Analysis of telomere length and function in radiosensitive mouse and human cells in response to DNA-PKcs inhibition

Abstract

BackgroundTelomeres, the physical ends of chromosomes, play an important role in preserving genomic integrity. This protection is supported by telomere binding proteins collectively known as the shelterin complex. The shelterin complex protects chromosome ends by suppressing DNA damage response and acting as a regulator of telomere length maintenance by telomerase, an enzyme that elongates telomeres. Telomere dysfunction manifests in different forms including chromosomal end-to-end fusion, telomere shortening and p53-dependent apoptosis and/or senescence. An important shelterin-associated protein with critical role in telomere protection in human and mouse cells is the catalytic subunit of DNA-protein kinase (DNA-PKcs). DNA-PKcs deficiency in mouse cells results in elevated levels of spontaneous telomeric fusion, a marker of telomere dysfunction, but does not cause telomere length shortening. Similarly, inhibition of DNA-PKcs with chemical inhibitor, IC86621, prevents chromosomal end protection through mechanism reminiscent of dominant-negative reduction in DNA-PKcs activity.ResultsWe demonstrate here that the IC86621 mediated inhibition of DNA-PKcs in two mouse lymphoma cell lines results not only in elevated frequencies of chromosome end-to-end fusions, but also accelerated telomere shortening in the presence of telomerase. Furthermore, we observed increased levels of spontaneous telomeric fusions in Artemis defective human primary fibroblasts in which DNA-PKcs was inhibited, but no significant changes in telomere length.ConclusionThese results confirm that DNA-PKcs plays an active role in chromosome end protection in mouse and human cells. Furthermore, it appears that DNA-PKcs is also involved in telomere length regulation, independently of telomerase activity, in mouse lymphoma cells but not in human cells.

DNA Damage Signalling and Repair Inhibitors: The Long-Sought-After Achilles’ Heel of Cancer

“…IC86621 inhibition of DNA-PKcs prevents chromosomal end protection through mechanism reminiscent of dominant-negative reduction in DNA-PKcs activity. [ 265 ].…”

Expression and localization of Artemis serine 516 phosphorylation in human scalp skin

“…Accepted for publication 10 August 2012 Background Previous studies have shown that Artemis, which belongs to SNM1 gene family (1)(2)(3), is involved in the repair of radiation-induced DNA double strand breaks as well as in the regulation of V(D)J recombination (4-7). Moreover, Artemis was shown to regulate genome stability (8)(9)(10)(11)(12)(13), telomeric region repair (14,15) and p53-dependent apoptosis (16,17). Artemis is phosphorylated at several serine residues (516,534,538,645) after ionizing radiation (IR) or ultraviolet (UV) exposure.…”

DNA–dependent protein kinase in telomere maintenance and protection

“…Whether DNA-PKcs overexpression contributes to telomere homeostasis during carcinogenesis requires further study. Nonetheless, a combination of anti-DNA-PKcs and anti-telomere strategies have proposed and might offer additional tools in combatting aggressive and radioresistant tumors [124][125][126][127]. Further investigation will help to determine the benefit of these combined modality approaches to cancer patients.…”