Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells.

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RESUMO

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

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