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• W1487941441 abstract "We have identified a novel c-Myc-responsive gene, named JPO1, by representational difference analysis.JPO1 responds to two inducible c-Myc systems and behaves as a direct c-Myc target gene. JPO1 mRNA expression is readily detectable in the thymus, small intestine, and colon, whereas expression is relatively low in spleen, bone marrow, and peripheral leukocytes. We cloned a full-length JPO1 cDNA that encodes a 47-kDa nuclear protein. To determine the role of JPO1 in Myc-mediated cellular phenotypes, stable Rat1a fibroblasts overexpressing JPO1 were tested and compared with transformed Rat1a-Myc cells. Although JPO1 has a diminished transforming activity as compared with c-Myc, JPO1 complements a transformation-defective Myc Box II mutant in the Rat1a transformation assay. This complementation provides evidence for a genetic link between c-Myc and JPO1. Similar to c-Myc, JPO1 overexpression enhances the clonogenicity of CB33 human lymphoblastoid cells in methylcellulose assays. These observations suggest that JPO1 participates in c-Myc-mediated transformation, supporting an emerging concept that c-Myc target genes constitute nodal points in a network of pathways that lead from c-Myc to various Myc-related phenotypes and ultimately to tumorigenesis. We have identified a novel c-Myc-responsive gene, named JPO1, by representational difference analysis.JPO1 responds to two inducible c-Myc systems and behaves as a direct c-Myc target gene. JPO1 mRNA expression is readily detectable in the thymus, small intestine, and colon, whereas expression is relatively low in spleen, bone marrow, and peripheral leukocytes. We cloned a full-length JPO1 cDNA that encodes a 47-kDa nuclear protein. To determine the role of JPO1 in Myc-mediated cellular phenotypes, stable Rat1a fibroblasts overexpressing JPO1 were tested and compared with transformed Rat1a-Myc cells. Although JPO1 has a diminished transforming activity as compared with c-Myc, JPO1 complements a transformation-defective Myc Box II mutant in the Rat1a transformation assay. This complementation provides evidence for a genetic link between c-Myc and JPO1. Similar to c-Myc, JPO1 overexpression enhances the clonogenicity of CB33 human lymphoblastoid cells in methylcellulose assays. These observations suggest that JPO1 participates in c-Myc-mediated transformation, supporting an emerging concept that c-Myc target genes constitute nodal points in a network of pathways that lead from c-Myc to various Myc-related phenotypes and ultimately to tumorigenesis. representational difference analysis Dulbecco's modified Eagle's medium Iscove's modified Dulbecco's medium 4-OH-tamoxifen cycloheximide endoplasmic reticulum ornithine decarboxylase murine Moloney leukemia virus c-myc was first identified as the cellular homologue of the v-myc oncogene in the avian myelocytomatosis retrovirus, MC29 (1Bishop J.M. Adv. Cancer Res. 1982; 37: 1-32Crossref PubMed Scopus (99) Google Scholar). Since its discovery, c-myc has been implicated in numerous human and animal tumors (2Nesbit C.E. Tersak J.M. Prochownik E.V. Oncogene. 1999; 18: 3004-3016Crossref PubMed Scopus (966) Google Scholar, 3Cole M.D. Annu. Rev. Genet. 1986; 20: 361-384Crossref PubMed Scopus (554) Google Scholar). In cases of Burkitt's lymphoma, the c-myc gene on chromosome 8 is translocated to chromosome 2, 14, or 22 placing c-myc under the regulation of the immunoglobulin regulatory elements and leading to sustained elevated levels of c-Myc expression (4Battey J. Moulding C. Taub R. Murphy W. Stewart T. Potter H. Lenoir G. Leder P. Cell. 1983; 34: 779-787Abstract Full Text PDF PubMed Scopus (446) Google Scholar, 5Dalla-Favera R. Bregni M. Erikson J. Patterson D. Gallo R.C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7824-7827Crossref PubMed Scopus (1280) Google Scholar). Elevated c-Myc expression also arises from genomic amplification of c-myc in breast cancer, lung, cervix, and colon carcinomas (6Little C.D. Nau M.M. Carney D.N. Gazdar A.F. Minna J.D. Nature. 