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Choudhary HB, Mandlik SK, Mandlik DS. Role of p53 suppression in the pathogenesis of hepatocellular carcinoma. World J Gastrointest Pathophysiol 2023; 14:46-70. [PMID: 37304923 PMCID: PMC10251250 DOI: 10.4291/wjgp.v14.i3.46] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Revised: 05/19/2023] [Accepted: 05/31/2023] [Indexed: 06/01/2023] Open
Abstract
In the world, hepatocellular carcinoma (HCC) is among the top 10 most prevalent malignancies. HCC formation has indeed been linked to numerous etiological factors, including alcohol usage, hepatitis viruses and liver cirrhosis. Among the most prevalent defects in a wide range of tumours, notably HCC, is the silencing of the p53 tumour suppressor gene. The control of the cell cycle and the preservation of gene function are both critically important functions of p53. In order to pinpoint the core mechanisms of HCC and find more efficient treatments, molecular research employing HCC tissues has been the main focus. Stimulated p53 triggers necessary reactions that achieve cell cycle arrest, genetic stability, DNA repair and the elimination of DNA-damaged cells’ responses to biological stressors (like oncogenes or DNA damage). To the contrary hand, the oncogene protein of the murine double minute 2 (MDM2) is a significant biological inhibitor of p53. MDM2 causes p53 protein degradation, which in turn adversely controls p53 function. Despite carrying wt-p53, the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway. High p53 in-vivo expression might have two clinical impacts on HCC: (1) Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways; and (2) Exogenous p53 makes HCC susceptible to various anticancer drugs. This review describes the functions and primary mechanisms of p53 in pathological mechanism, chemoresistance and therapeutic mechanisms of HCC.
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Affiliation(s)
- Heena B Choudhary
- Department of Pharmacology, BVDU, Poona College of Pharmacy, Pune 411038, Maharashtra, India
| | - Satish K Mandlik
- Department of Pharmaceutics, BVDU, Poona College of Pharmacy, Pune 411038, Maharashtra, India
| | - Deepa S Mandlik
- Department of Pharmacology, BVDU, Poona College of Pharmacy, Pune 411038, Maharashtra, India
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Molecular Targets of Active Anticancer Compounds Derived from Marine Sources. Mar Drugs 2018; 16:md16050175. [PMID: 29786660 PMCID: PMC5983306 DOI: 10.3390/md16050175] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2018] [Revised: 05/14/2018] [Accepted: 05/17/2018] [Indexed: 12/12/2022] Open
Abstract
Over the past decades, a number of novel compounds, which are produced in the marine environment, have been found to exhibit the anticancer effects. This review focuses on molecular targets of marine-derived anticancer candidates in clinical and preclinical studies. They are kinases, transcription factors, histone deacetylase, the ubiquitin-proteasome system, and so on. Specific emphasis of this review paper is to provide information on the optimization of new target compounds for future research and development of anticancer drugs, based on the identification of structures of these target molecules and parallel compounds.
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Vaughan CA, Pearsall I, Singh S, Windle B, Deb SP, Grossman SR, Yeudall WA, Deb S. Addiction of lung cancer cells to GOF p53 is promoted by up-regulation of epidermal growth factor receptor through multiple contacts with p53 transactivation domain and promoter. Oncotarget 2017; 7:12426-46. [PMID: 26820293 PMCID: PMC4914296 DOI: 10.18632/oncotarget.6998] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2016] [Accepted: 01/16/2016] [Indexed: 12/20/2022] Open
Abstract
Human lung cancers harboring gain-of-function (GOF) p53 alleles express higher levels of the epidermal growth factor receptor (EGFR). We demonstrate that a number of GOF p53 alleles directly upregulate EGFR. Knock-down of p53 in lung cancer cells lowers EGFR expression and reduces tumorigenicity and other GOF p53 properties. However, addiction of lung cancer cells to GOF p53 can be compensated by overexpressing EGFR, suggesting that EGFR plays a critical role in addiction. Chromatin immunoprecipitation (ChIP) using lung cancer cells expressing GOF p53 alleles showed that GOF p53 localized to the EGFR promoter. The sequence where GOF p53 is found to interact by ChIP seq can act as a GOF p53 response element. The presence of GOF p53 on the EGFR promoter increased histone H3 acetylation, indicating a mechanism whereby GOF p53 enhances chromatin opening for improved access to transcription factors (TFs). ChIP and ChIP-re-ChIP with p53, Sp1 and CBP histone acetylase (HAT) antibodies revealed docking of GOF p53 on Sp1, leading to increased binding of Sp1 and CBP to the EGFR promoter. Up-regulation of EGFR can occur via GOF p53 contact at other novel sites in the EGFR promoter even when TAD-I is inactivated; these sites are used by both intact and TAD-I mutated GOF p53 and might reflect redundancy in GOF p53 mechanisms for EGFR transactivation. Thus, the oncogenic action of GOF p53 in lung cancer is highly dependent on transactivation of the EGFR promoter via a novel transcriptional mechanism involving coordinated interactions of TFs, HATs and GOF p53.
