SV40-TRANSFORMED HUMAN-CELLS IN CRISIS EXHIBIT CHANGES THAT OCCUR IN NORMAL CELLULAR SENESCENCE

SV40 T antigen can induce senescent human diploid fibroblasts to synth esize DNA; however, the cells fail to go through mitosis. In this stud y, we examined the expression of the cdc2 and cyclin B genes, which ar e required for completion of mitosis, to determine whether defects in their expression occurred when SV40-transformed human cells entered th e phase of crisis. If defects were observed it would indicate that imm ortalization by the virus involved reexpression of these genes. We fou nd that the expression of cdc2 was unimpaired at both the RNA and prot ein levels, but that cyclin B expression was decreased in cells in cri sis when compared with precrisis (mortal) and postcrisis (immortal) ce lls. Tritiated thymidine uptake demonstrated that the majority of cell s in crisis were not actively cycling. Consistent with the latter obse rvation we found that cyclin A, which is required for cells to travers e through S to G(2), was downregulated in these cells. Since many of t he results obtained with cells in crisis were similar to what is obser ved in normal human cells when they become senescent, we analyzed the expression of the genes fibronectin and sdi1 (a gene recently cloned f rom senescent cells that codes for an inhibitor of DNA synthesis). Bot h genes were overexpressed in cells during crisis, as is the case with senescent cells. The results are discussed in terms of the two-stage model previously proposed to explain the process of immortalization of human diploid fibroblasts by SV40. (C) 1994 Academic Press, Inc.