1983; 306: 194-196Crossref PubMed Scopus (645) Google Scholar, 7Munzel P. Marx D. Kochel H. Schauer A. Bock K.W. J. Cancer Res. Clin. Oncol. 1991; 117: 603-607Crossref PubMed Scopus (37) Google Scholar, 8Deming S.L. Nass S.J. Dickson R.B. Trock B.J. Br. J. Cancer. 2000; 83: 1688-1695Crossref PubMed Scopus (229) Google Scholar). More recently, mutations in the adenomatous polyposis coli (APC) pathway that lead to increased Myc levels have been identified in colon cancers (9He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4083) Google Scholar, 10Kolligs F.T. Hu G. Dang C.V. Fearon E.R. Mol. Cell. Biol. 1999; 19: 5696-5706Crossref PubMed Google Scholar, 11Kolligs F.T. Kolligs B. Hajra K.M. Hu G. Tani M. Cho K.R. Fearon E.R. Genes Dev. 2000; 14: 1319-1331PubMed Google Scholar). c-Myc belongs to the family of basic helix-loop-helix leucine zipper transcription factors. c-Myc heterodimerizes with a partner protein, Max, through the C-terminal helix-loop-helix Zip domain (12Blackwood E.M. Eisenman R.N. Science. 1991; 251: 1211-1217Crossref PubMed Scopus (1473) Google Scholar, 13Luscher B. Larsson L.G. Oncogene. 1999; 18: 2955-2966Crossref PubMed Scopus (163) Google Scholar), and together Myc and Max bind DNA specifically (14Blackwell T.K. Kretzner L. Blackwood E.M. Eisenman R.N. Weintraub H. Science. 1990; 250: 1149-1151Crossref PubMed Scopus (700) Google Scholar, 15Blackwell T.K. Huang J. Ma A. Kretzner L. Alt F.W. Eisenman R.N. Weintraub H. Mol. Cell. Biol. 1993; 13: 5216-5224Crossref PubMed Scopus (332) Google Scholar) to activate transcription through the core sequence 5′-CA(C/T)GTG-3′, also known as an E box (16Kato G.J. Barrett J. Villa-Garcia M. Dang C.V. Mol. Cell. Biol. 1990; 10: 5914-5920Crossref PubMed Scopus (312) Google Scholar, 17Kretzner L. Blackwood E.M. Eisenman R.N. Curr. Top. Microbiol. Immunol. 1992; 182: 435-443PubMed Google Scholar). The N-terminal transactivation domain of c-Myc contains two highly conserved regions among the Myc family of proteins; these regions are termed Myc Box I (c-Myc residues 45–63) and Myc Box II (c-Myc residues 128–143) (16Kato G.J. Barrett J. Villa-Garcia M. Dang C.V. Mol. Cell. Biol. 1990; 10: 5914-5920Crossref PubMed Scopus (312) Google Scholar). Despite the wealth of knowledge that has accumulated about c-Myc, the connection between elevated c-Myc levels and oncogenesis remains incompletely understood (18Facchini L.M. Penn L.Z. FASEB J. 1998; 12: 633-651Crossref PubMed Scopus (333) Google Scholar, 19Zindy F. Eischen C.M. Randle D.H. Kamijo T. Cleveland J.L. Sherr C.J. Roussel M.F. Genes Dev. 1998; 12: 2424-2433Crossref PubMed Scopus (1059) Google Scholar). Presumably, the role of Myc as a transcription factor involves regulation of a set of genes that participate in the oncogenic process (20Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1383) Google Scholar, 21Grandori C. Eisenman R.N. Trends Biochem. Sci. 1997; 22: 177-181Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 22Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1038) Google Scholar). A number of c-Myc target genes have been identified, including CAD (23Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1997; 17: 2529-2537Crossref PubMed Scopus (139) Google Scholar), ODC (24Bello-Fernandez C. Packham G. Cleveland J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7804-7808Crossref PubMed Scopus (666) Google Scholar, 25Wu S. Pena A. Korcz A. Soprano D.R. Soprano K.J. Oncogene. 1996; 12: 621-629PubMed Google Scholar),LDH-A (26Shim H. Dolde C. Lewis B.C. Wu C.S. Dang G. Jungmann R.A. Dalla-Favera R. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6658-6663Crossref PubMed Scopus (857) Google Scholar, 27Hubank M. Schatz D.G. Nucleic Acids Res. 1994; 22: 5640-5648Crossref PubMed Scopus (780) Google Scholar), cyclin E (28Perez-Roger I. Solomon D.L. Sewing A. Land H. Oncogene. 1997; 14: 2373-2381Crossref PubMed Scopus (199) Google Scholar), MrDb (29Grandori C. Mac J. Siebelt F. Ayer D.E. Eisenman R.N. 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Biol. 1997; 17: 4967-4978Crossref PubMed Scopus (134) Google Scholar),IRP2 (37Wu K.J. Polack A. Dalla-Favera R. Science. 