MDM2 GENE AMPLIFICATION AND TRANSCRIPT LEVELS IN HUMAN SARCOMAS - RELATIONSHIP TO TP53 GENE STATUS

Background: Alterations of the TP53 tumor suppressor gene appear to be implicated in the tumorigenesis and progression of several types of h uman cancer, including different histologic subtypes of sarcomas. The MDM2 (murine double minute-2) gene encodes a nuclear phosphoprotein th at may interact with both mutant and wild-type p53 proteins, thereby i nhibiting p53-mediated transactivation in a dose-dependent manner. Rec ently it has been suggested that mdm2 and p53 proteins are components of an autoregulatory loop in which the MDM2 gene is transactivated by p53. Purpose: Our purpose was to examine the frequency of MDM2 amplifi cations in larger panels of sarcomas, determine if the mRNA level coul d be elevated in tumors without concomitant gene amplification, and re late MDM2 findings to the TP53 status of the tumors. Methods: Sarcoma tissue of different histologic subtypes was obtained from 68 patients at the time of surgery and from 26 human xenografts in nude mice. In a ddition, two human sarcoma cell lines (OSA and U2OS) mere studied. Gen omic DNA from tumor tissue. in vitro cell lines, and peripheral blood cells were isolated by Southern-blot analysis methods to determine MDM 2 gene amplification. Tumor DNA was analyzed for possible TP53 gene mu tations in exons 5, 7, and 8 by constant denaturing gel electrophoresi s. To determine the MDM2 and TP53 mRNA levels, Northern-blot analysis was performed. Results: Amplification of the MDM2 gene was detected in 10 tumors (10.3%). Whereas MDM2 amplification and/or overexpression w ere found only in two (U2OS and OSA cell lines) of 18 osteosarcomas, o ne of 20 malignant fibrous histiocytomas (MFHs), and in none of 14 lei omyosarcomas, such alterations were observed in two of two fibrosarcom as, three of six malignant schwannomas, three of 19 liposarcomas, and in the one hemangiopericytoma examined. MDM2 overexpression was found in all nine examined cases with and in three turners without amplifica tion. TP53 mutations were detected in 12 cases (five osteosarcomas, fo ur MFHs, and three leiomyosarcomas), of which none showed amplificatio n, but one had increased levels of MDM2 mRNA. None of the fibrosarcoma s, malignant schwannomas, and liposarcomas examined had mutated TP53. The six sarcomas that showed high TP53 mRNA expression in the absence of gene mutation also had elevated levels of MDM2 mRNA. Conclusions: T he present data provide further indications that increased MDM2 expres sion level, caused by gene amplification or altered regulation of tran scription, is involved in tumor progression of some, but not all, sarc oma subtypes.