FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2151665323> ?p ?o ?g. }

• W2151665323 endingPage "4886" @default.
• W2151665323 startingPage "4877" @default.
• W2151665323 abstract "The Mdm2 gene is amplified in approximately one-third of human sarcomas and overexpressed in a variety of other human cancers. Mdm2 functions as an oncoprotein, in part, by acting as a negative regulator of the p53 tumor suppressor protein. Multiple spliced forms of Mdm2 transcripts have been observed in human tumors; however, the contribution of these variant transcripts to tumorigenesis is unknown. In this report, we isolate alternative splice forms of Mdm2 transcripts from sarcomas that spontaneously arise in Mdm2-overexpressing mice, including Mdm2-b, the splice form most commonly observed in human cancers. Transduction of Mdm2-b into a variety of cell types reveals that Mdm2-b promotes p53-independent cell growth, inhibits apoptosis, and up-regulates the RelA subunit of NFκB. Furthermore, expression of Mdm2-b induces tumor formation in transgenic mice. These results identify a p53-independent role for Mdm2 and determine that an alternate spliced form of Mdm2 can contribute to formation of cancer via a p53-independent mechanism. These findings also provide a rationale for the poorer prognosis of those patients presenting with tumors harboring multiple Mdm2 transcripts. The Mdm2 gene is amplified in approximately one-third of human sarcomas and overexpressed in a variety of other human cancers. Mdm2 functions as an oncoprotein, in part, by acting as a negative regulator of the p53 tumor suppressor protein. Multiple spliced forms of Mdm2 transcripts have been observed in human tumors; however, the contribution of these variant transcripts to tumorigenesis is unknown. In this report, we isolate alternative splice forms of Mdm2 transcripts from sarcomas that spontaneously arise in Mdm2-overexpressing mice, including Mdm2-b, the splice form most commonly observed in human cancers. Transduction of Mdm2-b into a variety of cell types reveals that Mdm2-b promotes p53-independent cell growth, inhibits apoptosis, and up-regulates the RelA subunit of NFκB. Furthermore, expression of Mdm2-b induces tumor formation in transgenic mice. These results identify a p53-independent role for Mdm2 and determine that an alternate spliced form of Mdm2 can contribute to formation of cancer via a p53-independent mechanism. These findings also provide a rationale for the poorer prognosis of those patients presenting with tumors harboring multiple Mdm2 transcripts. Mdm2 was initially identified in a screen for genes amplified on double minute chromosomes found in spontaneously transformed BALB/c 3T3 cells (1Cahilly-Snyder L. Yang-Feng T. Francke U. George D.L. Somat. Cell Mol. Genet. 1987; 13: 235-244Crossref PubMed Scopus (305) Google Scholar). When overexpressed, the Mdm2 oncoprotein has been demonstrated to immortalize rodent primary fibroblasts, to increase the rate of cellular proliferation, and to induce cellular transformation (2Finlay C.A. Mol. Cell. Biol. 1993; 13: 301-306Crossref PubMed Scopus (310) Google Scholar). A large body of evidence has established the Mdm2 oncoprotein as a potent negative regulator of the p53 tumor suppressor protein. The N terminus of Mdm2 binds the transactivation domain of p53, and Mdm2-p53 complex formation can inhibit p53 modification and p53-mediated transcriptional regulation of heterologous gene expression (3Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2792) Google Scholar, 4Chen J. Marechal V. Levine A.J. Mol. Cell. Biol. 1993; 13: 4107-4114Crossref PubMed Scopus (624) Google Scholar, 5Oliner J.D. Pietenpol J.A. Thiagalingam S. Gyuris J. Kinzler K.W. Vogelstein B. Nature. 1993; 362: 857-860Crossref PubMed Scopus (1307) Google Scholar). In addition, Mdm2 contains a C-terminal zinc RING domain and functions as an E3-ligase to induce p53 ubiquitination and degradation (6Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3722) Google Scholar, 7Kubbatat M. