[CANCER RESEARCH 53, 4761-4763, October 15, 1993] Advances in Brief Homozygous Deletions within 9p21-p22 Identify a Small Critical Region of Chromosomal Loss in Human Malignant Mesotheliomas1 Jin Quan Cheng, Suresh C. Jhanwar, You Yong Lu, and Joseph R. Testa2 Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [J. Q. C., Y. Y. L., J. R. T.¡,and Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [S. C. J.] Abstract Previous DNA analyses have demonstrated that 9pl3-p22 is a frequent site of chromosomal loss in leukemia, glioma, melanoma, and lung and bladder carcinomas. Recent cytogenetic studies have revealed recurrent alterations of 9p in malignant mesothelioma (MM). We have performed gene dosage studies of 23 MM cell lines, using probes for several 9p21-p22 loci (IFNB, IFNA/IFNW, D9S3, D9S126, D9S169, and D9S171 ), to identify a common region of deletion. Homozygous and/or hemizygous deletions were identified in 19 (83%) cell lines. Homozygous losses (10 cell lines; 43%) occurred most often at the D9S171 and IFNAIIFNVÕ loci. In 8 cell lines, 2 or more of the 9p loci examined were found to be homozygously lost; 2 others displayed homozygous losses only at the D9S171 locus. Results from our deletion mapping analysis suggest that D9S171 is located between IFNA/IFNW and D9S126. The data presented here indicate that allelic loss from 9p21-p22 is a common occurrence in MM and further delineate the location of a putative 9p tumor suppressor gene(s) to a region between IFNA/IFNW and D9S17I. These MM cell lines may facilitate efforts to define an even smaller critically deleted region, leading to the eventual cloning and characterization of this gene. Introduction Neoplasia is thought to result from the accumulation of multiple genetic alterations that release cells from normal regulation of cell growth and differentiation. In addition to oncogene activation, the inactivation of tumor suppressor genes has been shown to play an important role in tumorigenesis (1). In many malignancies, two or more different tumor suppressor genes may be inactivated, and some suppressor genes are frequently altered in several different tumor types (2). Cytogenetic and restriction fragment length polymorphism analyses have led to the localization and eventual isolation of several tumor suppressor genes, including RBI, WT1, and DCC (3-5). MM3 is a rare cancer that is characterized by a long latency from onset of exposure to asbestos and by a short survival after diagnosis (6). The length of the latent period suggests that multiple genetic alterations may be required for tumorigenic conversion of a mesothe- lial cell (7). Recently, TP53 and KRAS mutations have been reported in several MM cell lines (8, 9). Previous karyotypic analyses have demonstrated that most MM have multiple numerical and structural changes. Several recurrent alterations have been found, particularly losses or structural rearrangements of Ip, 3p, 6q, 9p, and 22 (10-14). In a recent study, we found that loss of 9p21-p22 represents one of the most prominent cytogenetic alterations in MM (14). To map the mini mal region of deletion in 9p21-p22, we have performed gene dosage Received 7/28/93; accepted 8/23/93. 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. 1 Supported by National Cancer Institute Grants CA-45745 and CA-06927, and by an appropriation from the Commonwealth of Pennsylvania. 2 To whom correspondence should be addressed, at Fox Chase Cancer Center, Depart ment of Medical Oncology, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111. 3 The abbreviations used are: MM, malignant mesothelioma; IFNB1, interferon ß-1 locus; 1FNAHFNW, interferon a/interferon tu gene cluster locus; PCR, polymerase chain reaction. studies of 23 MM cell lines, using DNA markers for 6 different 9p loci. These analyses have resulted in the identification of a critical deleted region in 9p involved in the pathogenesis of many MM. Materials and Methods Tumor Cell Lines. All 23 MM cell lines were established from surgically explanted primary MM. The methods for establishing these cell lines have been described (15). None of the patients received previous cytotoxic therapy. DNA Isolation and Southern Blotting. DNA from cell lines was isolated as previously described (16). Approximately 10 fxg of purified DNA per sample were digested with restriction enzymes and separated by electropho- resis in a 0.8% agarose gel. DNA was transferred from the gels to nylon membranes (Gene Screen Plus; New England Nuclear, Boston, MA) (16). DNA Probes. The following cloned, 9p21-p22 DNA probes were used in Southern hybridizations: (a) pHFb, a genomic clone of the IFNB1 gene (17); (b) pL-FA, an IFNA2 clone that cross-hybridizes to all the genes of the IFNA/IFNW family (18); (c) p72-0.9, a genomic clone of the D9S126 locus (19); and (d) DR6 (D9S3). We used a genomic AKT2 clone (19ql3.1-ql3.2) as a control probe because numerical changes of chromosome 19 and structural alterations of 19q were relatively infrequent in our cytogenetic analyses of MM cell lines (14, 16). Gene Dosage Analysis. It was not possible to examine loss of heterozy- gosity in the 23 MM cell lines because matched normal tissues were available in only a few cases. The normal control was DNA from either placenta or normal lymphocytes. Autoradiograms exposed within the linear range of the film were scanned on an UltraScan XL laser densitometer (Pharmacia LKB, Piscataway, NJ). Absorbance of 9p signal intensities relative to a 19q standard (AKT2) was calculated using the formula described by Center et al. (20). A reduction in the autoradiographic signal intensity of 40-60% of that of controls was considered evidence of a hemizygous deletion. A reduction in signal intensity of >95% of that of controls constituted a homozygous deletion. PCR Amplification. Primers from D9S171 (5'-AGCTAAGTGAACCT- CATCTCTG TCT-3' and 5'-ACCCTAGCACTGATGGTATAGTCT-3') and D9S169 (5'-AGAGACAGAT CCAGATCCCA-3' and 5'-TAACAACTCACT- GATTATTTAA GGC-3') (Ref. 21; sequences obtained from Dr. J. Fountain, Massachusetts Institute of Technology, Cambridge, MA, and also available in the Genome Data Base) were used to amplify sequences containing (CA) repeats from genomic DNAs. PCR was performed in a MJ Research Minicy- cler (Boston, MA) with 0.5 fxg DNA in a total volume of 50 ¡i\.Thirty-five cycles of amplification were carried out with Taql DNA polymerase (Cetus, Norwalk, CT) and consisted of 30 s at 94°C,1 min at 58°C,and 30 s at 72°C. The PCR products were analyzed by electrophoresis in 2.5% agarose gels. Results and Discussion Southern blot analysis revealed overlapping homozygous deletions within 9p21-p22 in 8 MM cell lines (35%), each of which showed no detectable autoradiographic signal with 2 or more 9p probes (Fig. 1 and data not shown). The highest frequency of loss was at the IFNA/IFNW gene cluster (6 lines) and D9S126 (5 lines) loci. Two cell lines (M-l and M-4) displayed homozygous deletions of the en tire IFNA/IFNW gene cluster and the IFNBI gene, whereas cell line M-l2 had a deletion of the IFNA/IFNW gene cluster and a rearrange ment of the IFNBI locus. "Partial" homozygous deletions of the IFNA/IFNW gene cluster (i.e., losses of some, but not all, genes at 4761 on June 15, 2015. © 1993 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from