Human fibroblasts transformed by the early region of SV40 DNA: Analysis of “free” viral DNA sequences

Abstract

Human fibroblastic cells (HF) were transformed with the early region of the simian virus 40 genome (0.15 – 0.73 map units) by using the DNA-calcium phosphate coprecipitation technique of F. L. Graham and A. J. Van der Eb (1973, Virology 52, 456–467). Transformation resulted in altered morphology and ability to grow in agarose. The SV40-transformed human fibroblasts (SVHF-A) have a limited life span and reach “senescence” after 10–11 passages. Analysis of the low molecular weight DNA extracted from SVHF-A cells shows a relatively high amount of free viral DNA sequences in circular supercoiled form. These circular molecules are very heterogeneous in size and contain sequences corresponding to the early region of the SV40 genome. Part of them may contain cellular DNA sequences as well. In situ hybridization experiments indicate that a minority of the SVHF-A cells (2–3%) are spontaneously induced to synthesize free viral DNA molecules and their frequency is increased by mitomycin C treatment. Immunofluorescence staining for SV40 T antigens also indicates that the cells producing free viral DNA contain higher T-antigen levels than the rest of the population. Our data suggest that the “free” viral DNA molecules derive from integrated viral sequences following replication in a minority of the cells rather than originating from a persistent extrachromosomal replication in every cell.