita T.K. Oncogene. 2001; 20: 278-288Google Scholar). Briefly, protein concentrations of cell extracts were determined, and equal amounts of protein were used for the elongation process in which telomerase added telomeric repeats (TTAGGG) to the 3′ end of the biotin-labeled primer. These elongation products were amplified by PCR, and the PCR products were denatured and hybridized to digoxigenin-labeled detection probes, specific for the telomeric repeats. The resulting products were immobilized via the biotin label to a streptavidin-coated microtiter plate. Immobilized amplicons were detected with an antibody against digoxigenin that is conjugated to horseradish peroxidase and the sensitive peroxidase substrate 3,3′,5,5′-tetramethylbenzidine. The telomerase activity was quantified by measuring the absorbance of the samples at 450 nm (with a reference wavelength of 690 nm) using a microtiter plate reader. Statistical Analysis—Statistical analysis was performed using oneway analysis of variance, followed by Fisher's protected least significant difference analysis. Previous studies demonstrated that infection with Ad.hPNPaseold-35 inhibited colony formation in HO-1 melanoma cells (8Leszczyniecka M. Kang D.-C. Sarkar D. Su Z.Z. Holmes M. Valerie K. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16636-16641Google Scholar). The present studies were conducted to comprehend the molecular mechanism underlying the growth-arresting property of Ad.hPNPaseold-35. Different melanoma cell lines and SV40 T-Ag immortalized primary human melanocytes (FM-516-SV) were infected with Ad.hPNPaseold-35, and the growth of the cells was monitored by standard MTT assays. As shown in Fig. 1, infection with Ad.hPNPaseold-35 resulted in significant growth inhibition in all of the cells. The growth-inhibitory effect became significant from 4 days after infection, and, in certain cell lines (WM278 and MeWo), infection with Ad.hPNPaseold-35 completely inhibited cell growth. In addition, Ad.hPNPaseold-35 infection inhibited the growth of other cell types, including breast, prostate, colon and pancreatic carcinomas, glioblastoma multiforme, fibrosarcoma, and osteosarcoma, irrespective of their p53 or Rb status (data not shown). To investigate the mechanism of Ad.hPNPaseold-35-mediated growth inhibition, cell cycle analysis was performed following Ad.hPNPaseold-35 infection in HO-1 cells. When the cells were infected with Ad.hPNPaseold-35 at a high m.o.i. of 100 pfu/cell for 4 days, there was a significant increase in sub-G0 population of cells indicating apoptosis and a decrease in the S phase indicating inhibition of DNA synthesis (Fig. 2, A and B). When the kinetics of killing was slowed down by infecting cells at a low m.o.i. of 25 pfu/cell, there was an initial significant increase in cells in the G1 phase of the cell cycle (Fig. 2, C and D) at 3 and 6 days after infection. This increase was also accompanied by a marked decrease in the S phase. At later time points, the cells infected with Ad.hPNPaseold-35, but not control or Ad.vec-infected cells, started to die by apoptosis (Fig. 2D). The kinetics of cell death was very slow when HO-1 cells were infected with Ad.hPNPaseold-35 at a low m.o.i. It is worth noting that at 25 pfu/cell ∼90% of the cells are infected with adenovirus (data not shown). From these observations it might be inferred that infection with Ad.hPNPaseold-35 induces cell cycle arrest at the G1 phase that ultimately culminates in apoptosis. The inhibition of DNA synthesis following Ad.hPNPaseold-35 infection was confirmed using a [3H]thymidine incorporation assay. As shown in Fig. 3A, infection with Ad.vec did not have an impact on DNA synthesis. Infection with Ad.hPNPaseold-35 reduced DNA synthesis by ∼40% at an m.o.i. of 25 pfu/cell and by ∼75% at 50 pfu/cell 4 days after infection. Telomerase activity is decreased in both terminal differentiation and senescence. As shown in Fig. 3B, telomerase activity decreased in a time-dependent manner to ∼50% when HO-1 cells were treated with IFN-β + MEZ for up to 4 days. This treatment protocol results in the induction of irreversible growth arrest and terminal differentiation in HO-1 melanoma cells (2Fisher P.B. Prignoli D.R. Hermo Jr., H. Weinstein I.B. Pestka S. J. Interferon Res. 1985; 5: 11-22Google Scholar, 5Jiang H. Su Z.Z. Boyd J. Fisher P.B. Mol. Cell Differ. 