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2847) Google Scholar). The ability of p53 to induce the expression of the Mdm2 gene suggests that Mdm2 and p53 form a negative feedback loop to regulate p53 activity in the cell (8Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1178) Google Scholar, 9Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1136Crossref PubMed Scopus (1642) Google Scholar). Mdm2-mediated destabilization of p53 is regulated by binding of Mdm2 to the p19 (ARF) tumor suppressor protein and by ATM-induced phosphorylation of Mdm2 and p53 (10.11). The importance of Mdm2 in regulating p53 function has been demonstrated in mice, where the early embryonic-lethal phenotype of Mdm2-null mice is rescued by deletion of functional p53 (12Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1066) Google Scholar, 13Montes de Oca Luna R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1207) Google Scholar). Mice deficient for both Mdm2 and p53 undergo normal development, are viable, and are fertile, suggesting that any functions possessed by Mdm2 aside from its ability to regulate p53 are dispensable for normal cell growth and development. The human MDM2 gene is amplified to high copy numbers in approximately one-third of all human sarcomas (14Oliner J.D. Kinzler K.W. Meltzer P.S. George D.L. Vogelstein B. Nature. 1992; 358: 80-83Crossref PubMed Scopus (1802) Google Scholar), and is overexpressed in a wide range of human cancers (15Reifenberger G. Liu L. Ichimura K. Schmidt E.E. Collins V.P. Cancer Res. 1993; 53: 2736-2739PubMed Google Scholar, 16Bueso-Ramos C.E. Yang Y. deLeon E. McCown P. Stass S.A. Albitar M. Blood. 1993; 82: 2617-2623Crossref PubMed Google Scholar). As many of these tumors retain a wild-type p53 gene, it is presumed that overexpression of Mdm2 serves to inactivate p53 function in these tumors. However, tumors have been identified that have both Mdm2 amplification and p53 loss; a seemingly redundant set of mutations (17Cordon-Cardo C. Latres E. Drobnjak M. Oliva M.R. Pollack D. Woodruff J.M. Marechal V. Chen J. Brennan M.F. Levine A.J. Cancer Res. 1994; 54: 794-799PubMed Google Scholar). Interestingly, these rare sarcomas are much more aggressive than those tumors with alterations in only Mdm2 or p53, suggesting that there may exist a p53-independent role for Mdm2 when overexpressed in these tumors. Several additional lines of evidence suggest that Mdm2 may regulate growth not only by inhibiting p53 function, but through p53-independent mechanisms as well. Human MDM2 has been reported to form a complex with the major (p110) Rb 1The abbreviations used are: RbretinoblastomaGFAPglial fibrillary acidic proteinMEFmouse embryonic fibroblastsFACSfluorescence-activated cell sorterFITCfluorescein isothiocyanatePBSphosphate-buffered salineDAPI4′,6-diamidino-2-phenylindoleTNFtumor necrosis factor. tumor suppressor protein and with E2F1 and DP1 transcription factors (18Xiao Z.X. Chen J. Levine A.J. Modjtahedi N. Xing J. Sellers W.R. Livingston D.M. Nature. 1995; 375: 694-698Crossref PubMed Scopus (573) Google Scholar, 19Martin K. Trouche D. Hagemeier C. Sorensen T.S. La Thangue N.B. Kouzarides T. Nature. 1995; 375: 691-694Crossref PubMed Scopus (452) Google Scholar), and can alter transcription of E2F1-induced reporter genes in cell transfection assays. Other cell cycle regulatory proteins that bind with Mdm2 include Numb, MTBP, SMAD transcription factors, TIP60, and β-arrestin; a β2-adrenergic receptor regulator (20Juven-Gershon T. Shifman O Unger T. Elkeles A. Haupt Y. Oren M. Mol. Cell. Biol. 1998; 18: 3974-3982Crossref PubMed Scopus (120) Google Scholar, 21Boyd M.T. Vlatkovic N. Haines D.S. J. Biol. Chem. 2000; 275: 31883-31890Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22Yam C.H. Siu W.Y. Arooz T. Chiu C.H. Lau A. Wang X.Q. Poon R.Y. Cancer Res. 1999; 59: 5075-5078PubMed Google Scholar, 23Legube G. Linares L.K. Lemercier C. Scheffner M. Khochbin S. Trouche D. EMBO J. 2002; 21: 1704-1712Crossref PubMed Scopus (127) Google Scholar, 24Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (713) Google Scholar). Genetic evidence for a p53-independent role for Mdm2 in cell growth has been provided through analysis of transgenic mice. Overexpression of MDM2 cDNA in the mammary epithelium of transgenic mice was found to inhibit development of the mammary gland by inducing multiple rounds of S phase without completion of mitosis (25Lundgren K. Montes de Oca Luna R. McNeill Y.B. Emerick E.P. Spencer B. Barfield C.R. Lozano G. Rosenberg M.P. Finlay C.A. Genes Dev. 1997; 11: 714-725Crossref PubMed Scopus (212) Google