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Affiliation(s)
- Catherine A Vaughan
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA
| | - Isabella Pearsall
- Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA
| | - Shilpa Singh
- Integrated Life Sciences Program, Virginia Commonwealth University, Richmond, VA, USA
| | - Brad Windle
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.,Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA.,Philips Institute, Virginia Commonwealth University, Richmond, VA, USA
| | - Swati P Deb
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.,Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA.,Integrated Life Sciences Program, Virginia Commonwealth University, Richmond, VA, USA
| | - Steven R Grossman
- Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA.,Division of Hematology, Oncology and Palliative Care, Virginia Commonwealth University, Richmond, VA, USA
| | - W Andrew Yeudall
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.,Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA.,Philips Institute, Virginia Commonwealth University, Richmond, VA, USA.,Department of Oral Biology, Augusta University, Augusta, GA, USA
| | - Sumitra Deb
- Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.,Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA.,Integrated Life Sciences Program, Virginia Commonwealth University, Richmond, VA, USA
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4
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Deb SP, Singh S, Deb S. MDM2 overexpression, activation of signaling networks, and cell proliferation. Subcell Biochem 2014; 85:215-34. [PMID: 25201197 DOI: 10.1007/978-94-017-9211-0_12] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Frequent overexpression of MDM2 in human cancers suggests that the protein confers a survival advantage to cancer cells. However, overexpression of MDM2 in normal cells seems to restrict cell proliferation. This review discusses the cell growth regulatory functions of MDM2 in normal and genetically defective cells to assess how cancer cells evade the growth-restricting consequence of MDM2 overexpression. Similar to oncoproteins that induce a DNA damage response and oncogene induced senescence in non-transformed cells, MDM2 induces G1-arrest and intra-S phase checkpoint responses that control untimely DNA replication in the face of genetic challenges.
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Affiliation(s)
- Swati Palit Deb
- Department of Biochemistry and Molecular Biology, and the Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, 23298, USA,
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5
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Frum RA, Singh S, Vaughan C, Mukhopadhyay ND, Grossman SR, Windle B, Deb S, Deb SP. The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing. Nucleic Acids Res 2013; 42:926-40. [PMID: 24163099 PMCID: PMC3902934 DOI: 10.1093/nar/gkt944] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53−/−:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53−/− mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.
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Affiliation(s)
- Rebecca A Frum
- Division of Hematology, Oncology and Palliative Care, Department of Medicine, The Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA, Department of Biochemistry and Molecular Biology, The Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA, Department of Biostatistics, The Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA and Department of Medicinal Chemistry, The Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
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T antigen transformation reveals Tp53/RB-dependent route to PLAC1 transcription activation in primary fibroblasts. Oncogenesis 2013; 2:e67. [PMID: 23999628 PMCID: PMC3816221 DOI: 10.1038/oncsis.2013.31] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2013] [Accepted: 07/22/2013] [Indexed: 01/03/2023] Open
Abstract
PLAC1 (placenta-specific 1) is a gene that is placenta specific and transcribed very little, if at all, in any somatic tissue. It is nevertheless expressed in many cancer cell lines. To understand how cancer cells may activate the gene in nonexpressing cells, we found that a model is provided by classical transformation of normal fibroblasts by SV40 T antigen. T antigen derepressed the PLAC1 P1 promoter, with Tp53 and RB exerting critical and opposing actions and nuclear receptors, retinoid X receptor and liver X receptor, sharply increasing the level of expression.
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Thakur BK, Dittrich T, Chandra P, Becker A, Lippka Y, Selvakumar D, Klusmann JH, Reinhardt D, Welte K. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells. Biochem Biophys Res Commun 2012; 424:371-7. [DOI: 10.1016/j.bbrc.2012.06.075] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2012] [Accepted: 06/16/2012] [Indexed: 12/17/2022]
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Abstract
p53 is a potent tumor suppressor with a crucial role in preventing uncontrolled cell proliferation and is therefore frequently deleted or mutated in cancer. For tumors with wild-type p53, its function can be overcome by overactive cellular antagonists, such as the ubiquitin ligase murine double minute clone 2 (MDM2). Restoring p53 activity by inhibiting MDM2 in such cancers can eradicate tumors. Consequently, the MDM2-p53 interaction has been extensively targeted for inhibition by small molecules. In recent years, MDM2-like protein (MDMX), another key downregulator of p53, has gained increasing importance as an additional target for drug development, in order to provide a complementary approach to MDM2 inhibition. In this review, we describe how detailed structural knowledge of the MDM2-p53 interface and, more recently, of the MDMX-p53 interaction have helped advance the development of inhibitors against the two targets. We present a summary of the functional biochemistry of MDM2, MDMX and p53 as well as their interactions and examine recent progress in the development of inhibitors of MDM2 and MDMX.
