CARCINOGEN-INDUCED AMPLIFICATION OF SV40 DNA INSERTED AT 9Q12-21.1 ASSOCIATED WITH CHROMOSOME BREAKAGE, DELETIONS, AND TRANSLOCATIONS IN HUMAN UROEPITHELIAL CELL-TRANSFORMATION IN-VITRO

The fate of integrated SV40 viral genome in SV40-immortalized human uroepithelial cells (SV-HUC) during multistep chemical transformation in vitro was studied. We previously reported that exposure of SV-HUC at passage (P) 15 to the chemical carcinogens 3-methylcholanthrene (MCA), 4-aminobiphenyl (ABP), or the N-hydroxy metabolites of ABP causes tumorigenic transformation and/or neoplastic progression. We report now that these same chemical carcinogens induce amplification of SV40 DNA in SV-HUC We used fluorescence in situ hybridization (FISH) to show that this amplification occurs at the SV40 integration site, which was mapped near a common fragile site at 9q12-21.1 on the der(9)t(8;9) chromosome that is present in all SV-HUC at the earliest passage studied. Karyotypic analysis, along with FISH, also revealed that all carcinogen-induced tumors (T-SV-HUCs) had breaks at 9q12-21.1, deletions of 9q12-21.1 --> pter, and new derivative chromosomes containing SV40 in the segment 9q12-21.1 --> 9q34 = 8q22 --> 8qter. Southern blot analysis, along with FISH. confirmed SV40 genome rearrangements in T-SV-HUCs. In contrast, no 9q12-21.1 breaks were observed in control SV-HUC. Thus, these results associate 9q12-21.1 --> pter alterations with HUC tumorigenic transformation. In addition, these results indicate for the first time that (carcinogen-induced) amplification of chromosome-integrated viral genes may create sites that are prone to breakage, deletions, and translocations. These results suggest a new mechanism by which chemical carcinogens in synergy with a DNA tumor virus could initiate a cascade of events that contribute to the genomic instability associated with tumorigenesis. (C) 1993 Wiley-Liss, Inc.