| The telomere is a nucleoprotein complex protecting the ends of mammalian chromosomes. Telomere stability is required for proper cellular function as dysfunctional telomeres lead to chromosome end-to-end fusions, aneuploidy and cell death. Cells that divide beyond their normal proliferative lifespan exhibit telomeric shortening and telomeric and other genomic DNA damage terminating in a proliferative arrest termed replicative senescence. The enzyme telomerase synthesizes additional telomeric repeats and maintains a 3' telomeric overhang. Activation of the catalytic subunit of telomerase, hTERT, correlates with telomere stability and cell immortalization and is a hallmark of cancer. However, whether telomere shortening or 3' overhang loss triggers replicative senescence, and the roles hTERT plays in this process are not fully understood.To investigate triggers of replicative senescence, I performed a screen for proteins whose suppression allows cells to bypass replicative senescence. Nek4 was identified as being required for proper entry into replicative senescence in primary foreskin fibroblasts. Nat10, MCM7 and GNL3, three hTERT interacting proteins, were identified as Nek4 interactors. These proteins mutually interact and their suppression results in acute senescence. Reduction in Nek4 expression was identified in several lung cancer cell lines as was a mutation in Nek4 resulting from loss of heterozygosity.Finally, in an effort to investigate the prevailing notion that hTERT activation and telomere length stability are equivalent, I researched novel roles for hTERT in promoting a proper DNA damage response and normal chromatin configuration.POT1 is a 3' overhang binding protein implicated in chromosome end protection and regulation of telomerase function. In human cancer cells that exhibit constitutive hTERT activity, hPOT1 exerts control over telomere length. Herein I show that human diploid fibroblasts in which hPOT1 is suppressed harbor longer telomeres than control cells, delaying the onset of replicative senescence dependent upon S-phase restricted hTERT. These findings are consistent with the view that hPOT1 promotes a non-extendable telomere state resistant to extension by S-phase restricted telomerase. |
