Specific high frequency rearrangements induced by MNNG in SV4O-infected human keratinocytes

Abstract

In order to study carcinogen-induced changes in integrated viral sequences clonal sublines of SV40-transformed human keratinocytes were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at sublethal concentrations ranging up to 10 micrograms/ml for a period of 15 min and then examined by Southern blot hybridization using full-length SV40 DNA as a probe. Of the clonal sublines tested, one (AG34) was found to exhibit certain consistent, dose-dependent changes at 4 days post-treatment: (i) loss of at least two EcoRI fragments of approximately 4.4 and 3 kb, with the concomitant appearance of two bands migrating between 1.8 and 2.3 kb; and (ii) loss of a 1.5-kb fragment and the appearance of 2, 3.8 and 5 kb fragments in KpnI digests. Minor variable changes in restriction patterns were seen at 24 h post-treatment but consistent and pronounced effects were observed only at 4 days post-treatment. The altered restriction fragment patterns indicate that MNNG caused highly specific rearrangements in some subset of the DNA sequences associated with the integrated SV40 sequences in human epithelial cells. This differs from what has been found for SV40 sequences in other cell lines where amplification has been reported. These results suggest that (i) the site of SV40 integration may determine the response of integrated SV40 segments after carcinogen treatment, and (ii) carcinogen treatment can result in the induction of a common genetic event throughout the entire population of exposed cells. Genomic libraries created in a lambda phage vector have been used to isolate BamHI fragments containing SV40 sequences. Isolates have been found which exhibit EcoRI and KpnI restriction patterns consistent with the polymorphisms displayed in Southern blots.