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Kashuba E, Yurchenko M, Yenamandra SP, Snopok B, Szekely L, Bercovich B, Ciechanover A, Klein G. Epstein-Barr virus-encoded EBNA-5 forms trimolecular protein complexes with MDM2 and p53 and inhibits the transactivating function of p53. Int J Cancer 2010; 128:817-25. [DOI: 10.1002/ijc.25414] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
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Zhou R, Frum R, Deb S, Deb SP. The growth arrest function of the human oncoprotein mouse double minute-2 is disabled by downstream mutation in cancer cells. Cancer Res 2005; 65:1839-48. [PMID: 15753382 DOI: 10.1158/0008-5472.can-03-3755] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
We have reported earlier that ectopic expression of mouse double minute-2 (MDM2) induces G1 arrest in normal cells. To explain occasional overexpression of MDM2 in cancer cells, we searched for deletion or substitution mutation in the growth suppressor domains of MDM2 in several breast cancer cell lines that overexpress the oncoprotein. Our results suggest the absence of alteration (deletion or substitution) in the open reading frame of MDM2 transcripts in such cells. Because the breast cancer cell line MCF-7 overexpresses MDM2, we isolated the full-length MDM2 transcript from this cell line. The MDM2 cDNA synthesized from transcripts isolated from MCF-7 cells induced inhibition of G1 to S phase transition in normal human diploid cells such as WI38, suggesting that the genetic alterations in breast cancer cells that overexpress MDM2 disable the growth arrest function of the oncoprotein. Consistently, overexpression of full-length MDM2 in MCF-7 cells over its high endogenous level did not inhibit G1-S transition efficiently. Although MDM2 overexpression was accompanied by CDK4 overexpression or absence of cdk4 inhibitor p16 in most breast cancer cells, we found remarkably high levels of cyclin A rather than cyclin E in these cells. Ectopic expression of cyclin A released MDM2-mediated inhibition of G1-S transition in normal human diploid WI38 cells. We propose that cancer cells expressing high levels of cyclin A escape MDM2-mediated G1 arrest, which may account for a selective growth advantage over normal cells.
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Affiliation(s)
- Ruizhe Zhou
- Department of Biochemistry and the Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298-0614, USA
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11
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Abstract
OBJECTIVE To review the literature published in the past 6 years concerning the role of p53 tumor-suppressor protein in rheumatoid arthritis (RA). METHODS A MEDLINE search was performed to identify all publications that covered the role of p53 in RA. In addition, selected articles related to proto-oncogenes and matrix metalloproteinases were included in this review. RESULTS p53 protein is expressed in RA fibroblast-like synoviocytes (FLSs), and its overexpression is a characteristic feature of RA. The overexpression of p53 is probably induced by DNA strand breaks caused by the genotoxic environment of RA joints, in some cases because of p53 mutations. Independent studies from 3 groups indicated that p53 mutations can and do occur in RA synovial tissue samples derived from a subset of RA patients. Inactivation of p53 may contribute to the invasiveness of FLSs and to the high-level expression of cartilage degradation enzymes as well. Gene transfer or gene knockout studies using a collagen-II-induced RA animal model to examine the role of p53 in RA have been reported. Initial results are positive and indicate that gene transfer of p53 may be clinically useful for the management of RA. CONCLUSIONS p53 protein is expressed in RA FLSs, and its overexpression is a characteristic feature of RA. p53 mutations occur in the synovial tissues derived from a subset of RA patients. The clinical implications of p53 expression and the functional importance of somatic mutations in RA, however, are still unclear. Further research is needed to fully understand the implications of these findings and develop corresponding new therapeutic strategies.
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Affiliation(s)
- Yubo Sun
- Department of Medicine, University of Miami School of Medicine, FL, USA
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12
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Ali SH, DeCaprio JA. Cellular transformation by SV40 large T antigen: interaction with host proteins. Semin Cancer Biol 2001; 11:15-23. [PMID: 11243895 DOI: 10.1006/scbi.2000.0342] [Citation(s) in RCA: 277] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
SV40 large T antigen (TAg) is a powerful oncoprotein capable of transforming a variety of cell types. The transforming activity of TAg is due in large part to its perturbation of the retinoblastoma (pRB) and p53 tumor suppressor proteins. In addition, TAg binds to several other cellular factors, including the transcriptional co-activators p300 and CBP, which may contribute to its transformation function. Several other features of TAg that appear to contribute to its full transformation potential are yet to be completely understood. Study of TAg therefore continues to provide new insights into the mechanism of cellular transformation.
