BRCA2 is a highly penetrant breast cancer predisposing gene. The protein product of the BRCA2 gene mediates repair of breaks in DNA, through Homologous Recombination (HR). Understanding the mechanism(s) behind BRCA2 involvement in HR will help clarify its clinical importance and may pave the way for possible therapy. In this work we show that BRCA2 affects telomere maintenance in mammalian cells. Telomeres are physical ends of chromosomes implicated in cell senescence and carcinogenesis. In particular, the enzyme telomerase that synthesizes telomeric DNA is highly active in ~90% cancers and it is considered one of the cancer markers. The remaining 10% of cancers do not show telomerase activity and they maintain their telomeres by an alternative pathway known as Alternative Lengthening of Telomeres (ALT). We observed telomere shortening, loss of telomere function in the form of end chromosome fusions and increased incidence of Telomere Sister Chromatid Exchanges (T-SCE), one of the recognized markers of ALT, in 3 sets of Chinese hamster and human BRCA2 defective cell lines, all of which maintained telomeres by conventional mechanisms. We have also inhibited BRCA2 expression in ALT positive cells by transfecting them with si (short interfering) RNA oligonucleotides specific for BRCA2 and monitored its expression by Real Time-PCR and Western blot. Results indicate that BRCA2 knock-down in ALT positive human cells that causes reduction in T-SCE frequencies, thus suggesting that ALT cells and those that maintain telomeres by conventional mechanisms differ in this respect. One interesting scenario that emerges from these results is that BRCA2 deficiency could potentially suppress the ALT pathway. We wanted to explore this possibility further by creating a permanent BRCA2 knock-down. Our preliminary results suggest that our method for the permanent BRCA2 knock-down based on the SMARTvector 2.0 system and sh (short hairpin) iv RNA approach is still not working effectively. We identified hyper-methylation of the promoter within the vector as a possible cause. Finally, we examined repair kinetics of interstitial telomeric sites (ITSs) in BRCA2 deficient Chinese hamster cells in order to test the hypothesis that defective DNA double strand break repair may be responsible for their increased sensitivity to DNA damaging agents. Our results indicate that DNA damage within ITSs is repaired effectively thus disproving the above hypothesis. In conclusion, this work demonstrates the involvement of BRCA2 in telomere maintenance.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:607551 |
| Date | January 2014 |
| Creators | Gozaly Chianea, Yaghoub |
| Contributors | Slijepcevic, P. |
| Publisher | Brunel University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://bura.brunel.ac.uk/handle/2438/8724 |
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