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Background: Alterations of the TP53 tumor suppressor gene appear to be implicated in the tumorigenesis and progression of several types of human cancer, including different histologic subtypes of sarcomas. The MDM2 (murine double minute-2) gene encodes a nuclear phosphoprotein that may interact with both mutant and wild-type p53 proteins, thereby inhibiting p53-mediated transactivation in a dose-dependent manner. Recently 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 amplifications in larger panels of sarcomas, determine if the mRNA level could be elevated in tumors without concomitant gene amplification, and relate 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 addition, two human sarcoma cell lines (OSA and U2OS) were studied. Genomic DNA from tumor tissue, in vitro cell lines, and peripheral blood cells were isolated by Southern-blot analysis methods to determine MDM2 gene amplification. Tumor DNA was analyzed for possible TP53 gene mutations in exons 5, 7, and 8 by constant denaturing gel electrophoresis. 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 over-expression were found only in two (U2OS and OSA cell lines) of 18 osteosarcomas, one of 20 malignant fibrous histiocytomas (MFHs), and in none of 14 leiomvosarcomas, such alterations were observed in two of two ribrosarcomas, 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 tumors without amplification. TP53 mutations were detected in 12 cases (five osteosarcomas, four MFHs, and three leiomyosarcomas), of which none showed amplification, but one had increased levels of MDM2 mRNA. None of the fibrosarcomas, 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: The present data provide further indications that increased MDM2 expression level, caused by gene amplification or altered regulation of transcription, is involved in tumor progression of some, but not all, sarcoma subtypes. [J Natl Cancer Inst 86:1297-1302, 1994]

Protein tyrosine kinases (PTKs) have been implicated in the development of many common human tumours including melanoma. Previously we isolated PTK gene sequences expressed in normal melanocytes. Here we examined expression of 9 of these genes in cell lines derived from defined stages of melanoma progression, by Northern blotting and in some cases immunoblotting. We also tested cells from 2 animal models of particular stages in progression, as well as uncultured biopsies of metastatic melanoma. The expression of 2 receptor kinase family members found in melanocytes, PTK7/CCK-4 and SEK/TYRO1, was decreased or lost in advanced melanomas. PTK7 mRNA was found in only 54% of melanoma cell lines and 20% of melanoma biopsies. Similarly, expression was lost in 2 advanced cell lines selected from an early melanoma line that did express PTK7 mRNA. SEK/TYRO1 expression was observed in 75% and 17% of cell lines from primary and metastastic melanomas, respectively. Conversely, mRNA for the non-receptor kinase PTK6/BRK was not detected in normal melanocytes or primary melanoma lines, but was found in 9% of metastatic melanoma cell lines. Int. J. Cancer 71: 1061-1065, 1997. © 1997 Wiley-Liss Inc.