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Affiliation(s)
- S H Ali
- Department of Adult Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA
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13
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Zuzarte PC, Farrance IK, Simpson PC, Wildeman AG. Tumor cell splice variants of the transcription factor TEF-1 induced by SV40 T-antigen transformation. BIOCHIMICA ET BIOPHYSICA ACTA 2000; 1517:82-90. [PMID: 11118619 DOI: 10.1016/s0167-4781(00)00261-x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
The large tumor antigen (TAg) of simian virus 40 is able to transform cells through interactions with cellular proteins, notably p53 and Rb. Among the other proteins that form complexes with TAg is TEF-1, a transcription factor utilized by the viral enhancer to activate expression of the early gene which encodes TAg. We show that fibroblasts contain several alternately spliced TEF-1 mRNAs, the most abundant of which encodes a protein with an additional four amino acid exon compared to the database entry for Hela cell TEF-1. Transformation by TAg induces alternate splicing, producing a more abundant form lacking this exon and matching the published sequence. Splicing variants lacking this exon were detected in mouse pancreatic tumors and in cell lines derived from human pancreatic cancers, in contrast to a single isoform with the exon in normal mouse pancreas. A total of eight splice variants were identified, with the loss of the four amino acid exon typical of transformed cells. These and other data presented suggest that TAg 're-models' host cell transcription factors that are used early in viral infection, and thereby mimics an event that naturally occurs during transformation. The data indicate that TEF-1 alterations may be a hallmark feature of tumorigenesis.
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Affiliation(s)
- P C Zuzarte
- Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada
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14
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Burch LR, Midgley CA, Currie RA, Lane DP, Hupp TR. Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA. FEBS Lett 2000; 472:93-8. [PMID: 10781812 DOI: 10.1016/s0014-5793(00)01427-7] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Biochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full-length mdm2 does not bind stably to p53-DNA complexes, contrasting with C-terminal truncations of mdm2 which do bind stably to p53-DNA complexes. In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX-I domain of p53 becomes concealed when either p53 binds to DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C-terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full-length mdm2 with p53 lacking the BOX-I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX-I domain, addition of RNA activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53-mdm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein; (2) the C-terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA.
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Affiliation(s)
- L R Burch
- Department of Molecular and Cellular Pathology, University of Dundee, Dundee, UK.
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15
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Abstract
p53 is mutated in approximately 50% of human cancers, whereas mutations of the related p73 gene are rare. p73 can activate p53-responsive promoters and induce apoptosis when overexpressed in certain p53-deficient tumor cells. We show that p73 isoforms, p73alpha and p73beta, can each induce permanent growth arrest with markers of replicative senescence when overexpressed in a tetracycline-regulatable manner in human cancer cells lacking functional p53. Human homologue of mouse double minute 2 gene product (hMDM2), but not an NH(2)-terminal deletion mutant, coimmunoprecipitated with p73alpha or p73beta, and inhibited p73 transcriptional activity as with p53. In contrast to p53, ectopically expressed hemagglutinin (HA)-tagged p73 proteins were not stabilized by treatment with several DNA damaging agents. Furthermore, unlike normal p53, which increases in response to DNA damage due to enhanced protein stability in MCF7 cells, endogenous p73 protein levels were not increased in these cells under the same conditions. Thus, although p73 has an ability, comparable to that of p53, to suppress tumor cell growth in p53-deficient cells, p73 induction is regulated differently from p53. These findings suggest that the selective pressures for p53 rather than p73 inactivation in tumors may reflect their differential responses to stresses such as DNA damage, rather than their capacities to induce permanent growth arrest or apoptosis programs.
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Affiliation(s)
- Li Fang
- Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029
| | - Sam W. Lee
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine and Harvard Medical School, Boston, Massachusetts 02115
| | - Stuart A. Aaronson
- Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029
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Taniura H, Matsumoto K, Yoshikawa K. Physical and functional interactions of neuronal growth suppressor necdin with p53. J Biol Chem 1999; 274:16242-8. [PMID: 10347180 DOI: 10.1074/jbc.274.23.16242] [Citation(s) in RCA: 108] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Necdin is expressed in virtually all postmitotic neurons, and ectopic expression of this protein suppresses cell proliferation. Necdin, like the retinoblastoma protein, interacts with cell cycle promoting proteins such as simian virus 40 large T antigen, adenovirus E1A, and the transcription factor E2F1. Here we demonstrate that necdin interacts with the tumor suppressor protein p53 as well. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region (amino acids 35-62) located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed p53-dependent activation of the p21/WAF promoter. Necdin and p53 inhibited cell growth in an additive manner as assessed by the colony formation of SAOS-2 cells, suggesting that necdin does not affect p53-mediated growth suppression. On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons.