Scholar). The uncoupling of S phase from mitosis was seen in transgenic mice, which were either wild type or deficient for p53, indicating a p53-independent role for Mdm2 in the regulation of DNA synthesis. We have also provided genetic evidence for a p53-independent role for Mdm2 in sarcoma formation. Transgenic mice were generated using mouse genomic DNA encoding the entire Mdm2 gene under control of its native promoter region. These mice displayed a 4-fold increase in the level of Mdm2 expression and were found to have increased predisposition to spontaneous sarcoma formation regardless of the p53 status of the mice (26Jones S.N. Hancock A.R. Vogel H. Donehower L.A. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15608-15612Crossref PubMed Scopus (318) Google Scholar). retinoblastoma glial fibrillary acidic protein mouse embryonic fibroblasts fluorescence-activated cell sorter fluorescein isothiocyanate phosphate-buffered saline 4′,6-diamidino-2-phenylindole tumor necrosis factor. Analysis of a variety of human tumors that overexpress Mdm2 has revealed the presence of multiple, alternatively spliced forms of Mdm2 message (27Bartel F. Meye A. Wurl P. Kappler M. Bache M. Lautenschrunlager C. Grunbaum U. Schmidt H. Taubert H. Int. J. Cancer (Pred. Oncol.). 2001; 95: 168-175Crossref PubMed Scopus (77) Google Scholar). In some cases, the presence of these spliced Mdm2 forms has been correlated with a more aggressive disease state (28Matsumoto R. Tada M. Nozaki M. Zhang C.L. Sawamura Y. Abe H. Cancer Res. 1998; 58: 609-613PubMed Google Scholar, 29Bartel F. Pinkert D. Fiedler W. Kappler M. Wurl P. Schmidt H. Taubert H. Cancer Cell. 2002; 2: 9-15Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Interestingly, some of these transcripts encode Mdm2 proteins that lack the p53-binding domain and are incapable of complexing with p53, yet can induce foci formation in 3T3 cells in culture, suggesting that these tumor-isolated Mdm2 isoforms may contribute to transformation in a p53-independent manner (30Sigalas I. Calvert A.H. Anderson J.J. Neal D.E. Lunec J. Nat. Med. 1996; 2: 912-917Crossref PubMed Scopus (252) Google Scholar). More recently, several groups have characterized several spliced isoforms of Mdm2 transcripts isolated from mouse or human tumors and have reported that many of these isoforms appear to inhibit cell proliferation, though the precise mechanism of growth inhibition remains unclear (31Dang J. Kuo M.L. Eischen C.M. Stepanova L. Sherr C.J. Roussel M.F. Cancer Res. 2002; 62: 1222-1230PubMed Google Scholar, 32Evans S.C. Viswanathan M Grier J.D. Narayana M. El-Naggar A.K. Lozano G. Oncogene. 2001; 20: 4041-4049Crossref PubMed Scopus (121) Google Scholar). In order to assess the potential role of Mdm2 isoforms in tumorigenesis, we have analyzed sarcomas isolated from our Mdm2-transgenic mice. We have detected numerous spliced isoforms of Mdm2 transcripts in the tumors, including the murine equivalent of the B isoform; the most prevalent isoform observed in human cancers. Hdm2-B has been previously detected in high grade bladder and uterine cancers, lacks the p53-binding region present in full-length Mdm2, and was found to be incapable of complexing with the p53 protein (30Sigalas I. Calvert A.H. Anderson J.J. Neal D.E. Lunec J. Nat. Med. 1996; 2: 912-917Crossref PubMed Scopus (252) Google Scholar). In this report, we examine the functional significance of the Mdm2-b form in cells and in mice. Our results indicate that this Mdm2 isoform encodes a variant Mdm2 protein that lacks the p53-binding domain and contains only the C-terminal RING domain. This Mdm2 protein is found to induce cell proliferation and to interfere with apoptosis in a p53-independent manner in cultured cells, and induce spontaneous tumorigenesis in transgenic mice. Interestingly, expression of either Mdm2 or Mdm2-b increases the level of the RelA (p65) protein in cells, and Mdm2-b can increase NFκB-dependent transcription in transduced cells and potentiate the response of these cells to TNF-mediated apoptosis. These results identify a p53-independent role for Mdm2 in modulating cell proliferation and apoptosis, demonstrate that a splice isoform of Mdm2 can induce tumor formation in vivo, and further suggest that the presence of this splice isoform of Mdm2 contributes to the