Termination of lifespan of SV40-transformed human fibroblasts in crisis is due to apoptosis

Normal human fibroblasts in culture have a limited lifespan, ending in replicative senescence. Introduction of SV40 sequences encoding large T antigen and small t antigen into pre-senescent cells results in an extension of lifespan for an additional 20-30 population doublings. Rare clones of SV40-transformed cells are capable of indefinite growth and are described as immortal; however, the great majority of SV40-transformed cells terminate this extended lifespan in cell death, termed "crisis." We have examined the properties of cells in crisis to obtain further insights into mechanism of cell death and immortalization. Populations at the terminal cell passage show a balance between cell replication and cell death over a period of several weeks, with a progressive increase in cells undergoing cell death. During this period, there is less than a 3-fold increase in attached cell number, with two stages being identifiable on the basis of the focal pattern of cell survival. We also demonstrate that cells in crisis are undergoing apoptosis based on TUNEL assay, subG1 DNA content, annexin V reactivity, and activation of caspases 3 and 8. We suggest a model whereby SV40-transformed cells acquire increased sensitivity to apoptosis based on changes in properties which activate caspase 8 in addition to changes previously described involving shortening of telomeric sequences. While only telomere stabilization could be clearly shown to be essential for survival of cells through crisis, the extended period of cell replication and altered gene expression observed in SV40-transformed cells during crisis are compatible with other genetic alterations in immortal cells.