Inhibition of p53 and ATRX increases telomeric recombination in primary fibroblasts

Abstract

Telomere length can be maintained either by the telomerase enzyme or by alternative lengthening of telomeres (ALT), which is based on telomeric recombination. However, both mechanisms are inactive in most human somatic cells. ATRX has been previously identified as an ALT repressor gene. Nonetheless, TP53 is also deficient in most ALT cell lines, and previous works showed that it is an inhibitor of homologous recombination (HR). Despite this, the role of p53 as an ALT repressor has not been previously examined. Therefore, we investigated the effects of p53 and ATRX inhibition on normal human fibroblasts (devoid of any mutation), in the presence or absence of X‐ray‐induced telomeric damage. Performing immunofluorescence with antibodies for RAD51, H2AX, and TRF1 (for studying HR‐mediated DNA damage repair) and CO‐FISH (for telomeric sister chromatid exchanges), we observed that HR is a normal mechanism for the repair of telomeric damage, present also in noncancer cells. Moreover, we discovered that telomeric HR, as for HR in general, is significantly inhibited by p53. Indeed, we observed that inhibition of p53 drastically increases telomeric sister chromatid exchanges. We also confirmed that ATRX inhibition increases telomeric recombination. In particular, we observed an increase in crossover products, but a much higher increase in noncrossover products.

Effects of p53 and ATRX inhibition on telomeric recombination in aging fibroblasts

“…This confirms earlier works showing that p53 inhibition increases HR ( 21 ) and in our case, we showed that it increases telomeric HR. The same was observed in the first part of our work ( 35 ), in which we observed that p53 inhibition increased T-SCE frequencies in X-ray-treated young fibroblasts (which also have telomeric damage). Chronic p53 inhibition also produced a small but significant increase of telomere length ( Figure 7 ) or, more correctly, a decrease of telomere shortening; it could be hypothesized that this may be ascribable to the increased telomeric recombination, thus reminiscent of ALT activity.…”

“…In the case of siATRX treatment, transfections were performed during seeding and 24 hours later (see above). In the case of pft treatment, the compound at a concentration of 10 µM ( 34 ; 35 ) was added 24 hours after seeding. In the case of combined treatment, pft was added immediately after the second transfection.…”