neoplasia induced by Mdm2 overexpression in human cancers. Isolation of Alternatively Spliced Mdm2 Transcripts—Total RNA was isolated from dounce homogenized, snap frozen transgenic tumor tissue using TRIzol (Invitrogen). RT-PCR was performed using Superscript First-Strand Synthesis System (Invitrogen). RNA was reverse-transcribed using an oligo(dT) primer. The resulting cDNA was used in nested PCR utilizing primer pair Ex2forward (5′-CTGCTGGGCGAGCGGGAGACC-3′) and Ex12reverse (5′-GTGGACTAAGACAGTTTTCTGGC-3′) for the first amplification of 25 cycles followed by a second amplification with primer pair Ex2nest (5′-GACCCTCTCGGATCACCGCGC-3′) and Ex12nest (5′-GTGAGCAGGTCAGCTAGTTGA- 3′) for a total of 35 cycles of 94 °C for 2 min, 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s. PCR products were resolved on 1% agarose gels, excised, purified (GENECLEAN), and cloned into pGEM-T Easy Vector (Promega) for sequencing. PCR identification of specific Mdm2-b isoform was done using primer pair MBforward (5′-AAGAGACTCTGGACTATTGGAAGTG-3′) and 12reverse (5′-GCAGATCACACATGGTTCGATGGCA-3′). DNA sequencing of cDNAs was performed by the University of Massachusetts Nucleic Acid Facility to identify Mdm2-specific isoforms. Cloning and Expression of the Mdm2-b Isoform—Mdm2-b and Hdm2-B cDNAs were cloned into the EcoRI sites of pBabe-PURO and pcDNA3.1HisC expression plasmids (Invitrogen). Mdm2-b protein product was confirmed using an in vitro transcription and translation system in rabbit reticulocyte lysates (Promega). Cell Culture, Cell Lines, and Antibodies—NIH3T3 cells were purchased from the ATCC. Mouse embryonic fibroblasts (MEFs) null for pRB or p53 were generated using standard protocols. p19(ARF)-null MEFs were kindly provided by the Kowalik laboratory at the University of Massachusetts Medical School. All cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, (100 units/ml) of penicillin and (100 μg/ml) of streptomycin. Stable cell lines were passaged in media containing (3 μg/ml) puromycin (Sigma). Stable transfection of NIH3T3 cells were performed in 100-mm plates using 10 μg of linearized expression plasmid DNA along with FuGENE 6 reagent (Roche Applied Science), according to the manufacturer's protocol. Following puromycin drug selection, surviving colonies were pooled for the generation of stable cell lines. For immunolocalization studies, 2 × 105 cells were seeded onto coverslips in the bottom of 6-well plates. Transient transfections were performed using 1 μg of Express-Mdm2-b, Express-AML3, or Express-empty vector using LipofectAMINE (Invitrogen). Foci formation was scored in NIH3T3 stable cell lines following methanol fixation and 0.1% crystal violet after 2 weeks of culture. Bosc293 cells at 80% confluency were transfected with 10 μgs of pBabe-Mdm2-b, pBabe-Hdm2-B, pBabe-Mdm2-bssp1, or pBabe control using LipofectAMINE to generate recombinant retrovirus. Forty-eight hours following transfection, retroviral particles were collected and used for the viral transduction of primary MEFs or NIH3T3 cells seeded at 1 × 106 cells per 100-mm plate. Polyclonal antibodies against p65 (C-20) and against the C terminus of Mdm2 (C-18) were purchased from Santa Cruz Biotechnology, Inc. Primary polyclonal antibody Ab-7 (Oncogene Research Products) was used to detect p53, followed by secondary biotin-conjugated rabbit anti-sheep IgG (Oncogene Research Products) and tertiary horseradish peroxidase-conjugated Strepavidin (Zymed Laboratories Inc. Anti-Xpress-FITC antibody (Invitrogen) was used for immunolocalization studies. An anti-BrdUrd antibody (BD Biosciences) was used to label cells for FACS analysis. Anti-tubulin monoclonal antibody (Sigma) was used for protein loading control. Analysis of Cell Proliferation—Growth curves were performed with triplicate plating of either NIH3T3 stable cell lines, p53-/-, p19-/-, or pRB-/- PuroR early passage MEFs. Cells were seeded at a density of 2 × 105 cells per 60-mm plate and counted every 24 h using a Beckman Coulter Counter. For the determination of asynchronous S phase populations, cells were seeded at 1 × 106 cells per 100-mm plate and pulsed 24 h later with 10 μm BrdUrd for 1 h. FACS analysis was performed on cells stained for BrdUrd and propidium iodide. Analysis of Cell Death—NIH3T3 cell lines were 50-60% confluent when treated