Mutant telomerase RNAs induce DNA damage and apoptosis via the TRF2-ATM pathway in telomerase-overexpressing primary fibroblasts

Mutant template human telomerase RNAs (MT-hTers) have been shown to induce apoptosis in various cancer cells with high telomerase activity. However, the mechanism by which MT-hTers inhibit growth of cancer cells and their effects on normal cells remain unknown. To determine the effects of MT-hTers on normal cells, MT-hTer-47A and -AU5 were introduced into IMR90 lung fibroblasts that have low telomerase levels. Growth of IMR90 cells following MT-hTers infection was not significantly impaired; however, similar treatments in telomerase-overexpressing IMR90 (IMR90 WThTERT) cells inhibited cell proliferation and induced apoptosis. Confocal microscopy showed that MT-hTers induced DNA damage foci i.e. 53BP1 and γ-H2AX in IMR90 WThTERT cells. Microarray analysis revealed that GADD45γ was significantly elevated in MT-hTers treated IMR90 WThTERT cells. MT-hTers also induced ATM phosphorylation at Ser 1981 in IMR90 WThTERT cells, and Western blot analysis revealed high levels of phosphorylated p53 following down-regulation of cellular TRF2 expression in MT-hTers treated IMR90 WThTERT cells. Taken together, we have elucidated that MT-hTers induce double-stranded DNA breaks (DSBs)-like damages in telomerase-positive IMR90 WThTERT cells following phosphorylation of ATM and p53 via suppression of TRF2 which eventually may have led to apoptosis via elevation of GADD45γ. MT-hTer-47A and -AU5 were introduced into IMR90 primary lung fibroblasts and immortalized IMR90 WT-hTERT cells. Through microarray analysis, gene expression of MT-hTer-infected IMR90 WT-hTERT cells were compared against MT-hTer-infected IMR90 control cells.