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dc.contributor.advisorSgura, Antonella-
dc.contributor.authorBuonsante, Rossella-
dc.description.abstractTelomeres are nucleoprotein complexes that protect the ends of linear chromosomes and are required for a wide range of cellular processes, as apoptosis, aging, cancer and chromosome stability. Telomere shortens every time a somatic human cell divides. Furthermore human cancers, avoid the progressive loss of telomeric DNA by the telomerase or the telomerase-independent mechanism, termed ALT (Alternative Telomere Lengthening), result into indefinite cell proliferation. However, it has been demonstrated, that several physical or chemical agents are able to modulate telomere length; in particular oxidative stress accelerates telomere shortening. Literature data showed that telomeres are the preferential target for oxidative damage because of ROS able to induce 8-oxodG from triplet GGG present in human telomeric sequence TTAGGG. Nevertheless, the effects of prolonged oxidative stress on telomere metabolism are still poorly investigated. With the aim to investigate the effects of oxidative stress on telomere length and chromosome segregation and to test mitotic defects, human primary lung fibroblasts (MRC-5) were daily treated with 10μM H2O2 and analysed at regular intervals over period of 94 days. The telomere length analysed by Q-FISH (Quantitative-FISH), showed a telomere shortening at 9-21 days, and a telomere elongation between 21-48 days. Moreover we observed that this trend of “shortening-lengthening” was repeated on time up to 94 days. Based on this repeated trend, we focused our attention to the first 45 days, because at longer time several mechanisms, as senescence, not straight related to oxidative stress effects, could interfere with the results. At this interval of time, we saw a significant telomere shortening after 5 days and a telomere elongation at 15 days of treatment. Based on these results, we confirm that prolonged oxidative stress is responsible of telomere shortening even at low daily doses. Considering that telomere shortening could have an effect on cell viability, first of all, we analysed other endpoints: a) Possible cell cycle perturbation by Cytofluorimetric analysis. We noticed any differences between treated and own control samples. b) The entity of DNA damage induced by prolonged treatment with H2O2 by “Alkaline Comet Assay”. Moreover we used Trypan Blue Assay to evaluate cell viability and this analysis has relieved in all cases that cell death was less than 30%, value above which the mortality is considered significant. c) To understand if the DNA damage and telomere shortening observed could induce premature senescence, we assessed the number of β- galattosidase positive cells, typically expresses in senescence cells. As expected, we observed a light trend of senescence on time in treated and control samples, with a difference between the two only at 10-15 and 20-27 days. This led us to conclude that oxidative stress did not induce significant premature senescence in treated cells. Considering that telomere shortening did not alter cell viability, we studied the mechanism responsible of telomere elongation observed at 15 days. To evaluate this mechanism the analysis of telomerase activity was performed by RTQ-TRAP assay that showed no telomerase activation for all fixation times. On the other hand, to verify ALT activation, we assessed two ALT markers: telomeric-sister chromatid exchanges (T-SCE) by CO-FISH analysis and colocalization of telomeres-PML proteins by FISH and immunostainig. We showed a higher frequency of both these ALT markers at 15 days of treatment, corresponding to the time of telomere elongation observed. This led us to hypothesize Human Primary Fibroblast could activate ALT mechanism, known in literature be present in a little percentage of tumor cells. However we cannot exclude the presence of a cellular selection system that promotes cells with longer telomeres due their major viability. In literature is known that telomere shortening could alter chromosomes segregation by inducing chromosome bridges (end-to-end fusion). To analyse the relation between telomere length and chromosome segregation defects, like chromosome bridges, during my period at the Virginia Tech University we performed Time lapse on live cells and immunostainig (that detect Kinetochore and mitotic spindle) in mitotic cells in the range of 1-48 days. Data obtained were pulled and showed an increase of chromosome bridges in treated cells at 5, 27, 41 and 48 days of treatment. The induction of chromosome bridges at 5 days indicated a relation between telomere shortening and chromosome segregation defects. Moreover the reduction of chromosome bridges, observed at 15 days, was related to telomere lengthening saw at the same day. Based on these results, we could observe that telomere shortening induced by oxidative stress at 5 days of treatment could triggers the increase of chromosome bridges induction. Subsequently, we observed a chromosome bridges decrease corresponding to the time of telomere elongation and based on previous results obtained, we hypothesized that ALT mechanism restores telomere length inducing a reduction of chromosome segregation defects related to telomere shortening. In fact, according to literature data, the loss of chromosome ends could result in “sticky” chromosomes that will give rise to dicentric/ring chromosomes: when these dicentric/ring chromosomes will try to separate in anaphase, they will create a chromosome bridge that breaks before or in transition to telophase. Then, the breakage of bridge will create new sticky chromosome ends that could fuse with other sticky ends or that could determine the sister chromatid fusion at the following cell cycle, creating the so called “breakage/fusion/bridge cycle” (BFB). This BFB cycle will continue until the affected chromosome will acquire a new telomere, as in our case when ALT mechanism restores the telomere length. To further assess the effects of telomere length changes on chromosome instability, we evaluated abnormal nuclear structures at 1-20 days. We estimated the quantity of NBPs (nucleoplasmic bridge) and NBuds (nuclear Buds), markers of chromosome segregation defects, measuring a significant increase of these biomarkers at 5 days of treatment and confirming previous result obtained for chromosome bridges. These data have been supported by anaphase-lagging chromosomes, marker of chromosome loss. In both fixed- and live-cells. We observed an increase in anaphase lagging chromosomes frequencies over the control at 5, 20, 27 and 41 days of treatment. Additionally, kinetochore positive micronuclei (MN) were analysed by immunostainig. Kinetochore positive MN represents a marker of chromosome loss and aneuploidy (Fenech, M. and Morley, A.A. 1985). Our analysis resulted in a significant increase of kinetochore positive MN in almost all-fixing time. With the aim to evaluate if also other mitotic defects, in addition to chromosome segregation defects, were induced by prolonged oxidative stress, cells with tilted mitotic spindles, defined as the positioning of the spindle long axis at an angle instead of parallel to the substrate, were observed. We observed frequencies of tilted spindles above the control frequencies during the first 10 days, at 27, and 48 days of treatment. These results indicated that prolonged oxidative stress affected spindle structure and/or function. Thus, in addition to the telomeric damage, which causes chromosome rearrangements and can result in chromosome bridges in mitosis, prolonged oxidative stress could also induce telomere-independent mitotic defects, such as MN, lagging chromosomes, which can result in aneuploidy, and cells with tilted spindle. Taken together, these data indicated that prolonged oxidative stress could cause the two most common karyotype defects observed in cancer cells: chromosome rearrangements and aneuploidy.it_IT
dc.publisherUniversità degli studi Roma Treit_IT
dc.subjectoxidative stressit_IT
dc.titleTelomere alterations and chromosome segregation defects induced by oxidative stressit_IT
dc.title.alternativeAlterazioni al telomero e difetti nella segregazione cromosomica indotti dallo stress ossidativoit_IT
dc.typeDoctoral Thesisit_IT
dc.subject.miurSettori Disciplinari MIUR::Scienze biologiche::GENETICAit_IT
dc.subject.isicruiCategorie ISI-CRUI::Scienze biologiche::Molecular Biology & Geneticsit_IT
dc.subject.anagraferoma3Scienze biologicheit_IT
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