with (500 ng/ml) doxorubicin (Sigma). Triplicate samples of cells were harvested 24-36 h later and analyzed for propidium iodide uptake by FACS analysis. NFκB Activity Assays—293T cells were seeded into 6-well plates at a density of 5 × 105 cells in 2 mls of medium and transfected with 50 ng of each of an internal β-galactosidase transfection efficiency control plasmid and either a κB-responsive luciferase reporter plasmid containing two canonical κB sites or a control plasmid lacking κB sites together with Mdm2-b or control pcDNA3.1 expression plasmids. Cells were treated with recombinant TNF-α (Roche Applied Science) and 24-36 h following transfection, luciferase assays (Promega) were performed using a luminometer as previously described (33Duckett C.S. Gedrich R.W. Gilfillan M.C. Thompson C.B. Mol. Cell. Biol. 1997; 17: 1535-1542Crossref PubMed Google Scholar). Immunolocalization Assays—Forty-eight hours following transient transfection, cells on coverslips were fixed with (3.7%) formaldehyde in PBS, permeabilized with (0.25%) Triton X-100 in PBS, and blocked in 0.5% bovine serum albumin in PBS prior to a 1-h incubation with an anti-Xpress-FITC-conjugated antibody for the recognition of Mdm2-b or AML3. Cell nuclei were stained with DAPI (0.5 μg DAPI in 0.1% Triton X-100-PBSA). Cells were visualized using a Zeiss Confocal Microscope. Generation of Transgenic Mice—Mdm2-b cDNA was cloned into the EcoRI sites of transgene cassettes pCAGGs and glial fibrillary acidic protein (GFAP). Transgenic mice were generated via pronuclear injection using standard procedures. Identification of GFAP-Mdm2-b founder mice and transmission of the transgene was determined by PCR and Southern analyses. The PCR primers used for genotyping span the junction of cDNA to MP-1pA, Tgforward 5′-CCAATCCAAATGATTGTGCTA-3′ and TGreverse 5′-CATTGTTCCTTAGCAGGCTCC-3′. Southern analysis was performed on EcoRI-digested genomic tail DNA using Mdm2-b cDNA as a probe, and densitometry using a phosphorimager identified the relative copy number of transgenes in each line. Isolation and Characterization of Mdm2-b—To determine whether Mdm2 splice variant transcripts are present in our Mdm2 transgenic mouse tumors, RNA was extracted from 14 frozen tumor samples, and RT-PCR was performed using nested PCR amplification. PCR products were analyzed by gel electrophoresis (Fig. 1A), and Southern hybridization using various Mdm2 oligonucleotide probes spanning the Mdm2 coding sequences. The majority of spliced variants hybridized to 3′-probes corresponding to exon 12 of the Mdm2 gene (data not shown). In order to isolate individual spliced variants, nested Mdm2 PCR products were purified and 72 transcripts were subcloned into plasmid vectors. Subsequent DNA sequencing of the cDNA clones revealed a wide range of Mdm2 spliced variants and included both aberrant transcripts resulting from cryptic splice sites within introns and exons as well as transcripts generated from the donor and acceptor splice sites located at the Mdm2 intron-exon boundaries (28Matsumoto R. Tada M. Nozaki M. Zhang C.L. Sawamura Y. Abe H. Cancer Res. 1998; 58: 609-613PubMed Google Scholar). The most prevalent transcript observed is identical to Hdm2-B, the most frequently detected Mdm2 spliced variant found in human tumors (29Bartel F. Pinkert D. Fiedler W. Kappler M. Wurl P. Schmidt H. Taubert H. Cancer Cell. 2002; 2: 9-15Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 30Sigalas I. Calvert A.H. Anderson J.J. Neal D.E. Lunec J. Nat. Med. 1996; 2: 912-917Crossref PubMed Scopus (252) Google Scholar). This mouse Mdm2-b isoform was detected in all 14 analyzed tumor samples and was not detected in wild-type tissue in these experiments (Fig. 1B). The Mdm2-b transcript encodes for sequences present in Mdm2 exons 2-3 and exon 12, with RNA splicing between exons 3 and 12 occurring at the precise exon splice donor-acceptor motifs. The predicted protein alignment between Mdm2-b and Hdm2-B is illustrated (Fig. 1C). Amino acid identity between the two proteins is 82%. The encoded Mdm2-b protein lacks the p53-binding, p300-binding, pRb-binding, and p19(ARF) binding domains present on full-length Mdm2, as well as the Mdm2 nuclear localization and nuclear export signals. Mdm2-b does contain the complete C-terminal zinc finger, Ring finger domain, and Mdm2 residues that have been identified as targets for phosphorylation by ATM (11Maya R. Balass M. Kim S.T. Shkedy D. Leal J.F. Shifman O. Moas M. Buschmann T. Ronai Z. Shiloh Y. Kastan M.B. Katzir E. Oren M. Genes Dev. 2001; 15: 1067-1077Crossref PubMed Scopus (532) Google Scholar, 35de Toledo S.M. Azzam E.I. Dahlberg W.K. Gooding T.B. Little J.B. Oncogene. 