Escape from in vitro aging in SV40 large T antigen-transformed human diploid cells: a key event responsible for immortalization occurs during crisis.

A human diploid fibroblast strain MRC-5 was transfected with a replication origin-defective early region of SV40 containing the gene of large T antigen, and 48 clones of T antigen-transformed MRC-5 were isolated. Although T antigen prolonged the lifespan of MRC-5, all the transformed clones were still mortal. From two of the transformed MRC-5 clones, eight independent immortalized clones were obtained in triple experiments of immortalization. The transformed phenotypes of immortalized clones were widely varied and did not always retain those of the parental pre-immortalized clones. The immortalization occurred at the frequency of about 1-3 x 10(-7)/cell. From cell number and population doubling time of the immortalized clones, the immortalization was estimated to occur in or just before the crisis of parental mortal cells. The decreases of modal chromosome numbers and the loss of chromosomes 5 and 10 were found to be common in three independent immortalized clones examined. Thus, the escape from the cellular aging process seemed to be caused by certain genetic events including the loss of chromosomes at the end of lifespan in T antigen-transformed human diploid cells.