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Affiliation(s)
- H Taniura
- Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
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Pomerantz J, Schreiber-Agus N, Liégeois NJ, Silverman A, Alland L, Chin L, Potes J, Chen K, Orlow I, Lee HW, Cordon-Cardo C, DePinho RA. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 1998; 92:713-23. [PMID: 9529248 DOI: 10.1016/s0092-8674(00)81400-2] [Citation(s) in RCA: 1078] [Impact Index Per Article: 39.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The INK4a gene encodes two distinct growth inhibitors--the cyclin-dependent kinase inhibitor p16Ink4a, which is a component of the Rb pathway, and the tumor suppressor p19Arf, which has been functionally linked to p53. Here we show that p19Arf potently suppresses oncogenic transformation in primary cells and that this function is abrogated when p53 is neutralized by viral oncoproteins and dominant-negative mutants but not by the p53 antagonist MDM2. This finding, coupled with the observations that p19Arf and MDM2 physically interact and that p19Rrf blocks MDM2-induced p53 degradation and transactivational silencing, suggests that p19Arf functions mechanistically to prevent MDM2's neutralization of p53. Together, our findings ascribe INK4a's potent tumor suppressor activity to the cooperative actions of its two protein products and their relation to the two central growth control pathways, Rb and p53.
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Affiliation(s)
- J Pomerantz
- Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA
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18
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Léveillard T, Wasylyk B. The MDM2 C-terminal region binds to TAFII250 and is required for MDM2 regulation of the cyclin A promoter. J Biol Chem 1997; 272:30651-61. [PMID: 9388200 DOI: 10.1074/jbc.272.49.30651] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
MDM2 proto-oncogene expression is aberrant in many human tumors. Its normal role is to modulate the functions of p53. The N terminus of MDM2 interacts with p53, whereas the properties of the rest of the molecule are poorly understood. We show that MDM2 binds to the general transcription factor TFIID in vivo. The C-terminal Ring finger interacts with TAFII250/CCG1, and the central acidic domain interacts with TBP. Expression of MDM2 activates the cyclin A gene promoter but not c-fos, showing that the effects of MDM2 are specific. Deletion of the C-terminal region of MDM2 abolishes activation, showing that the C-terminal domain of MDM2 is functionally important. We found that increasing MDM2 expression to higher levels inhibits the cyclin A promoter. Inhibition appears to result from titration of general transcription factors because MDM2 overexpression inhibits c-fos as well as other promoters in vivo and basal transcription in vitro. The mechanisms of repression of the cyclin A and fos promoters appear to be different. Cyclin A repression is lost by deleting the C terminus, whereas that of c-fos is lost by removal of the acidic domain. These results reinforce the conclusion that the C terminus of MDM2 mediates effects on the cyclin A promoter. MDM2 transformed cells contain elevated levels of cyclin A mRNA, showing that activation occurs under physiological conditions. There is a positive correlation between MDM2 binding to TAFII250 and MDM2 activation of the cyclin A promoter. The C-terminal region of MDM2, which contains the Ring finger, interacts with TAFII250 and is required for regulation of the cyclin A promoter by MDM2. Our results link the activity of MDM2, a transforming protein implicated in many human tumors, with cyclin A, a regulator of the cell cycle.
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Affiliation(s)
- T Léveillard
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, ULP, 1 Rue Laurent Fries, BP 163, 67404 Illkirch cedex, France
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19
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Shvarts A, Bazuine M, Dekker P, Ramos YF, Steegenga WT, Merckx G, van Ham RC, van der Houven van Oordt W, van der Eb AJ, Jochemsen AG. Isolation and identification of the human homolog of a new p53-binding protein, Mdmx. Genomics 1997; 43:34-42. [PMID: 9226370 DOI: 10.1006/geno.1997.4775] [Citation(s) in RCA: 107] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
We recently reported the identification of a mouse cDNA encoding a new p53-associating protein that we called Mdmx because of its structural similarity to Mdm2, a well-known p53-binding protein. Here we report the isolation of a cDNA encoding the human homolog of Mdmx. The ORF of the cDNA encodes a protein of 490 amino acids, 90% similar to mouse Mdmx. The homology between Mdmx and Mdm2 is most prominent in the p53-binding domain and the putative metal-binding domains. The Mdmx protein, which, based on SDS-PAGE, has a MW of 80 kDa, can bind p53 in vitro. The human MDMX gene is transcribed in all tissues tested, with high levels in thymus. By fluorescence in situ hybridization analysis we mapped the mouse mdmx gene to chromosome 1 (region F-G) and the human MDMX gene to chromosome 1q32.