2000; 19: 6185-6193Crossref PubMed Scopus (57) Google Scholar) and c-Abl (36Sionov R.V. Moallem E. Berger M. Kazaz A. Gerlitz O. Ben-Neriah Y. Oren M. Haupt Y. J. Biol. Chem. 1999; 274: 8371-8374Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). To confirm that the Mdm2-b spliced transcript encodes for a protein product, Mdm2-b cDNA was cloned into pcDNA3.1 in frame with an N-terminal Xpress epitope tag (Invitrogen) and expressed the protein in an in vitro transcription/translation expression system (Promega). The Mdm2-b transcript encodes a protein product of ∼47 kDa in size, when the size of the 3.5-kDa Xpress tag is subtracted (Fig. 1D). To determine the cellular location of the Mdm2-b protein, the pcDNA-XpressMdm2-b vector was transiently transfected into NIH3T3 cells and immunofluorescence microscopy was performed using an α-XPRESS-FITC-conjugated antibody against XPRESS-Mdm2-b. Mdm2-b was determined to localize predominantly in the cytoplasm of the transfected NIH3T3 cells (Fig. 1E), in keeping with the absence of a nuclear localization signal on Mdm2-b. An XPRESS tagged-AML3 expression plasmid that encodes a protein that localizes to the nucleus was used in parallel as a control in this experiment. Expression of Mdm2-b Increases Cell Proliferation and Transformation—Numerous spliced forms of Mdm2, including the b isoform, have been identified previously in human tumors (30Sigalas I. Calvert A.H. Anderson J.J. Neal D.E. Lunec J. Nat. Med. 1996; 2: 912-917Crossref PubMed Scopus (252) Google Scholar, 32Evans S.C. Viswanathan M Grier J.D. Narayana M. El-Naggar A.K. Lozano G. Oncogene. 2001; 20: 4041-4049Crossref PubMed Scopus (121) Google Scholar). However, there have been contradictory reports as to the effect of the splice forms upon cell growth (30Sigalas I. Calvert A.H. Anderson J.J. Neal D.E. Lunec J. Nat. Med. 1996; 2: 912-917Crossref PubMed Scopus (252) Google Scholar, 31Dang J. Kuo M.L. Eischen C.M. Stepanova L. Sherr C.J. Roussel M.F. Cancer Res. 2002; 62: 1222-1230PubMed Google Scholar, 32Evans S.C. Viswanathan M Grier J.D. Narayana M. El-Naggar A.K. Lozano G. Oncogene. 2001; 20: 4041-4049Crossref PubMed Scopus (121) Google Scholar). Therefore, we sought to examine if the presence of the Mdm2-b isoform might contribute to the malignant phenotype of our Mdm2 transgenic mice. Hdm2-B and Mdm2-b cDNAs were cloned separately into the pBabe retroviral expression vector and stably transduced into NIH3T3 cells to examine if Hdm2-B or Mdm2-b is capable of altering cellular growth characteristics. Selection for puromycin-resistant clones indicated a transduction frequency of ∼90%. Following drug selection, the stable transfectants were pooled and the expression of spliced variants was confirmed with RT-PCR and Northern blot analysis (data not shown). Transduction of Mdm2-b into NIH3T3 cells was found to promote obvious rapid cell proliferation. BrdU staining of asynchronous growing cells transduced with Mdm2-b or with control (pBABE-empty vector) revealed an increase in the numbers of Mdm2-b-transduced cells present in S phase of the cell cycle relative to control transduced cells (Fig. 2A). To confirm the positive effects of the B splice form on cell growth, cell proliferation assays were performed using triplicate plates of NIH3T3 cells transduced with Hdm2-B, Mdm2-b, or empty vector (pBABE) (Fig. 2B). Three repeat experiments confirmed that the presence of either Hdm2-B or Mdm2-b increased the proliferation rate and saturation density of NIH3T3 cells. To determine whether Mdm2-b could contribute to cellular transformation, Mdm2-b NIH3T3 cells and control pBabe NIH3T3 cells were seeded onto 60-mm dishes and maintained in culture for 2 weeks. Following crystal violet staining, foci formation was scored from six representative plates of each cell line (Fig. 2C). Mdm2-b expression induced larger and more numerous foci in the monolayer (44.6 ± 4.5 foci per plate) than did transduction with pBabe