Acquisition of anti-apoptotic potential in the process of telomerase-mediated immortalizaion of human fetal lung fibroblasts

5000 Immortalization process is an essential step in the multiple stages of carcinogenesis. Most cancer cells are immortal as a consequence of constitutive expression of telomerase, which prevents senescence and crisis (replicative barriers) by maintaining the length of specialized chromosomal-end structures, called telomeres. We have previously shown that ectopic expression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase enzyme activity, elongates telomeres and increases the lifespan of primary human fetal lung fibroblasts (MRC5s) 1, 2 . Extensive clonal studies demonstrated that hTERT-transduced MRC5s (MRC5hTERT) had an extended lifespan, but underwent a telomere-independent crisis prior to immortalization. Immortalized MRC5hTERT cells had inactivated p16INK4a and retained a functional P53/p21 cip1 pathway. However, p16INK4a inactivation did not correspond with the transition through crisis, suggesting that additional events may have been involved in immortalization. The objective of the present study was to investigate the potential involvement of anti-apoptotic mechanisms in MRC5hTERT immortalization. Low passage of MRC5 cells, at 50 population doublings (PDs) and a series of MRC5hTERT cells at pre-crisis (60 and 100 PDs) and post-crisis (200 and 300 PDs) time points were tested for sensitivity to a variety of apoptosis-inducing agents, including okadaic acid (OA), actinomycin D (ActD), TNFa, H 2 O 2 , Taxol, and Vincristine. OA was the most potent inducer of apoptosis in MRC5 cells. However, Annexin V/propidium iodide staining and TUNEL assay demonstrated that all MRC5hTERT cells were significantly more resistant to OA than non-transduced MRC5 cells (p Exp Cell Res 259(2): 336-50.2. Taylor, L. M., et al (2004). J Biol Chem 279(42): 43634-45.