Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation

During the process of immortalization, at least two mortality checkpoints, M1 and M2, must be bypassed. Cells that have bypassed M1 (senescence) have an extended life span, but are not necessarily immortal. Recent studies hav e shown that ectopic expression of the catalytic subunit of telomerase (hTE RT) enables normal human cells to bypass senescence (M1) and oncogene trans formed cells to avert crisis (M2) and become immortal. However, it is uncle ar whether hTERT expression is sufficient for normal human fibroblasts to o vercome both M1 and M2 and become immortal. We have investigated the role o f telomerase in immortalization by maintaining mass cultures of hTERT-trans duced primary human fetal lung fibroblasts (MRC-5 cells) for very long peri ods of time (more than 2 years). In the present studies, up to 70% of MRC-5 cells were transduced with retroviral vectors that express hTERT. hTERT-tr ansduced cells exhibited high levels of telomerase activity, elongation of telomeres, and proliferation beyond senescence. However, after proliferatin g for more than 36 population doublings (PDLs) beyond senescence, the overa ll growth rate of hTERT-expressing cells declined. During theses periods of reduced growth, hTERT-transduced MRC-5 cells exhibited features typical of cells in crisis, including an increased rate of cell death and polyploidy. In some instances, very late passage cells acquired a senescence-like phen otype characterized by arrest in the G1 phase of the cell cycle and greatly reduced DNA synthesis. At the onset of crisis, hTERT-transduced cells expr essed high levels of telomerase and had very long telomeres, ranging up to 30 kb. Not all cells succumbed to crisis and, consequently, some cultures h ave proliferated beyond 240 PDLs, while another culture appears to be perma nently arrested at 160 PDLs. Late passage MRC-5 cells, including postcrisis cells, displayed no signs of malignant transformation. Our results are con sistent with the model in which telomerase and telomere elongation greatly extends cellular life span without inducing malignant changes. However, the se investigations also indicate that hTERT-expressing cells may undergo cri sis following an extended life span and that immortality is not the univers al outcome of hTERT expression in normal diploid fibroblasts. (C) 2000 Acad emic Press.