1993; 1: 41-66Google Scholar). Based on these findings, telomerase activity was also determined following Ad.hPNPaseold-35 infection. As shown in Fig. 3C, infection with Ad.hPNPaseold-35 at an m.o.i. of 100 pfu/cell, but not with Ad.vec, inhibited telomerase activity by almost 60% at day 4 after infection. One of the factors that facilitate entry into the S phase of the cell cycle is Myc. During terminal differentiation of melanoma cells, c-myc mRNA expression is down-regulated (35Jiang H. Lin J. Young S.M. Goldstein N.I. Waxman S. Davila V. Chellappan S.P. Fisher P.B. Oncogene. 1995; 11: 1179-1189Google Scholar). The expression level of c-myc mRNA following Ad.hPNPaseold-35 infection was, therefore, determined by Northern blot analysis. The expression of c-myc mRNA began decreasing 2 days after Ad.hPNPaseold-35 infection but not in uninfected or Ad.vec-infected cells even at 4 days after infection (Fig. 4A). This decrease correlates with the expression of hPNPaseold-35 mRNA that was also detected 2 days after infection. It should be noted that, under basal condition, hPNPaseold-35 mRNA is undetectable in HO-1 cells. The expression of the GAPDH housekeeping gene remained unchanged following Ad.hPNPaseold-35 infection. Down-regulation of Myc protein by different stimuli is usually accompanied by up-regulation of Mad1, the transcriptional repressor belonging to the Max family of transcription factors (9Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar). In this context, the expressions of Myc, its heterodimer partner Max, and Mad1 were determined by Western blot analysis following Ad.hPNPaseold-35 infection. As anticipated from Northern blot analysis, Myc expression started decreasing 2 days after Ad.hPNPaseold-35 infection but not in uninfected or Ad.vec-infected cells at 4 days after infection (Fig. 4B). This down-regulation was accompanied by up-regulation of Mad1 protein. The level of Max protein remained unchanged, indicating that infection with Ad.hPNPaseold-35 switches the Myc-Max transcriptional activator to Mad1-Max transcriptional repressor. The expression level of the EF1α housekeeping gene did not change under any condition. We next addressed whether c-myc overexpression could protect HO-1 cells from Ad.hPNPaseold-35-mediated cell death. At first we determined whether the Myc expression plasmid generates the appropriate protein. For this assay, HEK-293 cells were used because transfection efficiency in these cells is very high, permitting easy detection of expressed protein by Western blot analysis. As shown in Fig. 4C, transfection of p290-myc (2Fisher P.B. Prignoli D.R. Hermo Jr., H. Weinstein I.B. Pestka S. J. Interferon Res. 1985; 5: 11-22Google Scholar, 3Graham G.M. Guarini L. Moulton T.A. Datta S. Ferrone S. Giacomini P. Kerbel R.S. Fisher P.B. Cancer Immunol. Immunother. 1991; 32: 382-390Google Scholar) in HEK-293 cells resulted in significant overexpression of Myc in comparison with the cells transfected with empty vector. For protection assays, HO-1 cells were transfected with p290-myc (2Fisher P.B. Prignoli D.R. Hermo Jr., H. Weinstein I.B. Pestka S. J. Interferon Res. 1985; 5: 11-22Google Scholar, 3Graham G.M. Guarini L. Moulton T.A. Datta S. Ferrone S. Giacomini P. Kerbel R.S. Fisher P.B. Cancer Immunol. Immunother. 1991; 32: 382-390Google Scholar), infected with Ad.hPNPaseold-35, and the growth of the cells were analyzed by colony formation assays. Overexpression of Myc provided partial but significant protection against Ad.hPNPaseold-35-mediated cell death (Fig. 4, D and E) consistent with the possibility that one pathway by which Ad.hPNPaseold-35 induces growth suppression and cell death is by down-regulation of Myc. hPNPaseold-35 is a 3′,5′-exoribonuclease, prompting us to determine whether it can directly degrade c-myc mRNA. For this analysis a C-terminal HA-tagged hPNPaseold-35-expressing construct (hPNPaseold-35-HA) was created. The authenticity of the construct was first confirmed by transfecting it into HEK-293 cells. As shown in Fig. 5A, Western blot analysis using anti-HA antibody detected a single protein of ∼90 kDa in size only in the hPNPaseold-35-HA-transfected cells. To check whether this construct has functional similarity to Ad.hPNPaseold-35, HO-1 cells were transfected
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