ALTERNATIVE LENGTHENING OF TELOMERE (ALT) IMPLICATED IN TELOMERE LENGTH MODULATION INDUCED BY X-RAYS IN HUMAN PRIMARY FIBROBLASTS

Abstract

Telomeres are nucleoprotein structures localized at the end of chromosomes, organized in heterochromatic domains and involved in genome and chromosome stability. Human telomeres consist of 2-30 Kbp of repeated TTAGGG sequences and telomeric proteins. At every round of replication, telomeres become shorter and when they reach a threshold length, the cell goes through senescence and apoptosis, preventing DNA repair mechanisms activation at telomeres (which could induce chromosome and genome instability) and thus avoiding cellular transformation. Sometimes cells could escape from senescence and apoptosis activating some mechanisms that can elongate telomeres again, counteracting the natural and physiological shortening. These mechanisms are called telomeres maintenance mechanisms (TMMs). Two TMMs are mainly known: telomerase and alternative lengthening of telomere (ALT). Telomerase is the most common TMM used by cells to elongate and maintain telomeres and it is well known from a molecular point of view. It consists of a ribonucleoproteic complex with an enzymatic activity that uses an RNA template to add telomeric TTAGGG sequences at the 3’-overhang ends of chromosomes. Telomerase is activated in germinal cells, stem cells and in the 80- 85% of tumors. The alternative lengthening of telomere (ALT) pathway has been found in the 15-20% of the remaining tumors and molecular factors and causes of its activation are still not well known. Based on homologous recombination, this mechanism is able to add telomeric sequences at chromosome ends by interaction of telomeres, which belong to sister chromatids or different chromosomes. The most important target of ionizing radiations (IRs) in eukaryotic cells is the DNA. In fact, IRs can interact and induce DNA damage by a direct or an indirect effect, depending on the ionizing radiation typology. X-rays can interact with DNA by an indirect way, giving rise to water radiolysis and generating reactive oxygen species (ROSs) that are able 9 to induce DNA damage. It has been demonstrated that oxidative stress can cause telomere shortening. In fact, 8-oxoguanine (8-oxoG) is the most common oxidative DNA damage in human cells and telomere shows high presence of guanine residues and low DNA repair efficiency. In this way, telomere is able to accumulate oxidized bases, which can induce telomeric breaks. Previous studies demonstrated that particle ionizing radiations (like protons) are able to induce telomere lengthening by activation of an ALT-like mechanism, while literature data about X-rays indicate just the capability of this kind of IR to induce telomere length modulation without deepen the mechanisms behind such modulation. To better understand the cause and the kinetics of this modulation human primary fetal foreskin fibroblasts (HFFF2) were treated with 4 Gy dose of X-rays and it was performed an analysis for telomere length by Q-FISH at 3,4,5,6,7,8,10 and 13 days after treatment. Results showed a telomere length modulation with a trend of shortening and elongation during this time period. A TIFs (telomere dysfunctional induced foci) analysis performed by colocalization between telomere, yH2AX and 53BP1 proteins (as a telomere damage index), showed a close correlation between telomere shortening and telomere damage. We first supposed that the main cause for this telomere modulation could be telomerase activation after X-rays treatment. But an analysis performed by RTQ-TRAP assay did not show any activity of this enzyme. This was expected, according to many literature data. In fact, it has been fully demonstrated that cells with mesenchymal embryonic origin (like fibroblasts) do not activate telomerase as TMM, probably because genes involved in telomerase activation and regulation are tightly silenced during cellular differentiation. The possibility of ALT activation was then investigated, analyzing some proteins involved and two different hallmarks of ALT: telomere sister chromatid exchanges (T-SCEs) by CO-FISH and the ALT associated PML bodies (APBs) by immunoFISH. APBs and T-SCEs showed a correlation with telomere elongation. Also the analysis of ALT related proteins such as ATRX, RAD51 and RPA2 by western blot showed a pattern similar to the ALT-positive cell lines. These results proved that X-rays treatment is able to activate an ALT pathway similar to the one found in ALT-positive tumors. Finally, the way by X-rays are able to induce telomere length 10 modulation and ALT activation was investigated. Thus, a dichlorofluorescein-diacetate (DCFH-DA) analysis for ROS put in evidence a persistent oxidative stress during the first 4 days after X-rays treatment, that was the same time period in which telomere shortening and the highest telomere damage was detected. On the contrary, administration to HFFF2 of N-acetyl-cysteine (NAC), an antioxidant molecule, 30 minutes prior and every 24 hours after X-rays treatment, did not show any oxidative stress and any modulation of telomere length observed by Q-FISH. All these results allow to conclude that X-rays are able to induce a persistent oxidative stress, which can induce DNA damage, in a particular way at telomeres. Thus, cells characterized by telomeric damage can activate the ALT pathway in order to elongate telomeres again. Finally, ALT activation inside primary cells has to be considered an interesting result. Since ALT has been found in tumor cells, this PhD project describes (together with other few and recent works in literature) a new role for this pathway. In fact, if in tumors ALT is involved in telomere maintenance as well as in cellular immortalization and cancer development (probably because it is completely deregulated); inside primary cell lines, ALT could act as a strictly regulated pathway involved in damage-induced telomere repair.