An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (C060) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated C060 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 pSVK,. (15,17,20,22,35,45). This process is regulated by cellular factors, is not dependent on DNA replication, and is controlled at the levels of transcription and posttranscription (20, 22; T. Kleinberger, manuscript in preparation). The nature of the factors involved in the enhanced expression and their possible role in the overreplication phenomenon is not yet known.To facilitate analysis of the trans-acting factors responsible for viral overreplication, an in vitro system for DNA amplification was established in our laboratory (4). This system is based on the in vitro system for SV40 replication developed recently by Li and Kelly (27). By using the in vitro system for SV40 replication, it was demonstrated by several laboratories that cytosolic extracts from permissive cells supplemented with purified T antigen efficiently replicate exogenous SV40 DNA templates containing a functional origin of replication, yielding complete closed circular DNA molecules (16,18,27,43,49). Cytosolic extracts from Chinese hamster and other nonpermissive cell lines failed to support significant viral DNA replication (28).The in vitro amplification system described here consists of cytosolic extracts from drug-treated SV40-transformed Chinese hamster (C060) cells (24), template SV40 DNA molecules, and exogenous T antigen. This system provides an excellent tool for analysis of the mode of DNA amplification, characterization of the amplified DNA, and purification of the cellular factors responsible for the amplification process. The availability of this system will facilitate studies on the molecular mechanisms of gene amplification, on the control of cellular permissivity to viral replication, and on the putative existence of carcinogen-induced SOS-like responses.

The ability of the glucocorticoid receptor (GR) to induce gene expression in embryonic chicken retinal tissue increases dramatically during development, although the quantity of the receptor molecules does not change greatly with age. This study examines the possible involvement of c‐Jun in the developmental control of GR activity. Expression of c‐Jun in retinal tissue was high at early embryonic ages and declined during development. Elevation of c‐Jun expression in retina of mid‐developmental ages by treatment with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), or by introduction of a c‐Jun expression vector, caused a pronounced decline in the inducibility of the endogenous glutamine synthetase gene and the transiently transfected CAT constructs p delta G46TCO and pGS2.1CAT, that are controlled by a minimal consensus glucocorticoid response element (GRE) promoter and the glutamine synthetase promoter, respectively. The effect of c‐Jun was dose dependent and could be reversed by overexpression of GR. C‐Jun‐evoked repression of GR activity could be relieved by overexpression of Jun D. Overexpression of Jun D could also elevate the responsiveness of early embryonic retina to glucocorticoids and cause a 5‐fold increase in p delta G46TCO induction. The effect of Jun D could be reversed by overexpression of c‐Jun. Expression of c‐Jun might therefore be important for repression of GR activity at early embryonic ages.

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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.