DNA damage and genetic polymorphisms : influence on individual radiosensitivity

Abstract

Polymorphisms represent the main source of genetic inter-individual variability. The presence of polymorphic alleles in DNA repair genes may alter repair capacity and thus modify the biological responses to exogenous and endogenous DNA insults, both at the cellular and tissue level. In addition to impaired DNA repair capacity and increased mutagenesis, polymorphisms in DNA repair genes may also result in a modified risk of developing cancer. Radio-induced DNA damage and its repair also play a critical role in the susceptibility of patients to develop side effects after radiotherapy (RT). Therefore, the development of in vitro cellular radiosensitivity tests and genetic markers, that can be used as biomarkers for the extent of patients’ normal tissue reactions, is of great interest. Such markers could be used to adjust RT protocols for both radio-sensitive and radio-resistant patients. The aim of this PhD project was to analyse the relationship between induced DNA damage, the DNA damage responses and the individual’s genetic background. In particular the influence of variant alleles in damage signalling (RAD51) and repair (XRCC1, OGG1 and XRCC3) genes on individual susceptibility to developing cancer and on sensitivity to IRexposure, were assessed analysing both in vivo/ex vivo and in vitro systems. Ex vivo studies were focused on breast cancer patients, enrolled in Italian and French Oncology Units and in order to investigate the cellular response to IR exposure and to find a possible explanation for differences in radiosensitivity, we conducted in vitro assays on lymphoblastoid cell lines (LCLs) established from BC subjects, peripheral blood mononuclear cells (PBMCs) isolated from healthy donors and the hamster (CHO) cell lines AA8 and EM9, that represent a model to study the functional role of the XRCC1 gene. In the research part on BC patients, the Comet assay revealed that the cases exhibited a higher level of basal and X-ray (2Gy) induced-DNA damage than healthy controls. Moreover, in patients showing no adverse reactions (G0) the DNA damage significantly decreased from 30 to 60 min of repair times, unlike BC subjects showing acute skin reactions (G1-G3). With respect to the polymorphisms in the XRCC1 gene, XRCC1-399 (rs25487) was significantly associated with an increased risk of developing sporadic breast cancer. The 399-Gln may act as a dominant allele and when combined with the wild type allele at codon 194 and the variant allele at the position -77, was associated with a significantly higher BC risk. On the contrary, XRCC1-77, XRCC1-194, OGG1-326, XRCC3-241, RAD51-01, RAD51-52 as individual SNPs did not show any association with BC risk. However, carrying combination of SNPs in several genes, involved in different repair mechanisms, increased the risk of developing breast cancer. We found a significantly higher BC risk for subjects with ≥3 variant alleles compared to those with <3 variants, suggesting a joint or additive effects of genetic variants in multiple repair pathways. Using LCLs we demonstrated that RAD51 mRNA and the microRNA (miR) 34a* were expressed constitutively and that after IR exposure (5Gy of γ- rays) they appeared induced at 2h and 4h respectively, but this induction was independent of the RAD51-52 (rs11855560) genotype. Furthermore, by 4h to 8h post-irradiation a decrease in RAD51 mRNA expression was noted in all the LCLs. Differences in the constitutive levels of RAD51 protein levels were found in the four LCLs examined that also appeared to be independent of the RAD51-52 genotype, however p53 protein levels were similar. Following IR treatment, as expected p53 levels increased reaching a maximum at 4h post-treatment, however no marked differences in RAD51 protein levels were observed. Using the two hamster cell lines, AA8 and EM9, we investigated the impact of irradiation on XRCC1 levels. Immediately after exposure to 1.25, 2.5 and 5Gy no significant change in XRCC1 mRNA levels was found indicating that these doses of X-rays did not cause a direct damage to RNA molecules. In contrast, western blotting analysis conducted on protein extracts from AA8 cells revealed that XRCC1 protein levels seemed to be unchanged immediately after irradiation with 1.25 and 2.5Gy but reduced immediately after 5Gy treatment. In all extracts from EM9 cells the XRCC1 protein was completely absent confirming its status as a null mutant line. In EM9 cells, which are capable of expressing XRCC1 mRNA, the XRCC1 protein is absent as result of a C T substitution at nucleotide 661 that introduces a termination codon thus producing a truncated polypeptide lacking two thirds of the normal sequence. However, by comparing the XRCC1 mRNA levels in AA8 and EM9 cells, the null mutant EM9 displayed significantly lower levels of XRCC1 transcript than the wild type AA8, both before and immediately after treatments. It is likely that the lack of functional XRCC1 protein influenced XRCC1 gene expression or that the small amount of XRCC1 transcript was a consequence of a nonsense-mediated mRNA decay in EM9 cells. Using synchronized cell lines we examined XRCC1 mRNA levels in different cell cycle phases; in untreated AA8 cells, we observed significantly higher levels of XRCC1 transcript in S phase compared to G0 and G1 and significantly reduced levels in G1 phase when compared to S and G0 phases. The EM9 cells also showed a significant decrease of XRCC1 mRNA levels in G1 as regards G0. In contrast, the EM9 cells did not show an increase of XRCC1 mRNA during the replicative phase and instead they showed a decrease when compared to G0 cells. The treatment of cells with IR (2Gy of X-rays) did not influence XRCC1 mRNA levels in the different cell cycle phases either in AA8 or EM9 cells, except for a significant decrease in S phase in AA8 cells. In quiescent PBMCs, we observed that IR treatment specifically caused a XRCC1 induction in a time-dependent manner; 90 min after irradiation a significant increase of XRCC1 mRNA levels was found in comparison to control level. However, already at 60 min post-treatment a significant, but less pronounced, enhancement in XRCC1 expression was noted. With respect to the repair kinetics of radio-induced DNA damage, in G0 PBMCs from 15 to 90 min after treatment a gradual and significant decrease of Tail DNA (TD) mean value, measured using the Comet assay, was detected. This trend indicated that radio-induced DNA damage is repaired very quickly after IR exposure. In summary, we highlight the potential of XRCC1 as a possible genetic marker to assess the risk of developing sporadic breast cancer and we suggest studying it in combination with other SNPs. The in vitro CHO studies allow us to conclude that XRCC1 is expressed differentially through the cell cycle and maximally in S phase during which the XRCC1 protein assists in DNA replication. Furthermore, by doseresponse analysis, we show that the average X-ray dose generally used as a single fraction dose in radiotherapy does not affect XRCC1 mRNA and protein levels. Investigating the response to IR in quiescent peripheral blood mononuclear cells, we can confirm that X-ray treatment causes an induction of XRCC1 gene expression and that the DNA radio-induced damage is quickly repaired, mainly by global rapid SSBR pathway in which XRCC1 operates as a scaffold protein. In LCLs, we conclude that the miR34a* binding in the 3’UTR of RAD51 is not influenced by the RAD51-52 SNP, and it does not modify RAD51 mRNA levels. The IR activation of p53 is responsible for the induction of the miR34a* expression, seen 4h post-treatment, and for the decrease in the RAD51 mRNA levels, observed starting from 4h post-irradiation.