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Affiliation(s)
- A Shvarts
- Laboratory of Molecular Carcinogenesis, Leiden University, The Netherlands
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20
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Abstract
Leukemia in the soft-shell clam, Mya arenaria, is characterized by tumor cells which are detected initially in the hemolymph. This disease is much more common in clams inhabiting polluted waters, suggesting an environmental component to its pathogenesis. In this study, leukemia cells were identified using a murine monoclonal antibody, 1E10, which recognizes a leukemia-specific protein expressed by tumor cells. Mutant p53 protein was detected using a murine monoclonal antibody (PAb 240) which reacts with mutant p53. Using immunofluorescence, the reactivity of clam cells to the 1E10 antibody was evaluated along with mutant p53 protein reactivity. Reverse transcriptase-polymerase chain reactions followed by sequence analyses were utilized to examine clams with hemocytes reacting with the p53 antibody for possible p53 gene mutations. Mutant p53 protein was expressed by tumor cells from five animals with advanced disease (in which greater than 90% of cells reacted with 1E10). A C-->G transversion was detected at the end of exon 6 from two of the five animals that reacted with both the mutant p53 antibody and 1E10. This substitution changes the amino acid of this codon from proline to alanine. Overall, our results suggest that environmentally induced alterations in p53 can contribute to the pathogenesis of leukemia in soft-shell clams inhabiting polluted water and/or sediment.
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Affiliation(s)
- C M Barker
- Department of Comparative Medicine, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA
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21
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Haidar MA, El-Hajj H, Bueso-Ramos CE, Manshouri T, Glassman A, Keating MJ, Maher A. Expression profile of MDM-2 proteins in chronic lymphocytic leukemia and their clinical relevance. Am J Hematol 1997; 54:189-95. [PMID: 9067496 DOI: 10.1002/(sici)1096-8652(199703)54:3<189::aid-ajh3>3.0.co;2-s] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The MDM-2 oncoprotein exists in an autoregulatory feedback loop with the tumor suppressor protein p53. Therefore, intracellular levels of these two proteins may play important roles in cell proliferation and tumorigenesis. Several MDM-2 proteins (Mr 35-100 Kd) have been demonstrated in human cell lines. We report here the expression profile of MDM-2 and p53 proteins in 87 cases of chronic lymphocytic leukemia (CLL) as detected by immunoblot analysis. The MDM-2 proteins (p57, p59, p67, and p90) were found to be overexpressed in different combinations in 56/87 (64%) of cases of CLL when compared with normal volunteers. The MDM-2 protein p57 was predominantly overexpressed 46/87 (53%) in CLL. In 22/87 (25%) cases of CLL p57 was overexpressed alone, and in 24/87 (28%) cases it was co-overexpressed with other MDM-2 proteins p59/p67/p90. Six of the 87 cases of CLL showed overexpression of the tumor suppressor protein p53 by immunoblot analysis, and five of those cases also co-overexpress MDM-2 protein p57. No statistically significant correlation of MDM-2 protein overexpression to clinical disease stage and history of previous chemotherapy of CLL patients has been found. However, considering the oncogenic potential of overexpressed MDM-2 proteins, a possible role of MDM-2 proteins in the promotion of CLL disease remains to be evaluated.
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Affiliation(s)
- M A Haidar
- Hematopathology Program, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA
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22
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Kido A, Tsujiuchi T, Tsutsumi M, Takahama M, Okajima E, Kobitsu K, Miyauchi Y, Mii Y, Tamai S, Konishi Y. p53 mutation and absence of mdm2 amplification and Ki-ras mutation in 4-hydroxyamino quinoline 1-oxide induced transplantable osteosarcomas in rats. Cancer Lett 1997; 112:5-10. [PMID: 9029163 DOI: 10.1016/s0304-3835(96)04537-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Previously, we reported the establishment of two transplantable osteosarcomas, one induced by local application of a carcinogen, 4-hydroxyamino quinoline 1-oxide(4-HAQO), and another which developed spontaneously in rats, and their subdivision into four lines with high and low metastatic potential to the lung. In the present study, mutations of p53 and Ki-ras genes were investigated by PCR and SSCP followed by direct sequencing, and the amplification of the mdm2 gene was assessed by Southern blot analysis. Mutations of p53 in exon 7 were detected in 4-HAQO-induced transplantable osteosarcomas, but not their spontaneous counterparts, irrespective of the metastatic potentials. Direct sequencing revealed a CGC to CAC transition with an amino acid change of Arg to His, at codon 246. Neither Ki-ras mutations nor mdm2 amplification were detected in any of the transplantable tumors. The results suggest that while p53 mutations occurred during osteosarcoma development by 4-HAQO without mdm2 amplification and Ki-ras mutation does not contribute to osteosarcoma development in rats.
