Mitochondrial hTERT exacerbates free‐radical‐mediated mtDNA damage

Abstract

SummaryTelomerase is often re-activated in human cancers and is widely used to immortalize cells in culture. In addition to the maintenance of telomeres, telomerase has been implicated in cell proliferation, genomic instability and apoptosis. Here we show that human telomerase reverse transcriptase (hTERT) is targeted to the mitochondria by an N-terminal leader sequence, and that mitochondrial extracts contain telomerase activity. In seven different human cell lines, mitochondrial telomerase increases hydrogen-peroxide-mediated mitochondrial DNA damage. hTERT expression did not alter the rate of hydrogen peroxide breakdown or endogenous cellular levels . Because the damaging effects of hydrogen peroxide are mediated by divalent metal ions (Fenton chemistry), we examined the levels of bioavailable metals. In all cases, higher levels of chelatable metals were found in hTERTexpressing cells. These results suggest that mitochondrial telomerase sensitizes cells to oxidative stress, which can lead to apoptotic cell death, and imply a novel function of telomerase in mitochondrial DNA transactions.

The p53/p21WAF/CIP Pathway Mediates Oxidative Stress and Senescence in Dyskeratosis Congenita Cells with Telomerase Insufficiency

“…Although our results do not clearly identify the source of increased steady-state levels of superoxide in the DC cells, our findings showing MitoSOX oxidation being altered, coupled with the differential effects seen with manipulations of O 2 tension, support the hypothesis that mitochondria may represent a significant source of superoxide in DC cells containing dysfunctional telomerase and telomeres. This is of interest because recent studies have implicated TERT in localizing to the mitochondria and modulating ROS (1,42,43). However, our studies showing that a TERT mutant without a mitochondria localization signal was still able to reduce steady-state levels of superoxide in DC cells do not support this model.…”

DNA Protein Kinase–Dependent G2 Checkpoint Revealed following Knockdown of Ataxia-Telangiectasia Mutated in Human Mammary Epithelial Cells

“…The immortalized human mammary epithelial cell line hTERT184 was derived from the normal human mammary epithelial 184 cell line (18,19) and was a kind gift from J. Carl Barrett (National Cancer Institute, Bethesda, MD). Cells were grown in defined MEGM media (Cambrex) supplemented with 5 Ag/mL transferrin (Sigma), 10 À5 mol/L isoproterenol (Sigma), and 250 Ag/mL geneticin (Invitrogen) in a humidified 2% CO 2 incubator.…”

Actions of human telomerase beyond telomeres

“…However, due to the lack of highly specific antibodies against hTERT and low expression of endogenous hTERT in cells, it has not been examined whether endogenous hTERT localizes in mitochondria under physiological condition. Further analysis shows that the mitochondrial telomerase sensitizes the damage effects of H 2 O 2 on mtDNA, presumably via modulation of metal homeostasis [28]. In addition, studies indicate that mitochondrial localization of telomerase functions is a determinant of hydrogen peroxide-induced mitochondrial DNA damage and apoptosis [29].…”

“…It has been shown that the catalytic subunit of telomerase hTERT contains a mitochondrial localization signal peptides at its N-terminal 20 amino acids which targets hTERT to mitochondria, resulting in active telomerase in this organelle [28]. These experiments were done by transiently over-expression of In all case tested, a mitochondrial localization of GFP-hTERT was observed by confocal microscopy and by the telomeric repeat amplification protocol (TRAP).…”