Metabolismo telomerico in risposta al danno al DNA indotto da radiazioni ionizzanti di diversa qualità

Abstract

Ionizing radiations are a well known genotoxic agents, widely studied for the great impact of their applications (i.e., radiotherapy and hadrontherapy) and effects (i.e., exposure risk for astronauts in space missions). Exposure to ionising radiations (IR) can result in the deposition of energy to DNA molecules, thus leading to DNA damage. IR-induced DNA damage is localized, and the level of localization is believed to increase with increasing linear energy transfer (LET) values of the radiation. Because LET is a measure of the energy released to an object along the path of the radiation, high-LET radiation can deposit more energy than low-LET one. Condensed or concentrated energy deposition results in cluster of ionization events. When the target is the DNA, the site of such lesions is termed "clustered DNA damage" or "locally multiply damaged site", which consists in two or more lesions localized in close proximity on the DNA duplex. In order to study the biological effects of high-LET radiations, several endpoints have been evaluated both in rodent- and in human-irradiated cells, including chromosomal aberrations, micronuclei (MN), chromosomal non-disjunction, mutations, DNA fragmentation, clonogenic survival, and cell cycle effects. However, aspects related to telomere length modulation and telomere metabolism have been so far poorly investigated both in primary and in immortalized cells exposed to low- and high-LET radiations. The aim of the first part of the study was to analyze the DNA-damage and the genotoxic effects induced by graded doses (0, 25-2 Gy) of low-energy protons (high-LET radiation), and X-rays (low- LET radiation) in human primary fibroblasts. DSB induction and repair as mesured by scoring for -H2AX foci indicated that 3MeV protons, with respect to X-rays, yielded a lower number of DSBs per Gy, which showed a slower kinetics of disappearance in the first hours from irradiations. Furthermore, irrespective of dose delivered, a higher fraction of unrejoined DSBs persisted in sample harvested 24 hours from exposure to protons. The higher clastogenic effect of protons was in agreement with the extent of micronuclei (MN) induction in binucleated cells up to 1, 5 Gy. Our results support the notion that DNA DNA damage produced by 28.5 keV/µm protons appears less amenable to be repaired and could be transformed in cytogenetic damage in the form of MN in the first cell cycle from irradiation . After confirming the greater biological effectiveness of high-LET radiations compared to low-LET ones, we focused our attention on studying telomere metabolism within 24 hours from the exposure to both types of radiations. Interestingly, data obtained showed a different kinetics of telomere length modulation in cells exposed to low- or high-LET radiations. Moreover, the phenomenon observed appeared to be conserved both in primary and in immortalized cell lines. Interestingly, exposure of human primary fibroblasts to 4Gy high-LET radiation determined a telomere elongation respect to untreated cells, whereas no telomere length modulation was observed in low- LET treated fibroblasts. In order to investigate the molecular mechanism underlying the observed elongation, the expression levels of the telomerase (i.e., hTERT) and its enzymatic activity were evaluated. Results obtained excluded the involvement of the telomerase in the observed telomere lengthening induced by high-LET radiation, thus supporting the activation of a telomerase- independent mechanism. Some mammalian cells lacking in any telomerase activity are able to maintain the length of their telomeres for many population doublings (PDs). This indicated the existence of one or more non-telomerase mechanism(s) for telomere maintenance, further termed Alternative Lengthening of Telomeres (ALT). To date, clear evidences of the existence of an ALT activity has been demonstrated only in human tumours and immortalized cell lines, and in telomerase-null mouse cell lines. To analyze whether a recombinational mechanism could be responsible for the high-LET-induced telomere lengthening observed in human primary fibroblasts, two types of experiments were performed. On one side, the incidence of recombinational events at telomeres (T-SCE) was measured, and on the other side the colocalization of telomeres and PML bodies (that are considered as an hallmark of cells with activated ALT pathway), was analyzed. Strikingly, our results indicated that the DNA damage induced by high-LET radiation is somehow able to induce telomere lengthening through the transient activation of an ALT recombinational pathway. Recent reports demonstrated that NBS1 is essential for the correct functioning of the ALT pathway. NBS1 gene, mutated in the NBS human chromosome instability disorder, encodes for the NBS1 protein, a central player in the response to the ionizing radiation-induced DNA damage, as well as in the homologous recombination repair. In order to confirm the high-LET-induced recombinational ALT pathway, telomere length was evaluated in Lymphoblastoid Cell Lines (LCLs) heterozygous (NBS1+/- ) and homozygous (NBS1-/- ) for a mutation of the NBS1 gene, as well as in normal cells (NBS1+/+ ) exposed to 4 Gy of carbon ions (39keV/m). Remarkably, a telomere elongation was observed in NBS1+/+ and NBS1+/- cells, but not in NBS1-/- ones. These data evidenced that the process of telomere lengthening induced by high-LET radiation is NBS1- dependent, thus supporting the hypothesis that telomere elongation is mediated by recombinational mechanisms. Beside the analysis performed at 24 hours, telomere length modulation was followed up to 15 days from the irradiation of both human primary fibroblasts and LCLs. Dynamics of telomere lengths modulation appeared to be different after low- and high-LET irradiation. Our data showed that the telomere lengthening observed in high-LET-treated cells seems to be maintained at 3-4 days, as well as 15 days after exposure. Interestingly, the time-course of the low-LET radiation-induced telomere length modulation appeared to be more complex than the high-LET one. In fact, after 3-4 days telomere erosion was reported, whereas after 15 days from the exposure a telomere lengthening was observed in primary as well as in immortalized cell lines. To explain the time course of low-LET-induced telomere length modulation we have hypothesized that a direct correlation between telomere length and radioresistance/radiosensitivity could account for this phenomenon. To test our hypothesis, we decided to perform experiments in TK6 lymphoblast cells, since they represent a good and widely used radiobiological cellular model. Data obtained brought us to suggest a model: the radioresistance of cells with longer telomeres drives a selection process that led to an increased telomere length in clones survived to low-LET radiation exposure. A direct correlation between telomere length and radisensitivity/radiresistance has already been proposed in some published reports and imply that telomeres length measurement could be potentially used as a tool to predict clinical radiation response in radiotherapy.