Activities of SV40 T antigen necessary for the induction of tetraploid DNA content in permissive CV‐1 cells

To determine the role of SV40 T antigen in stimulating multiple rounds of DNA synthesis in permissive cells, CV‐1 cells were transfected with plasmids expressing mutant or wt T antigen in the presence or absence of the SV40 origin of replication. Induction of cells with >G2 DNA content (tetraploid DNA content) and levels of T antigen protein were detected and analyzed by flow cytometry. The mutant T antigen proteins demonstrated the expected phenotypes as determined by immunoprecipitation. Elevated levels of T antigen protein were detected in each transfection, but full‐length T antigen alone was responsible for the tetraploid DNA content. The studies show that full‐length T antigen with point mutations to reduce binding to the cellular proteins p53 and/or Rb were capable of inducing >G2 DNA content though the induction by these mutants was greatly enhanced by the presence of the SV40 origin of replication. Truncated T antigen (aa 1–259) could induce cells with tetraploid DNA content only in the presence of the SV40 origin of replication and the absence of Rb binidng. These studies suggest that multiple functions of T antigen are involved in the stimulation of the second round of cellular DNA synthesis. Cytometry 31:251–259, 1998. © 1998 Wiley‐Liss, Inc.