Abstract 2121: Nonerythroid alpha spectrin prevents telomere fragility after DNA interstrand crosslink damage

Abstract

Abstract Telomere dysfunction and increased telomere fragility are important contributing factors to chromosome instability. We have previously demonstrated that the structural protein, nonerythroid -spectrin(IISp), is critical for maintenance of chromosome stability after DNA interstrand crosslink (ICL) damage. However, whether IISp plays an important role in maintaining telomere stability after DNA damage is an extremely important question and the goal of the present study. IISp in normal human lymphoblastoid cells was depleted by siRNA and cells treated with an ICL agent, mitomycin C (MMC). Cells were analyzed using immunofluorescence and in situ hybridization (IF-FISH) and probed with anti–spectrin and a telomeric Cy3-conjugated-PNA probe or antibodies against two telomeric proteins, TRF1 or TRF2. Metaphase spreads of these cells were also examined and stained with the PNA probe and scored for telomeric abnormalities. In cells damaged with MMC but not in undamaged cells, a portion of IISp (50%) was found to co-localize with the PNA probe in the telomeres along with TRF1 and TRF2, indicating that IISp localizes to telomeres after ICL damage. To assess whether there was increased telomere fragility after ICL damage, chromosome ends were examined for loss of telomeric signal. After treatment with MMC, 28% of the chromatid ends showed a loss of telomeric signal compared to 2% in nondamaged cells. However, after depletion of IISp, 74% of the chromatid ends in MMC damaged cells displayed loss of telomeric signal compared to 18% in the nondamaged cells. This indicates that there was increased telomere fragility in MMC treated cells after knockdown of IISp. In addition to an increase in fragile telomeres in these cells, the chromosomes in the IISp siRNA treated cells damaged with MMC showed a significant increase in sister chromatid end fusions compared to the undamaged cells, which is indicative of aberrant telomere structure.These results on telomere fragility after loss of IISp are similar to those obtained using cells from the cancer prone genetic disease, Fanconi anemia complementation group A (FA-A), in which we have shown that there is a deficiency in IISp levels which correlates with a defect in ability to repair DNA ICLs. Examination of FA-A lymphoblastoid cells showed that there is an increase in fragile telomeres after MMC treatment: 76% of the chromatid ends showed loss of telomeric signal after MMC damage compared to 24% in undamaged cells. When levels of IISp were restored to normal in the FA-A cells by knocking down µ-calpain, a protease that cleaves IISp, the percentage of chromatid ends showing loss of telomeric signal (26%) after MMC was similar to that in normal cells. These studies collectively show that IISp plays an important and central role in maintenance of telomere stability after DNA ICL damage and suggests that it is an important factor in telomere function and chromosome stability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2121. doi:1538-7445.AM2012-2121