Evaluation of dna damage in myotonic dystrophy cells after treatment with different genotoxic agents

Abstract

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by an unstable trinucleotide (CTG) repeat motif expansion in the 3’ untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene, located on chromosome 19q13.3 (1-3). It has a frequency of 1 of 8000 individuals worldwide (2-5). The length of (CTG)n repeats is inversely correlated with the age of onset of the disease and consequently with its severity (1-3, 6-8). DM1 disease is characterized by somatic mosaicism: the existence of cells with different expansion length within an organism. In fact, some tissues and cell types possess a higher tendency to extend these tandem repeats sequences and the longest tandem repeats have been found in severely affected tissues (2, 6-10). Furthermore, DM1 has been proposed as a disease due to an increased susceptibility to oxidative stress (OS) with high level of free radicals and reduced cellular antioxidant activity and premature aging (2, 11, 12) and there are evidences that the susceptibility to OS is (CTG)n repeats number-dependent (13, 14). Mismatch Repair (MMR) protein complex in particular MSH2, MSH3 and PMS2, and Base Excision Repair (BER) components such us OGG1 can interact and cooperate to increase trinucleotide repeats instability (15-20). In particular MMR pathway is involved in repairing secondary structure acquired by DNA, while BER pathway is involved in repairing modified basis such as 8-oxoGuanine (8-oxoG) prevalently induced by OS. A direct prove of the implication of MMR component in trinucleotide expansion in DM1 has been demonstrated by Seriola et al., (20) who for the first time demonstrated the down-regulation of MMR components in differentiated DM1 human Embryonic Stem Cells (hESCs) correlated to triplet expansion stabilization (20). On the other hand, there are no direct evidence about an involvement of BER pathway in this disease. Starting from this knowledge, we wanted to understand the main mechanisms involved in DNA damage response (DDR) in this disease. In order to reach this purpose we first characterized our fibroblasts. After having quantified the (CTG)n expansion size we evaluated the gene expression profile of MMR and BER mechanism components: we found them both downregulated respect to the WT fibroblasts. In order to study the effects of the expression profile analyzed and then the repair systems, we tried to evaluate the response to two different genotoxic agents: X-rays and H2O2. We evaluated different markers of chromosome instability (CIN), and we found a higher basal level for all of them in DM1 cells respect to the WT, according to the lower level of MMR and BER gene expression they have. Furthermore, X-rays treatment (2 Gy) allowed us to evaluate Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) working by the analysis of breaks, fragments and translocations. After 24 hrs from treatment the analysis of karyotype of DM1 and WT cells allowed us to exclude a possible misregulation of both HR and NHEJ, in fact the increase of chromosome aberration was similar in DM1 and WT fibroblasts, for all the targets evaluated. The treatment with H2O2 (200 µM, 1 hr) allowed us to evaluate the activation of both MMR and BER pathways. By FPG-modified comet assay we demonstrated that, both in DM1 both in WT, genomic DNA damage induced by OS, in particular 8-oxoG, were completely repaired within 24 hrs, suggesting a similar trend of repair kinetic in our samples, although oxidative DNA damage induced in DM1 cells was higher. These data are correlated with the gene expression profile in both our samples that showed an activation of both pathways already at 45 mins of treatment. Surprisingly, the analysis of Abnormal Nuclear Morphologies (ANMs) showed a greater increase of all the markers analyzed (micronuclei, MN; nucleoplasmic bridge, NPBs and nuclear Buds, NBUDs) in DM1 fibroblasts respect to WT at 48 hrs (especially in DM cells) persisting up to 72 hrs. Recently, some authors have demonstrated that ANMs could be associated also with telomere dysfunction (21, 22). In fact, Pampalona et al., (21) have proposed that in particular the NPBs derive from end-to-end fusions, affecting chromosomes with critically short telomeres that leads to chromosome-end-fusions, visible as NPBs in interphase. For this reason, we investigated telomere length in DM1 and WT fibroblasts. As expected (23, 24), DM1 fibroblasts showed shorter telomeres respect to WT fibroblasts. After H2O2 (200 µM, 1 hr) treatment, WT and DM1 fibroblasts showed a telomere length reduction, as expected (22). In order to understand if DM1 telomeres are dysfunctional we evaluated the frequency of co-localization between TRF1, a shelterin complex protein, and γH2AX, a marker of double strand breaks (DSBs). In fact, in somatic cells telomeres that reach the critical length could be recognized as DSBs and activate the DDR pathway, leading to senescence (25). The analysis of telomere dysfunction-induced foci (TIFs) showed a significant increase of γH2AX TIFs 48 hrs after treatment for DM1 fibroblasts, while a lower increase in WT ones. These results well correlate with the increased number of NPBs we found at 48 hrs from treatment and with the consequent increase of MN and NBUDs at 72hrs due to the NPBs breaks. The short telomere length we found in DM1 untreated fibroblasts also justify the higher basal levels of NPBs in these cells. All these data strongly correlate with the finding reported in Coluzzi et al, 2014 (22), in which in MRC-5 fibroblasts treated with H2O2 an increase of γH2AX TIFs was correlated with a telomere shortening and with an increase of NPBs at 48 hrs that give rise to MN and NBUDs at 72 hrs (22). As in our case, also in this case the genomic FPG-comet assay showed a total rescue of the oxidative DNA damage within 24 hrs, but the FPG-sensitive base lesions within telomeric DNA revealed a significant persistence of oxidative DNA damage at telomere 24 hrs from treatment, suggesting that oxidative DNA damage repair may be less effective in telomeres (22). This allow us to hypothesize that also in our cells this phenomenon could occur and that at telomere level probably the oxidative damage could persist inducing the telomere dysfunction and the related ANMs observed (in DM1 more than in WT). Because of the role of telomeres in cellular growth and senescence, these two end-points were studied. We observed a strong proliferative arrest that persist until 168 hrs for both WT and DM samples. These results were strongly supported by the senescence β-galactosidase staining that revealed a percentage of senescent cells from 50 to 65%. Therefore, considering the greater damage observed in DM cells but a similar trend of cellular growth and senescence, this leads us to hypothesize that probably DM1 fibroblasts could have a less regulated cell cycle checkpoint that allow a DNA damage that normal cells would not tolerate. On the other hand, considering the high rate of senescent cells, it could be interesting to analyze the destiny of these cells both in DM1 and WT at longer time, in order to evaluate if they are able to recover the proliferative block or if they die.