Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

Abstract

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori-transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SVWHF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre-and postimmortalization, should be useful in defining molecular changes associated with immortalization.

SEN6, a locus for SV40-mediated immortalization of human cells, maps to 6q26-27

“…Additional loci may also be a ected in SV40-immortalized cells as more extensive LOH on chromosome 6 is evident on prolonged passage, as shown for derivatives of SV/HF-5/39 in this study. Karyotypic analyses of SV/HF-5/39 have also been consistent with a mixed population of rearrangements involving 6q (Neufeld et al, 1987;Patsalis, unpublished data). Mutations involving multiple loci a ecting cell proliferation might be expected to be selected for by passage of immortal SV/HF under conventional culture conditions as re¯ected in increased e ciency of colony formation and shorter generation times (Neufeld et al, 1987), accumulation of chromosome rearrangements (Neufeld et al, 1987;Hubbard-Smith et al, 1992) and progressive lengthening of telomeric sequences (Small et al, 1996).…”

“…Introduction of the gene for SV40 large T antigen transiently overcomes senescence through its ability to interfere with the growth suppressors pRB and p53. However, such SV40-transformed (preimmortal) cells nonetheless die after a period of extended lifespan (Neufeld et al, 1987;Ray and Kraemer, 1992). Permanent reversal of senescence requires one or more cellular mutations as well as persistent expression of large T antigen (Radna et al, 1989;Wright et al, 1989).…”

“…Expression of large T antigen is, however, insu cient to ensure immortalization of human ®broblasts. Rather such SV40-transformed cells (SV/HF) have an extended lifespan and only a rare pre-immortal SV/HF becomes immortal (Neufeld et al, 1987;Shay and Wright, 1989). Sequences on the long arm of chromosome 6 have been speci®cally associated with immortalization of such SV/HF.…”

Integration of SV40 in human osteosarcoma DNA

“…The result of this is that integrated SV40 DNA is usually rearranged. By contrast, Origin defective SV40 DNA, used to transform cells, is usually integrated as a tandem repeat (Neufeld et al, 1987).…”

“…Persistent expression of T-antigen could contribute to the oncogenic phenotype by its e ect on tumor suppressor genes and other cellular genes (Fanning and Knippers, 1992). Constitutive expression of T antigen plays an important role in the transformation of cells in culture by SV40 (Neufeld et al, 1987). Integration of SV40 could both activate genes by providing a strong promotor, and inactivate endogenous genes by direct disruption.…”