Repression of telomerase components in isogenic normal, immortal and tumorigenic cells

The telomere maintenance enzyme telomerase is a ribonuclear protein complex that is active in 80-90% of immortal cancer cells. Three essential components of the active telomerase holoenzyme include the reverse transcriptase, hTERT, an RNA component (hTR) that has a template region for reverse transcription and the RNA binding and modifying protein, dyskerin. There is evidence indicating that these components may contribute to the survival and/or proliferation of tumour cells via mechanisms that are independent of their roles within the telomerase holoenzyme. The aim of these investigations was to compare the effects of abolishing these individual components of telomerase to determine the most effective and least toxic approach to targeting telomerase.The functional and molecular consequences of repressing the telomerase components hTERT, hTR and dyskerin were compared in isogenic normal, immortal (pre-malignant) and tumorigenic human foetal lung (MRC5) myofibroblasts, as well as a fibrosarcoma derived cell line, Ht1080. siRNA-mediated inhibition of dyskerin or hTERT had a potent anti-proliferative effect on immortal and tumorigenic cells, but had no overt effect on the proliferation of isogenic normal cells. Suppression of dyskerin or hTERT also impaired anchorage-independent growth of tumorigenic cells. Treatment with hTR siRNA or a small molecule weight telomerase enzyme inhibitor had no significant effect on any of the cell lines in these short-term assays. Together, these results indicate immortal cells depend on hTERT or dyskerin for their proliferation, independent of their role of the telomerase holoenzyme. To identify pathways associated with the specific and potent effects of targeting dyskerin and hTERT, microarray gene expression analysis and Gene Set Enrichment Analyses (GSEA) of siRNA transfected normal, immortal and tumorigenic cells was performed. Inhibition of hTERT resulted in the activation of the DNA damage response, which was confirmed by immunofluorescence showing the activation of γH2AX, while the Rb/E2F pathway was altered upon dyskerin repression. These results underscore the therapeutic potential of targeting these telomerase components. Data from the microarray and bioinformatics analyses provide insight to downstream effector pathways that may be exploited in the development of new molecular-targeted therapeutics that halt the replication of immortal cancer cells