Upregulation of survivin confers resistance to apoptosis in human fibroblasts immortalized by overexpression of the catalytic component of telomerase (hTERT) Free

1223

We have previously shown that ectopic expression of telomerase reverse transcriptase (hTERT) is not sufficient for immortalization of primary human fetal lung fibroblasts (MRC5). During immortalization, hTERT-transduced MRC5 cells (MRC5hTERT) underwent a transient growth crisis that was characterized by increased cell death. Immortalized MRC5hTERT cells had inactivated p16^INK4a and retained a functional p53/p21^cip1 DNA damage response pathway. Cells in crisis exhibited morphologic features of apoptotic cells and were positive in the TdT-mediated dUTP nick end-labeling (TUNEL) assay. We therefore hypothesized that the cells that escaped crisis and were eventually immortalized had activated anti-apoptotic mechanisms. Consistent with this hypothesis, post-crisis MRC5hTERTs were found to be more resistant to apoptosis induced by okadaic acid (OA) or actinomycin (ActD), as detected by Annexin V/propidium iodide staining and capase-3/7 activity assay. Furthermore, investigation of anti-apoptosis candidates revealed that survivin, a member of the inhibitor-of-apoptosis family that is broadly overexpressed in human cancers, was progressively up-regulated during immortalization of MRC5hTERT cells. Upregulation of survivin mRNA and protein was not accompanied by an increased rate of proliferation, but did correspond with increased resistance to apoptosis. In contrast, there was no survivin expression in the parental MRC5 cells at any stage of their replicative lifespan, nor at any phase of the cell cycle. Upregulation of survivin was also demonstrated in hTERT-immortalized JFCF6 and WI38 fibroblasts. In immortal MRC5hTERT cells, survivin was localized to the mitochondria and nucleus. Mitochondrial localization of survivin is consistent with a role in the intrinsic cell death pathway. Infection with replicative-defective adenoviral vector encoding dominant-negative survivin reversed resistance to OA and ActD in immortal MRC5hTERT cells. These studies demonstrate that upregulation of survivin confers resistance to apoptosis in telomerase-immortalized human fibroblasts.