alone (18.2 ± 5.7 foci per plate). Thus, expression of Mdm2-b in NIH3T3 cells accelerates the rate of cell proliferation and interferes with growth suppression induced by contact inhibition. Mdm2-b Increases Cell Proliferation Independent of p53, p19 (ARF), and Rb—Unlike full-length Mdm2, Mdm2-b lacks the p53 binding domain of Mdm2, and Hdm2-B has been previously demonstrated to be incapable of complexing with p53 (30Sigalas I. Calvert A.H. Anderson J.J. Neal D.E. Lunec J. Nat. Med. 1996; 2: 912-917Crossref PubMed Scopus (252) Google Scholar). However, it remains possible that Mdm2-b still alters p53 functions, possibly by complexing with full-length Mdm2 (32Evans S.C. Viswanathan M Grier J.D. Narayana M. El-Naggar A.K. Lozano G. Oncogene. 2001; 20: 4041-4049Crossref PubMed Scopus (121) Google Scholar). To determine if Mdm2-b functions through p53 to increase cell proliferation, recombinant retroviruses were used to transduce early passage, primary mouse embryonic fibroblasts (MEFs) derived from p53-null mice. Transient selection of the MEFs in puromycin indicated a 95% transduction frequency. The pooled MEFs were triplicate plated in 60-mm dishes, and growth rates were monitored for each cell type over a period of 5 days in culture. Results of the growth curves clearly demonstrate the ability of both Hdm2-B and Mdm2-b to increase the rate of cellular proliferation when p53 is absent (Fig. 2D). In addition to the p53-binding region, Mdm2-b lacks both p19ARF and pRb binding domains. In order to determine the proliferative effect this spliced variant has on cells lacking either p19ARF or pRb, we infected early passage p19ARF-null MEFs or Rb-null MEFs with Hdm2-B or pBabe-control retrovirus, pooled those cells surviving drug selection, and used resulting cells for proliferation curves. Similar to results obtained with p53-null cells, Hdm2-B accelerates growth in the absence of either p19(ARF) or pRb (Fig. 2E). These data indicates that the b form does not depend upon the presence of p53, p19, or Rb to increase the rate of cell growth. Expression of Mdm2-b Interferes with Apoptosis—To determine if Mdm2-b is interfering with p53-mediated apoptosis, we examined the levels of p53 in the transduced 3T3 cells following treatment with the topoisomerase inhibitor, doxorubicin. Doxorubicin is an anthracycline analogue reported to induce apoptosis through p53-dependent and p53-independent mechanisms (37Gerwirtz D.A. Biochem. Pharm. 1999; 57: 727-741Crossref PubMed Scopus (1819) Google Scholar). Although p53 protein levels are elevated in the control-transduced cells, Mdm2-b-transduced cells, and in the Hdm2-B-transduced cells 18 h after doxorubicin treatment, no reduction was observed in p53 protein levels in cells transduced with the b isoforms relative to the control cells, indicating that Mdm2-b or Hdm2-B does not alter p53 levels in these cells (Fig. 3A). Furthermore, the presence of Mdm2-b in 3T3 cells does not inhibit p53-mediated induction of genes such as p21 (Waf/Cip) following treatment with 8Gy ionizing radiation (IR) and Mdm2-b-transduced cells undergo a G1 arrest in response to IR (data not shown). These data suggest that the Mdm2-b splice variant does not alter p53 stability or activity. Another potent regulator of apoptosis is the NFκB transcription factor, a dimeric complex composed of the transcriptionally inactive p50 subunit and the p65 (RelA) subunit, which contains a potent transactivation domain (38Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (666) Google Scholar). The activity of NFκB is suppressed by interaction with IκB proteins that sequester NFκB in the cytoplasm (39Malek S. Huxford T. Ghosh G. J. Biol. Chem. 1998; 273: 25427-25435Abstract Full Text Full Text PDF PubMed" @default.
• W2151665323 created "2016-06-24" @default.
• W2151665323 creator A5003098266 @default.
• W2151665323 creator A5004184410 @default.
• W2151665323 creator A5008832493 @default.
• W2151665323 creator A5015802052 @default.
• W2151665323 creator A5029509818 @default.
• W2151665323 creator A5046594267 @default.
• W2151665323 creator A5054946822 @default.
• W2151665323 date "2004-02-01" @default.
• W2151665323 modified "2023-10-02" @default.
• W2151665323 title "An Alternative Splice Form of Mdm2 Induces p53-independent Cell Growth and Tumorigenesis" @default.