1999; 283: 676-679Crossref PubMed Scopus (278) Google Scholar), and cdc25A (38Galaktionov K. Chen X. Beach D. Nature. 1996; 382: 511-517Crossref PubMed Scopus (648) Google Scholar). The use of cDNA microarrays has enlarged the list of potential c-Myc-responsive genes (39Schuhmacher M. Kohlhuber F. Holzel M. Kaiser C. Burtscher H. Jarsch M. Bornkamm G.W. Laux G. Polack A. Weidle U.H. Eick D. Nucleic Acids Res. 2001; 29: 397-406Crossref PubMed Scopus (266) Google Scholar, 40Coller 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: 3260-3265Crossref PubMed Scopus (709) Google Scholar, 41Guo Q.M. Malek R.L. Kim S. Chiao C. He M. Ruffy M. Sanka K. Lee N.H. Dang C.V. Liu E.T. Cancer Res. 2000; 60: 5922-5928PubMed Google Scholar, 42O'Hagan R.C. Schreiber-Agus N. Chen K. David G. Engelman J.A. Schwab R. Alland L. Thomson C. Ronning D.R. Sacchettini J.C. Meltzer P. DePinho R.A. Nat. Genet. 2000; 24: 113-119Crossref PubMed Scopus (120) Google Scholar). Despite the long list of c-Myc-responsive genes, the biological effects of these genes in c-Myc related phenotypes are largely unknown. Initial work in our laboratory relied on representational difference analysis (RDA)1 to identify novel putative c-Myc target genes that could be involved in cellular transformation. The screen made use of Rat1a fibroblasts that can be transformed by c-Myc alone (43Small M.B. Hay N. Schwab M. Bishop J.M. Mol. Cell. Biol. 1987; 7: 1638-1645Crossref PubMed Scopus (97) Google Scholar). Unlike parental cells, Rat1a-Myc cells have an increased growth rate, are capable of anchorage-independent growth, form colonies in a soft agar assay (44Hoang A.T. Lutterbach B. Lewis B.C. Yano T. Chou T.Y. Barrett J.F. Raffeld M. Hann S.R. Dang C.V. Mol. Cell. Biol. 1995; 15: 4031-4042Crossref PubMed Scopus (124) Google Scholar), are tumorigenic, and undergo apoptosis in response to growth factor or glucose withdrawal (45Evan G.I. Wyllie A.H. Gilbert C.S. Littlewood T.D. Land H. Brooks M. Waters C.M. Penn L.Z. Hancock D.C. Cell. 1992; 69: 119-128Abstract Full Text PDF PubMed Scopus (2773) Google Scholar, 46Shim H. Chun Y.S. Lewis B.C. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1511-1516Crossref PubMed Scopus (254) Google Scholar). Our original RDA screen identified 20 differentially expressed genes (36Lewis B.C. Shim H. Li Q. Wu C.S. Lee L.A. Maity A. Dang C.V. Mol. Cell. Biol. 1997; 17: 4967-4978Crossref PubMed Scopus (134) Google Scholar). We have named the most highly differentially expressed of these clones JPO1. Here we describe the cloning of human JPO1 and its characterization in the context of c-Myc-associated phenotypes. The role of JPO1 in transformation and anchorage-independent growth is underscored by its complementation with a transformation-defective c-Myc Box II mutant in Rat1a transformation assays. An oligo(dT)- and random hexamer-primed liver and spleen cDNA library (CLONTECH, Palo Alto, CA) was screened with human EST 59390 that contained 912 bp ofJPO1 cDNA. Of 4 × 105 colonies screened, three independent overlapping positive clones were identified and purified according to the manufacturer’s directions, with the exception that it was necessary to reduce the temperature of washes to 50 °C. The partial rat JPO1 and rat vimentin sequences cloned by RDA (36Lewis B.C. Shim H. Li Q. Wu C.S. Lee L.A. Maity A. Dang C.V. Mol. Cell. Biol. 1997; 17: 4967-4978Crossref PubMed Scopus (134) Google Scholar) were used as templates for RNA riboprobes. Probes were transcribed from linearized DNA using the T7 promoters in the pCRII and pBluescript plasmids. Hybridization and RNase digestion were performed in accordance with an RPA kit from Ambion (Austin, TX). Digested samples were subjected to electrophoresis on an 8 m urea, 6% polyacrylamide denaturing gel with Century Markers (Ambion) as size standards. JPO1 transcript levels were quantified relative to vimentin by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). 