Systematic variables affecting simian virus 40-induced T-antigen expression and transformation in human cells

Abstract

Simian virus 40 (SV40) infection of human skin fibroblast and human tumor cells resulted in the expression of T-antigen and transformed foci. By examining various conditions of input virus multiplicity and initial cell density, the systematic variation of T-antigen determination was minimized. The most uniform results were obtained at multiplicities of about 275 plaque-forming units/cell. Within limits (5 X 10(4) to 2 X 10(5) cells/dish), initial cell density had little effect on T-antigen expression. Volume of virus inoculum was critical for some cell lines, but not for others. Cell passage level had no general effect on T-antigen expression, although specific cell lines demonstrated increased or decreased levels of T-antigen expression with serial passage for no apparent reason. T-antigen expression correlated with virus-induced cell transformation (focus formation) at two different multiplicities. In addition, T-antigen assays at 3 days gave consistently more reproducible results than transformation assays at 21 days in seven cell lines tested at two multiplicities of infection. These results defined input multiplicity as the major source of systematic variability and will permit development of a more reproducible tool in the evaluation of individuals at high risk of cancer.