Abstract 2261: Natural redistribution of end-protection proteins in aging cells as telomeres shorten

Abstract

Abstract Telomeres are the nucleoprotein structures at the ends of linear chromosomes. In mammalian cells, a six-member protein complex, called shelterin, protects telomeres from being recognized as DNA double-strand breaks and from nucleolytic degradation. It has been speculated that as telomeres become progressively shorter with each round of replication, the number of binding sites for the components of shelterin would decrease as well. In an attempt to determine whether there is a relationship between the length of telomeres and shelterin binding, primary human dermal fibroblasts from neonatal foreskin (HDFn) were grown in culture, and the levels of a number of shelterin components were measured over time. Unlike other previous studies where artificially constructed telomeres were used to address this question, we are following the natural progression of aging of a population of cultured cells. Comparisons were made between young cells at a population doubling (PDL) between 3 and 6 and aged cells at a PDL of 28 to 38. Southern blotting using a telomeric probe showed that the aged cells possessed significantly shorter telomeres than their younger counterparts, supporting the notion that there will be fewer shelterin binding sites. Using immunoblotting to compare the levels of several shelterin subunits (TRF2, hRAP1, and POT1), we found that TRF2, the DNA binding subunit that targets hRAP1 to the telomeres, and POT1 decrease in level as would be expected when fewer binding sites are present for the complex. Interestingly, the levels of hRAP1 did not decrease proportionally and remained relatively high compared to other subunits. These data suggest that as telomeres decrease in length and shelterin binding sites become less numerous, most shelterin subunits are degraded, but hRAP1 most likely plays an additional role. We have stressed young HDFn cells with hydrogen peroxide to age them prematurely. Upon treatment, the young cells (PDL of 8) became similar in appearance to the untreated, aged cells at PDL greater than 25. In the treated, young cells, hRAP1 levels in the nucleus decreased in a manner that was unaffected by inhibition of the proteasome, suggesting that during the oxidative stress, hRAP1 translocates from the nucleus to the cytoplasm. Taken together, our data suggest that RAP1 plays a role in stress signaling or response to cellular aging. Citation Format: Michelle E. Baribault, Mark J. Swanson, Nancy S. Bae. Natural redistribution of end-protection proteins in aging cells as telomeres shorten. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2261. doi:10.1158/1538-7445.AM2014-2261