15 μg of total RNA isolated by Trizol (Life Technologies, Inc.) was subjected to electrophoresis on a 6% formaldehyde, 0.9% agarose gel and transferred to Nytran. Blots were hybridized with ≥106 counts/ml cDNA probes labeled with the Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA). Rat JPO1 and rat vimentin probes were labeled from the partial clones identified by RDA. Human JPO1 probe was labeled from the partial cDNA clone contained in EST H59390. Rat c-myc was labeled from partial cDNAs. Hybridization was quantified by PhosphorImager (Molecular Dynamics). Protein lysates were made by lysing cells in 10% SDS (∼107 cells/ml) and boiling lysates at 95 °C for 5 min. Protein content was assayed by a BCA kit (Pierce), and equal amounts of protein were resuspended in 2× Laemmli buffer. Equal relative amounts of protein, ∼10 μg, were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to nitrocellulose. Protein blots were blocked with 5% milk, TBST and probed with antibodies diluted in the same. Anti-Myc 9E10 antibodies were diluted 1:7,000. Anti-Myc antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse antibodies diluted 1:10,000 in 5% milk/TBST. Purified polyclonal anti-human JPO1 antibodies, raised against RGRHPLPGSDSQSRRPR (amino acids 144–160) (Zymed Laboratories Inc. Laboratories, South San Francisco, CA), were diluted 1:4000. Anti-JPO1 antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit antibodies diluted 1:10,000 in 5% milk/TBST. Blots were treated with enhanced chemiluminescent reagents and exposed to film. Equal loading was confirmed with cross-reacting bands and by Coomassie Blue staining of the gels. Rat1a cells were grown in low glucose DMEM. TGR (myc +/+) and HO15 (myc −/−) cells (47Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048PubMed Google Scholar) were grown in high glucose DMEM. Rat1a-MycER cells (48Eilers M. Picard D. Yamamoto K.R. Bishop J.M. Nature. 1989; 340: 66-68Crossref PubMed Scopus (390) Google Scholar, 49Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (702) Google Scholar) were grown in phenol red-free DMEM supplemented with 10% charcoal-stripped fetal calf serum. CB33 cells (gift of R. Dalla-Favera, Columbia University, New York), an Epstein-Barr virus immortalized B lymphoblast line, CB33-derived cells, and Burkitt’s lymphoma cell lines (Ramos, Thomas, and Wynn) were grown in IMDM. COS7 cells and PG13 cells, a murine 3T3-based retroviral packaging cell line, were grown in high glucose DMEM. All media were supplemented with 10% heat-inactivated fetal bovine serum and 10 mg/ml penicillin and streptomycin. Cells were forced to grow non-adherently by seeding cells in liquid media onto a layer of media containing 0.7% agarose and growing them at 37 °C for 48 h prior to collection. Cells were then collected at discrete time points following reintroduction into serum. Rat1a-MT-Myc cells were induced by the addition of 100 μmzinc sulfate. The MycER fusion protein is induced by addition of 0.2 μm 4-OH-tamoxifen (4-OHT) to the media. Protein synthesis is blocked by addition of 10 μm cycloheximide (CHX) to the media 30 min prior to 4-OHT induction. COS7 cells were grown in high glucose DMEM to 50–70% confluence on glass coverslips and were transiently transfected with 2 μg/ml DNA and 100 μmchloroquine in DEAE-dextran/high glucose DMEM. After 5 h at 37 °C, cells were given fresh media and were incubated at 37 °C for 48 h prior to antibody staining. Cells were fixed in 3.5% paraformaldehyde/phosphate-buffered saline for 8 min and then permeabilized in 0.1% Nonidet P-40/phosphate-buffered saline for 15 min. Fixed cells were blocked for ≥20 min in 10% fetal calf serum/phosphate-buffered saline before staining with antibodies diluted in the same. Polyclonal peptide antibodies to JPO1 were diluted 1:200; the secondary antibody was a tetramethylrhodamine B isothiocyanate-labeled goat anti-rabbit antibody diluted 1:200. Coverslips were then mounted onto glass microscope slides with glycerol-containing n-propyl gallate and fixed in place with clear fingernail polish. Slides were visualized by confocal fluorescent microscopy. Full-length JPO1 cDNA was created by ligating the 5′ end from one clone identified in the library screen onto the 3′ end from another clone at the uniqueEco RI site internal to JPO1. This full-lengthJPO1 cDNA was subcloned into the Not I sites of a pSG5 expression plasmid (50Green S. Issemann I. Sheer E. Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (546) Google Scholar) with a modified polylinker site. Expression of JPO1 in pSG5 is from the early SV40 promoter. The entire 1.2-kb JPO1 coding region was amplified with primers containing Sal I sites on the ends for ligation into the expression plasmid pBabePuro. PCR-amplified JPO1 sequence was confirmed by sequencing. Expression in pBabePuro is from the retroviral long terminal repeat promoter. Rat1a-derived cells were transfected by lipofection (Life Technologies, Inc.). Cells transfected with pSG5 or MLV expression plasmids were co-transfected with an empty selection plasmid, pBabePuro or pSVneo, at a ratio of 1:10 (selection plasmid:expression plasmid). Cells were moved into selection antibiotics 48 h after transfection. CB33 B lymphoblast-derived cells were transduced 4–18 h in the presence of 8 μg/ml Polybrene with control pBabePuro retrovirus or JPO1-expressing retrovirus produced from PG13 cells. Cells were moved into puromycin selection 1–2 days after retroviral transduction. Rat1a-transfected cells and CB33 transduced cells were selected in either 750 ng/ml puromycin or 400 μg/ml G418. Soft agar assays were performed by seeding 1.2 × 105 cells in a layer of 0.4% agarose/DMEM over a layer of 0.7% agarose/DMEM in a 100-mm2 plate. A top layer of liquid DMEM was added and changed every 3rd day. Colony formation was assayed at day 14 by light microscopy or by the use of an automated UVP imaging system that recognizes colonies and the UVP LabWorks colony counting program (UVP, Upland, CA). Methylcellulose assays were performed by adding 104 cells and 2.7-ml supplement (1× IMDM, 26.6% fetal calf serum) to 2.2 ml of a 3% methylcellulose/IMDM mixture and vortexing vigorously. Bubbles were allowed to settle for 5–10 min, and 1 ml of the mixture was added per 35-mm2 non-treated suspension plate (2000 cells/plate) from a syringe with a 16-guage needle. Individual 35-mm2 plates were placed in a larger dish with an extra 35-mm2 plate containing sterile water for the purpose of humidification. Plates were maintained in a humidified 37 °C incubator for 10 days prior to assaying colony formation. 5 × 106cells were injected subcutaneously into the right flank of male homozygous nude mice, 4–6 weeks of age (Taconic, Germantown, NY). Tumors were established and weighed at 6 weeks after injection or until tumor mass reaches 1500 mg. Experiments were approved by The Johns Hopkins School of Medicine Animal Care and Use Committee. JPO1 was identified as a differentially expressed gene in an RDA of non-adherent Rat1aversus Rat1a-Myc fibroblasts (36Lewis B.C. Shim H. Li Q. Wu C.S. Lee L.A. Maity A. Dang C.V. Mol. Cell. Biol. 1997; 17: 4967-4978Crossref PubMed Scopus (134) Google Scholar). The differentially expressed rat clone had significant homology to human EST H59390. Full-length human JPO1 cDNA was cloned from a human spleen cDNA library using this EST as a probe. The longest of the clones identified in the library screen was 2.4 kb in length. Primer extension was performed from the 5′ end to determine that full-lengthJPO1 transcript is 2.5 kb (data not shown). Attempts to clone the additional sequence by 5′-rapid amplification of cDNA ends were unsuccessful, so the 5′ sequence was determined by sequencing a P1 clone that contained genomic JPO1 DNA. The composite sequence of full-length JPO1 cDNA is shown in Fig.1 A. Theoretical translation of the cDNA predicts a 371-amino acid protein that contains a putative leucine zipper and a cysteine-rich region (Fig. 1 B). A BLASTP search of the non-redundant data base revealed no known proteins with significant homology to JPO1. The JPO1 GenBank™ accession number is AY029179. Rat1a cells are an immortalized, non-transformed line. With enforced c-Myc expression, these cells are able to grow non-adherently in semi-solid media and cause tumor formation in immunocompromised mice. The