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Affiliation(s)
- A Kido
- Department of Oncological Pathology, Cancer Center, Nara Medical University, Shijo-cho, Kashihara, Japan
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23
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Shvarts A, Steegenga WT, Riteco N, van Laar T, Dekker P, Bazuine M, van Ham RC, van der Houven van Oordt W, Hateboer G, van der Eb AJ, Jochemsen AG. MDMX: a novel p53-binding protein with some functional properties of MDM2. EMBO J 1996; 15:5349-57. [PMID: 8895579 PMCID: PMC452278] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
Here we report the isolation of a cDNA encoding a new p53-associating protein. This new protein has been called MDMX on the basis of its structural similarity to MDM2, which is especially notable in the p53-binding domain. In addition, the putative metal binding domains in the C-terminal part of MDM2 are completely conserved in MDMX. The middle part of the MDMX and MDM2 proteins shows a low degree of conservation. We can show by co-immunoprecipitation that the MDMX protein interacts specifically with p53 in vivo. This interaction probably occurs with the N-terminal part of p53, because the activity of the transcription activation domain of p53 was inhibited by co-transfection of MDMX. Northern blotting showed that MDMX, like MDM2, is expressed in all tissues tested, and that several mRNAs for MDMX can be detected. Interestingly, the level of MDMX mRNA is unchanged after UV irradiation, in contrast to MDM2 transcription. This observation suggests that MDMX may be a differently regulated modifier of p53 activity in comparison with MDM2. Our study indicates that at least one additional member of the MDM protein family exists which can modulate p53 function.
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Affiliation(s)
- A Shvarts
- Laboratory of Molecular Carcinogenesis, Sylvius Laboratories, Leiden University, The Netherlands
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24
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Affiliation(s)
- T M Gottlieb
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
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25
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Wertz IE, Deitch AD, Gumerlock PH, Gandour-Edwards R, Chi SG, de Vere White RW. Correlation of genetic and immunodetection of TP53 mutations in malignant and benign prostate tissues. Hum Pathol 1996; 27:573-80. [PMID: 8666367 DOI: 10.1016/s0046-8177(96)90164-1] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
The prognostic value of the p53 gene (TP53), the most commonly mutated gene in human cancers, has been well established for several cancer types. However, because varying frequencies of TP53 mutations have been identified in prostatic adenocarcinoma (CaP) by genetic and immunohistochemical (IHC) studies, the role of TP53 in CaP tumorigenesis is currently unresolved. These experimental discrepancies could be caused by tissue heterogeneity within prostatic neoplasms, variations in experimental protocols, or other factors. Thus, the goal of this study was to develop a reliable IHC approach for the detection of p53 in archival prostate tissue. The authors evaluated four p53 antibodies, CM-1, 1801, DO-1, and DO-7, for their ability to reveal p53. They chose two reference CaP cell lines, 26 patient specimens (including eight benign prostatic hyperplasias (BPHs), 16 CaPs, and two lymph node metastases), one prostate and nine kidney cell lines for p53 analysis. The TP53 status of these samples was characterized using single-strand conformational polymorphism (SSCP) analysis of RNA/PCR products and sequencing. IHC detection of p53 was markedly enhanced by using the combination of microwave heat-induced antigen unmasking and a cocktail of the DO-1 and DO-7 antibodies. This approach identified 14 of 15 (93%) cell lines and patient samples having TP53 missense mutations in the exons 5 to 8 region. Of the 21 patient samples and cell lines that were either normal by SSCP or expressed p53 mutations that are not expected to stain, 18 (86%) were immunonegative. Because of this good correlation between molecular and IHC analysis, this approach may help to resolve the uncertainty about TP53 in CaP tumorigenesis.
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Affiliation(s)
- I E Wertz
- Department of Urology, Cancer and Molecular Research Laboratory, University of California, Davis, Sacramento, USA
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26
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Haupt Y, Barak Y, Oren M. Cell type-specific inhibition of p53-mediated apoptosis by mdm2. EMBO J 1996; 15:1596-606. [PMID: 8612583 PMCID: PMC450069] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
The effect of excess mdm2 on p53-mediated apoptosis was investigated in two human-derived cell lines, H1299 and HeLa. In H1299 cells, overexpression of mdm2 resulted in effective protection from apoptosis. This protective effect was seen only under conditions allowing the formation of p53-Mdm2 complexes. In contrast, excess mdm2 failed to abolish p53-mediated apoptosis in HeLa cells, despite a complete abrogation of p53-dependent sequence-specific transcriptional activation (SST). These data strongly support the contention that SST is dispensable for at least some types of p53-mediated apoptosis. Further, they suggest that one of the roles of mdm2 may be to modulate the apoptotic activity of p53, in a manner which is dictated by the pathway through which p53 induced apoptosis in a given cell type
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Affiliation(s)
- Y Haupt
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
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27
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Abstract
Mutation of the p53 gene is among the most common lesions in a variety of human tumors, including those of the central nervous system. In most instances, mutation of one p53 allele is followed by loss of the remaining wild-type allele, resulting in cells with a complete absence of functional wild-type p53 protein. However, in some situations, such as at initiation of spontaneously arising gliomas or as the germline configuration of patients with the Li-Fraumeni syndrome, cells clearly carry both wild-type and mutant p53 alleles. These observations lead to the hypothesis that p53 mutations can give rise to loss of tumor suppressor functions as well as to gain of oncogenic transformation capabilities. In this review, we define the types of mutations that occur in the p53 gene in various glial tumors, contrast that with the spectra described in other human tumor types, and discuss the biochemistry and physiology of the p53 protein and its ability to regulate and be regulated by other gene products. We use this information to propose roles for p53 in the initiation and progression of human gliomas.