• W2151665323 cites W1526999767 @default.
• W2151665323 cites W1591281137 @default.
• W2151665323 cites W1603650405 @default.
• W2151665323 cites W1965173366 @default.
• W2151665323 cites W1968766467 @default.
• W2151665323 cites W1969044026 @default.
• W2151665323 cites W1971775560 @default.
• W2151665323 cites W1983398917 @default.
• W2151665323 cites W1986512406 @default.
• W2151665323 cites W1993864973 @default.
• W2151665323 cites W1997068466 @default.
• W2151665323 cites W2003925068 @default.
• W2151665323 cites W2007456673 @default.
• W2151665323 cites W2008072603 @default.
• W2151665323 cites W2010183849 @default.
• W2151665323 cites W2034177818 @default.
• W2151665323 cites W2039021150 @default.
• W2151665323 cites W2039335985 @default.
• W2151665323 cites W2039463309 @default.
• W2151665323 cites W2047814166 @default.
• W2151665323 cites W2054322190 @default.
• W2151665323 cites W2055121057 @default.
• W2151665323 cites W2056530510 @default.
• W2151665323 cites W2058485582 @default.
• W2151665323 cites W2061675994 @default.
• W2151665323 cites W2070403360 @default.
• W2151665323 cites W2073391655 @default.
• W2151665323 cites W2073936529 @default.
• W2151665323 cites W2082979850 @default.
• W2151665323 cites W2091151265 @default.
• W2151665323 cites W2091366475 @default.
• W2151665323 cites W2099907467 @default.
• W2151665323 cites W2106048601 @default.
• W2151665323 cites W2106402455 @default.
• W2151665323 cites W2137138278 @default.
• W2151665323 cites W2147541997 @default.
• W2151665323 cites W2155710216 @default.
• W2151665323 cites W2158005145 @default.
• W2151665323 cites W2158341017 @default.
• W2151665323 cites W2163515708 @default.
• W2151665323 cites W2169724245 @default.
• W2151665323 cites W2170494099 @default.
• W2151665323 cites W2322220262 @default.
• W2151665323 cites W4247685543 @default.
• W2151665323 doi "https://doi.org/10.1074/jbc.m305966200" @default.
• W2151665323 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14612455" @default.
• W2151665323 hasPublicationYear "2004" @default.
• W2151665323 type Work @default.
• W2151665323 sameAs 2151665323 @default.
• W2151665323 citedByCount "86" @default.
• W2151665323 countsByYear W21516653232012 @default.
• W2151665323 countsByYear W21516653232013 @default.
• W2151665323 countsByYear W21516653232014 @default.
• W2151665323 countsByYear W21516653232015 @default.
• W2151665323 countsByYear W21516653232016 @default.
• W2151665323 countsByYear W21516653232017 @default.
• W2151665323 countsByYear W21516653232018 @default.
• W2151665323 countsByYear W21516653232019 @default.
• W2151665323 countsByYear W21516653232020 @default.
• W2151665323 countsByYear W21516653232021 @default.
• W2151665323 countsByYear W21516653232022 @default.
• W2151665323 crossrefType "journal-article" @default.
• W2151665323 hasAuthorship W2151665323A5003098266 @default.
• W2151665323 hasAuthorship W2151665323A5004184410 @default.
• W2151665323 hasAuthorship W2151665323A5008832493 @default.
• W2151665323 hasAuthorship W2151665323A5015802052 @default.
• W2151665323 hasAuthorship W2151665323A5029509818 @default.
• W2151665323 hasAuthorship W2151665323A5046594267 @default.
• W2151665323 hasAuthorship W2151665323A5054946822 @default.
• W2151665323 hasBestOaLocation W21516653231 @default.
• W2151665323 hasConcept C104317684 @default.
• W2151665323 hasConcept C105580179 @default.
• W2151665323 hasConcept C185592680 @default.
• W2151665323 hasConcept C190283241 @default.
• W2151665323 hasConcept C194583182 @default.
• W2151665323 hasConcept C2776192174 @default.
• W2151665323 hasConcept C2780989783 @default.
• W2151665323 hasConcept C502942594 @default.
• W2151665323 hasConcept C55493867 @default.
• W2151665323 hasConcept C555283112 @default.
• W2151665323 hasConcept C62112901 @default.
• W2151665323 hasConcept C70721500 @default.
• W2151665323 hasConcept C86803240 @default.
• W2151665323 hasConcept C95444343 @default.
• W2151665323 hasConceptScore W2151665323C104317684 @default.
• W2151665323 hasConceptScore W2151665323C105580179 @default.

• next