effect of cellular adherence on JPO1 transcript levels was examined by Northern blot analysis of mRNA collected from both adherent and non-adherent Rat1a and Rat1a-Myc cell lines (Fig.2 A). We chose vimentin as an internal standard for mRNA sample loading, because we found that c-Myc does not affect vimentin levels in Rat1 fibroblasts (36Lewis B.C. Shim H. Li Q. Wu C.S. Lee L.A. Maity A. Dang C.V. Mol. Cell. Biol. 1997; 17: 4967-4978Crossref PubMed Scopus (134) Google Scholar).JPO1 transcript levels are higher in attached cells as compared with unattached cells. To confirm further the differences inJPO1 expression in non-adherent cells, we used the more sensitive RNase protection assay that revealed a 20-fold higherJPO1 transcript level in non-adherent Rat1a-Myc fibroblasts than in the non-adherent Rat1a fibroblasts (Fig. 2 B). Hence, enforced c-Myc expression increases the levels of JPO1 in both attached and unattached cells, although the differential expression is magnified in unattached cells. JPO1 transcript levels were compared with c-Myc levels in additional cell lines. The TGR (myc +/+) cell line is a derivative of the Rat1 cell line. HO15 (myc −/−) cells were created by targeted deletion of both c-myc gene copies from TGR (myc +/+) cells resulting in a severe cell cycle delay (47Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048PubMed Google Scholar). mRNA analyses of the myc −/− cells have been hampered by a global change in transcript levels such that comparison with wild-type cells has been performed on the basis of total RNA as reflected by 28 S ribosomal RNA (47Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048PubMed Google Scholar). Hence for load control, we used 28 S ribosomal RNA. Logarithmically growing HO15 (myc −/−) cells have markedly lower levels of theJPO1 transcript than wild-type TGR (myc +/+) cells, but expression of exogenous c-Myc in HO15 cells is sufficient for restoration of JPO1 expression (Fig. 2 C). The correlation between c-Myc and JPO1 expression also extends to human lymphoid cell lines. Although we have observed a correlation between c-Myc levels and JPO1 mRNA levels in these cells (data not shown), we sought to determine whether there is a correlation at the protein level. We generated a specific polyclonal anti-human JPO1 peptide antibody that only recognizes the human form. As shown by Western blot analysis, we found the correlation between c-Myc and JPO1 protein levels in human lymphoblastoid CB33 cells, CB33 overexpressing c-Myc (CB33-Myc), three Burkitt's lymphoma cell lines (Fig.2 D), and in a series of breast cancer cell lines (data not shown). A Myc-inducible cell line was used to determine whether JPO1 levels were elevated directly as a consequence of c-Myc induction. Rat1a-MT-Myc cells, in which c-Myc is under the control of the metallothionein promoter, were induced with 150 μm zinc. The c-Myc protein is elevated within 1 h and peaks maximally from 2 to 6 h post-zinc induction (Fig.3 A). RNA collected from zinc-induced cells over the same time course shows that JPO1 expression, as assayed by RNase protection assay, is elevated 2.5-fold at 6 h (Fig. 3 A), adding additional support for the hypothesis that JPO1 is a c-Myc target. To determine whether JPO1 is a direct or an indirect target of c-Myc, the Rat1-MycER system was utilized. These Rat1 cells are stably transfected with a chimeric fusion protein composed of c-Myc and the hormone binding domain of the estrogen receptor (49Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (702) Google Scholar). This MycER fusion protein remains sequestered in the cytoplasm by heat shock proteins except upon exposure to the estrogen analogue 4-OHT. 4-OHT binds the ER portion of the fusion protein, initiating its translocation into the nucleus where it then binds to and transactivates endogenous c-Myc target loci. Pretreatment of MycER cells with the protein synthesis inhibitor, CHX, allows the identification of only those genes whose expression depends directly on c-Myc transactivation and not on an intermediate step that requires protein synthesis. Thus by measuring mRNA induction in cells treated with only 4-OHT and in cells treated with both 4-OHT and CHX, one can distinguish between direct and indirect targets. JPO1 transcript levels were measured in MycER cells over a 6-h time course after treatment with either CHX alone, 4-OHT alone, or both (Fig. 3 B). We chose HuPO (3B4) as a loading control, because we and others (46Shim H. Chun Y.S. Lewis B.C. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1511-1516Crossref PubMed Scopus (254) Google Scholar) have found that HuPO mRNA levels do not vary with stimulation of MycER cells. In 4-OHT-treated cells,JPO1 levels rose steadily to 6 h, showing nearly a 3-fold induction. Cells treated with CHX alone show no JPO1 induction; instead JPO1 levels dropped by 6 h. Cells treated with both 4-OHT and CHX showed approximately a 1.7-fold induction in JPO1 transcript levels at 4 h that is not sustained at 6 h perhaps due to the inhibitory effect seen with CHX alone. These results indicate that JPO1 behaves as a direct c-Myc target gene. The expression levels ofJPO1 in various human tissues were assayed by probing a normalized human RNA Master Blot (CLONTECH).JPO1 levels were found to be highest in small intestine and thymus (Fig. 4). Additionally,JPO1 was highly expressed in fetal thymus, fetal lung, colon, stomach, appendix, and testis. In contrast to the high expression level seen in thymus, JPO1 levels were low in bone marrow, spleen, lymph node, and peripheral leukocytes. Two independent P1 clones containing JPO1 genomic DNA were used as probes for fluorescent in situ hybridization to map the chromosomal localization of JPO1. JPO1 maps to chromosome 2q31 as the center of the signals by proportional length (data not shown). Mapping data from the UniGene cluster (Hs. 333893; previously assigned to Hs. 47378) that corresponds to JPO1 has subsequently confirmed this map location. Western blot analysis of JPO1-transfected COS7 lysates indicates that JPO1 is a 47-kDa protein (data not shown). The subcellular localization of JPO1 was determined by immunofluorescent microscopy of transiently transfected COS7 cells. Polyclonal peptide antibodies to JPO1 detected strong nuclear staining in JPO1-transfected COS7 cells. Two patterns of nuclear staining are seen. The majority of JPO1-transfected cells showed diffuse nuclear staining exclusive of the nucleolus (Fig.5 F). Additional cells had a punctate nuclear pattern (Fig. 5 D) that did not co-localize with proliferating cell nuclear antigen (data not shown). Empty vector transfected cells (Fig. 5 B) and untransfected cells showed punctate nucleolar staining, which is also seen with pre-immune sera (data not shown) and is dependent on the method of fixation used. Since JPO1 expression is highly elevated in Myc-transformed cells, we sought to determine whether JPO1 overexpression is sufficient to induce Myc-related phenotypes. Rat1a cells were transfected with humanJPO1 cDNA expressed from the early SV40 promoter in the pSG5 plasmid vector. CB33 human B lymphoid cells were transduced with pBabe-JPO1 retrovirus that expresses JPO1 from the retroviral long terminal repeat. Two independent pools of stable Rat1a-JPO1 cells were generated with high levels of exogenous JPO1 expression (Fig.6 A), and one stable CB33-JPO1 pooled cell line was identified that overexpresses JPO1 3-fold (Fig.6 D). Overexpression of JPO1 did not alter cellular growth rates in either Rat1a or CB33 cells (data not shown). Involvement of JPO1 in the predisposition of cells to apoptosis was studied in Rat1a-JPO1 cells. Neither growth in 0.1% serum nor growth in low glucose led to any change in levels of apoptosis as compared with the parental control (data not shown). The ability of Rat1a cells to grow anchorage-independently and the clonogenicity of CB33 lymphoid cells were tested in the soft agar colony growt" @default.
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• W1487941441 title "A Novel c-Myc- responsive Gene, JPO1, Participates in Neoplastic Transformation" @default.
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