The Role of Oxidative Stress on Telomere Shortening, Telomere Dysfunction and Damage to Telomere G- Strand Overhang - A Putative Model of Carcinogenesis in Fanconi Anemia Cells.

Abstract

Fanconi Anemia is an autosomal recessive disease characterized by various inborn abnormalities and severe genetic instability, which may lead to bone marrow failure and increased incidence of cancer. One possible explanation for this genomic instability may be the cellular hypersensitivity to oxidative stress, leading to chromosomal breaks. The goal of our study was to explore the possible oxidative damage caused by H2O2 to telomeres of Fanconi Anemia (FA) lymphocyte cell line, HSC536/N and its possible relation to carcinogenesis. We surmised that combination of oxygenative damage to telomere function with Overexpression of telomerase may provide a model for malignant transformation in those cells.The cells were grown in the presence of hTR anti-sense (GRN163) and exposed for one month to hydrogen peroxide combined with a variety of antioxidants. One month of exposure caused marked shortening of telomere (TRF) length (measured by Southern blot), and damage to the single stranded overhang of the telomere [using the newly developed telomeres oligonucleotide ligation assay (T-OLA)]. The telomere and single stranded overhang shortening was associated with significant telomere dysfunction (assessed by karyotype analysis). Control cells did not exhibit this sensitivity to H2O2. Telomere dysfunction did not evoke damage response in FA cells, in contrast to normal p53 up regulation in control cells. Reconstitution of telomerase activity protected FA telomeres from further oxygenative damage. Several antioxidants prevented the H2O2 exerted damage. Based on our data we suggest a model of malignant transformation in FA in which oxidative stress led to a significant shortening of the overall length of the telomere and the single stranded overhang, thus causing telomere dysfunction. P53 mediated damage control was absent in these cells, preventing apoptosis or cycle arrest. Reconstitution of telomerase activity further stabilizes the damaged telomeres preventing crisis and forming the basis for future carcinogenesis. The differential effects of the antioxidants and the significance of our findings to malignancies will be discussed.