Subject Review MDM2 and Its Splice Variant Messenger RNAs: Expression in Tumors and Down-Regulation Using Antisense Oligonucleotides Frank Bartel, 1 Linda C. Harris, 2 Peter Wu ¨ rl, 3 and Helge Taubert 1 1 Institute of Pathology, Faculty of Medicine, University of Halle-Wittenberg, Halle/Saale, Germany; 2 Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis TN; and 3 Surgical Clinic I, Faculty of Medicine, University of Ulm, Ulm, Germany Abstract Alternative splicing has an important role in expanding protein diversity. An example of a gene with more than one transcript is the MDM2 oncogene. To date, more than 40 different splice variants have been isolated from both tumor and normal tissues. Here, we review what is known about the alteration of MDM2 mRNA expression, focusing on alternative splicing and potential functions of different MDM2 isoforms. We also discuss the progress that has been made in the development of antisense oligonucleotides targeted to MDM2 for use as a potential cancer therapy. Introduction MDM2 1 appears to play a role in many normal physiological and pathological pathways. It has been shown that MDM2 not only acts as an oncogene (1 – 3) but also displays growth- inhibitory functions (4, 5). MDM2 can also function indepen- dently of p53; for example, MDM2 interacts with transcription factors of the E2F family and the human TATA binding protein (6, 7) and inhibits retinoblastoma growth-regulatory function (8). A question remains whether the same MDM2 protein is responsible for the diverse functions reported or whether different isoforms or post-translationally modified MDM2 proteins could be involved. We examine here the different MDM2 variants that are likely translated from alternatively spliced MDM2 pre-mRNAs and how antisense oligonucleotide (AS-ODN) therapies might be useful for down-regulating the expression of both full-length and alternatively spliced variants of MDM2 . Down-regulation of full-length MDM2 expression in tumors that overexpress MDM2 would be predicted to result in p53 protein stability and sensitization to DNA-damaging agents (chemotherapeutic drugs and radiation) that act via p53-dependent pathways (Fig. 1). In addition, p53-independent functions of MDM2, such as in regulating the cell cycle, would also be abrogated, thereby restoring growth control in the tumor (Fig. 1). Progress has been made in the in vitro and in vivo uses of MDM2 antisense to down-regulate MDM2 expression. Splicing of the MDM2 Messenger RNA Three human MDM2 mRNA transcripts of 6.7, 4.7, and 1.9 kb long were reported by Pinkas et al. (9) in breast carcinoma cells, with the 1.9-kb mRNA having lost exon 12. In addition, several truncated MDM2 isoforms of 85, 76, and 57 kDa have been described in a panel of human breast carcinomas, together with the full-length 90-kDa protein (10). During the last few years, detailed expression analyses of the MDM2 mRNA in various cancer types and in normal tissue have revealed alternative as well as aberrant splicing (Fig. 2; reviewed in Ref. 11). The tumor types investigated to date include ovarian and bladder cancer (12), glioblastomas (13), glioblastoma cell lines (14), breast carcinomas (15, 16), soft tissue sarcomas (17–19), giant cell tumors of the bone (20), and Hodgkin’s lymphoma (21). The majority of the greater than 40 splice variants that have been detected to date lack sequences that encode at least part of the p53 binding domain, the nuclear localization and export sequences, the p300 binding domain, and the acidic domain. In vitro expression studies confirmed that the protein isoforms encoded by at least four of these splice variants (MDM2 -A, -B, -C, and -D; 12) are unable to bind p53. The splice form MDM2 -B, which is the most frequently detected transcript variant, has been described in various tumor types as well as in normal tissue, whereas many of the other variants have only been found in one particular cancer type. A problem that has developed, however, is that some investigators have chosen to give different names to previously published isoforms, although most of the sequences of published splice variants are in Genbank. For example, the recently detected splice form MDM2 -HD1 (accession no. AJ5505169) corresponds to the PM2 form (accession no. AJ278977), and another splice variant, MDM2 -Del.G (15), which lacks the sequence between nucleotides 182 and 1432 of the coding region of the MDM2 mRNA, exactly corresponds to the 219bp form Received 10/1/03; revised 11/11/03; accepted 11/13/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: NIH grants CA92401 and CA21765, American Lebanese Syrian Associated Charities (L. C. H.), Deutsche Krebshilfe e.V. grant 2130-Ta2, Land Sachsen-Anhalt grant 3347A/0021B, and GSGTe.V. (F. B. and H. T.). Requests for reprints: Frank Bartel, Institute of Pathology, Faculty of Medicine, University of Halle-Wittenberg, D-06097 Halle/Saale, Germany. Phone: 49-345- 557-4272; Fax: 49-345-557-1295. E-mail: frank.bartel@medizin.uni-halle.de Copyright D 2004 American Association for Cancer Research. 1 The abbreviations used are MDM2, human gene and oncogene; MDM2, human protein and isoform; mdm2, mouse gene; Mdm2, mouse protein. 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