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Affiliation(s)
- O Bögler
- Ludwig Institute for Cancer Research, University of California San Diego, La Jolla 92093-0660, USA
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28
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Lund T, Medveczky MM, Geck P, Medveczky PG. A herpesvirus saimiri protein required for interleukin-2 independence is associated with membranes of transformed T cells. J Virol 1995; 69:4495-9. [PMID: 7769710 PMCID: PMC189192 DOI: 10.1128/jvi.69.7.4495-4499.1995] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
A region of the herpesvirus saimiri genome encoding an mRNA with two open reading frames (ORFs) has been identified to be essential for transformation of T cells. Deletion of either ORF resulted in the loss of transforming ability. ORF-1 has been shown to code for a collagen-like oncoprotein. This study shows for the first time that the bicistronic mRNA can translate a 32-kDa protein from ORF-2. Polyclonal serum to ORF-2 was generated by using a glutathione fusion protein. Using this antiserum, ORF-2 was localized in cell membranes and is expressed on the outer cell membrane. The half-life of this membrane protein was found to be about 5.5 h. Limited sequence similarity was found between ORF-2 and interleukin-11; however, no secretion of ORF-2 protein was detected in supernatants from transformed cells. Further studies are required to investigate the potential interaction with the interleukin-11 receptor.
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Affiliation(s)
- T Lund
- Department of Microbiology and Immunology, University of South Florida, Tampa 33612-4799, USA
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29
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Martin K, Trouche D, Hagemeier C, Sørensen TS, La Thangue NB, Kouzarides T. Stimulation of E2F1/DP1 transcriptional activity by MDM2 oncoprotein. Nature 1995; 375:691-4. [PMID: 7791903 DOI: 10.1038/375691a0] [Citation(s) in RCA: 348] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The MDM2 proto-oncogene is found amplified in a variety of tumours. The oncogenic capacity of the MDM2 protein is attributed to its ability to bind the p53 tumour-suppressor protein and mask its transcriptional activation potential. Here we show that MDM2 makes a functional contact with two cooperating transcription factors, E2F1 and DP1 (refs 4,5), which are involved in S-phase progression. MDM2 contacts the activation domain of E2F1 using residues conserved in the activation domain of p53. However, in contrast to its repression of p53 activity, MDM2 stimulates the activation capacity of E2F1/DP1. These results indicate that MDM2 not only releases a proliferative block by silencing the tumour suppressor p53, it also positively augments proliferation by stimulating the S-phase inducing transcription factors E2F1/DP1.
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Affiliation(s)
- K Martin
- Wellcome/CRC Institute, Cambridge, UK
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30
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Bueso-Ramos CE, Manshouri T, Haidar MA, Huh YO, Keating MJ, Albitar M. Multiple patterns of MDM-2 deregulation in human leukemias: implications in leukemogenesis and prognosis. Leuk Lymphoma 1995; 17:13-8. [PMID: 7773150 DOI: 10.3109/10428199509051698] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The human analogue of the mouse double minute-2 (MDM-2) protein binds to p53 protein and abrogates its tumor-suppressing activity. MDM-2 overexpression may represent an alternative mechanism to p53 mutation for escaping the p53-mediated growth control. Interestingly, multiple MDM-2 protein isoforms have been described and the possibility of functional differences between various isoforms has been raised. Previously, we demonstrated significant MDM-2 mRNA overexpression in human leukemias and suggested that MDM-2 overexpression may be a marker of aggressiveness of the disease. Polyclonal antibodies (Ab) have been generated to detect various isoforms of the MDM-2 protein. Using these Abs, we confirmed MDM-2 protein overexpression in leukemias. Furthermore, we observed heterogeneity in the isoforms expressed in various types of leukemias. In addition, we demonstrated that analysis by flow cytometry could be used as a diagnostic tool for detecting altered MDM-2 protein expression in leukemias. Here we review and expand our initial observations and confirm MDM-2 mRNA and protein overexpression by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and western blot analyses. Understanding the possible role of MDM-2 oncogene expression in leukemias may establish the scientific basis for new therapeutic approaches.
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Affiliation(s)
- C E Bueso-Ramos
- Hematopathology Program, University of Texas M. D. Anderson Cancer Center, Houston 77030-4095, USA
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31
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Affiliation(s)
- G Selivanova
- Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden
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