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

Abstract

Background: Telomeres, 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.

Results: We 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.

Conclusion: These 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.

Figure 1

Examples of chromosomal aberrations in LY-R and LY-S mouse cells. A| No evidence of chromosomal aberrations in untreated mouse LY-R cells. Telomeric signals are in red with DNA counter stained in blue with DAPI. B| Elevated levels of Robertsonian fusion (white arrow) and end-to-end telomeric fusions in DNA-PKcs inhibited mouse LY-S. C| Similar observation in DNA-PKcs inhibited treated mouse LY-R. Note the difference in telomeric signal strength between LY-S (weaker signal) and LY-R (stronger) suggesting differences in telomeric length of the two mouse cell lines.

Figure 2

Flow-FISH analysis of telomere length of mouse cell lines following inhibition of DNA-PKcs activity. A reduction in TFI unit where observed when both mouse cell lines were treated with DNA-PKcs inhibitor. The difference between the percentage reduction between the treated and untreated were statistically significant (p < 0.0001). TFI unit were measured from at least 1000 cells in G0/G1 phase of cell cycle in each experiment from a total of seven independent experiments. The error bars represent s.d.

Figure 3

Telo-FISH result following DNA-PKcs inhibition in human Artemis defective primary cell lines. Two Artemis defective (CJ179, F01/240) and normal (GM08399) human primary cell lines were subjected to 200μM of DNA-PKcs inhibitor, IC86621, for twenty-four hour period. Total levels of telomeric fusions, including chromosome type, chromatid type, sister chromatic unions and chromosome ring fusions were scored in three independent experiments. Panel (A) is DMSO treated and panel (B) is DNA-PKcs inhibited. Levels of DNA-DSB chromatid fragments were also scored as breaks/fragments. Error bars indicate standard error of mean (SEM).

Figure 4

Flow-FISH analysis of telomere lengths in human primary fibroblasts. TFI units measured from at least four independent experiments (two for F01-240) with DMSO served as untreated control and IC86621 as inhibitor of DNA-PKcs. Error bars represent s.d. The difference in telomere length was not statistically significant.