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

Abstract

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.

Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification.

“…C060 cells were treated with MNNG 24 h after being seeded as previously described (4). Log-phase cells were plated at a density of 5 X 106/14-cm-diameter plate.…”

“…It has been previously demonstrated 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 (22,23,31,50,59), while extracts from Chinese hamster and other nonpermissive cell lines failed to support significant viral DNA replication (32 (4). Using the in vitro system, we have shown that partial replication of the SV40-containing plasmid is catalyzed by the carcinogentreated C060 cytosolic extracts, while HeLa extracts yielded full replication (4). The pattern of viral replication in the carcinogen-treated C060 extracts was unique, allowing restricted overreplication of the origin region (i.e., amplification), whereas distal sequences were not replicated significantly.…”

“…To identify these factors and to elucidate the structure of the initial amplification products, we have constructed an in vitro system for SV40 DNA amplification based on the in vitro SV40 replication system developed by Li and Kelly (31). It has been previously demonstrated 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 (22,23,31,50,59), while extracts from Chinese hamster and other nonpermissive cell lines failed to support significant viral DNA replication (32 (4). Using the in vitro system, we have shown that partial replication of the SV40-containing plasmid is catalyzed by the carcinogentreated C060 cytosolic extracts, while HeLa extracts yielded full replication (4).…”

Binding of a sequence-specific single-stranded DNA-binding factor to the simian virus 40 core origin inverted repeat domain is cell cycle regulated.

“…1A) in response to cell exposure to UV radiation and correlated this increase with DNA damage-induced viral DNA replication (28). Furthermore, cytosolic extracts from MNNG-treated SV40-transformed Chinese hamster cells supported SV40 DNA replication in vitro in the presence of exogeneous T antigen, whereas replication by extracts from untreated cells was inefficient (4). Treatment of irradiated cells with duplex oligonucleotides corresponding to the SV40 IR domain inhibited DNA amplification, presumably because the duplex oligonucleotides sequestered a DNA damage-activated factor needed for amplification in vivo (28).…”

Small polydispersed circular DNA (spcDNA) in human cells: association with genomic instability

“…CO60 cells were treated with MNNG 24 h after being seeded as was previously described (Berko-Flint et al, 1990). Log phase cells were plated at a density of 5610 6 / 14 cm-diameter plate.…”