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Characterizing canonical and non-canonical roles of telomerase reverse transcriptase in transformed human cells and cancer

University of British Columbia

Telomerase is the ribonucleoprotein reverse transcriptase that catalyzes the synthesis of TTAGGG nucleotide repeats at the ends of linear chromosomes, contributing to proper telomeric structure and cap formation. Most human somatic cells have low or undetectable telomerase expression. In contrast, telomerase overexpression is found in over 85% of human cancers allowing cancer cells to replicate indefinitely. Telomerase inhibition by GRN163L (Imetelstat) has previously been observed to potentiate genotoxic stress in a cell-cycle (S/G2) specific manner, through an unknown mechanism. We hypothesized that GRN163L treatment alters cell-cycle kinetics and that this effect depends upon active signaling through ataxia telangiectasia mutated (ATM). Here we tested the effects of combining GRN163L and the topoisomerase II inhibitor etoposide, together with pharmacological ATM inhibition on MCF-7 breast cancer cells, to assess dependence of telomerase’s cyto-protective function on this DNA-damage repair transducer. Additive increased cytotoxicity and cell-cycle profile alterations depended upon the order of treatment addition. Investigating possible causes of these cell-cycle distribution changes we observed that telomerase inhibition alone induces γH2AX DNA-damage foci in a subset of telomerase-positive cells but not telomerase-negative primary human fibroblasts. Additional FACS and immunocytochemistry experiments indicate that GRN163L-treated cells were reversibly stalled but not arrested at G2/M. Our results suggest that treatment with GRN163L sensitizes telomerase-positive cells to cell-cycle specific DNA-damaging agents through the engagement of an ATM-dependent DNA-damage signal, which may represent a separate mechanism by which telomerase inhibition could affect DNA repair homeostasis in telomerase-positive cancer cells. In addition to its telomere-maintenance function telomerase has recently been reported to participate in non-canonical activities such as protection from DNA-damaging agents, apoptosis, cellular proliferation rate, and resistance to oxidative stress. In a separate study, we hypothesized that overexpression of telomerase in transformed human cells would increase their survival following exposure to DNA-damaging agents. Our results indicate that telomerase expression protects cells from a variety of DNA-damaging drugs by improving the kinetics of DNA-repair. Telomerase expression also allows surviving cells to tolerate increased levels of chromosomal instability following drug exposure. This work has implications on improving the design of future telomerase inhibition strategies to also target non-canonical effects of this enzyme.

Text

2017-08-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/59034

Pharmaceutical Sciences

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/