The cellular ribonucleoprotein telomerase synthesizes telomeric DNA at the ends of chromosomes by copying an intrinsic RNA template. The long term goals of the proposed research are to understand specific roles in cell proliferation that have been recently uncovered for telomeres and telomerase, and investigate how subverting these roles can be used to impact on cancer cells and aging. In most cancer cells, telomerase is highly active. We recently reported that even a low threshold level of expression of human telomerase RNA gene constructs containing seven different mutant templates (but not the control wild-type template) was sufficient to decrease cellular viability, increase apoptosis and slow tumor growth of human cancer cells, We are interested in the potential feasibility of using this mutant-template telomerase RNA expression as an anti-neoplastic strategy for human cancers. The first three Specific Aims address the mechanism of the response to mutant-template telomerase RNA in normal and cancer cells. They are: #1: Test whether the seven specific template-mutated telomerase RNAs assemble into core enzyme with the ability to synthesize mutant repeats in vitro; #2: Determine whether expression of such mutant-template telomerase RNA in cultured human cells causes mutant telomeric DNA sequences to be incorporated into the telomeres, and test whether the binding of human telomeric proteins is affected by the mutations. Test specific models for the mechanism of action of one particular mutant-template telomerase RNA; #3: Use improved vector systems for delivery of mutant-template telomerase RNA genes, and short term assays for cell growth and apoptosis, to analyze which DNA damage checkpoint and apoptotic pathways are involved in the responses to mutant template telomerase RNA expression. Determine the involvement of specific genes implicated in human telomere functions, DNA damage and other response pathways. Accumulating recent evidence suggests that telornerase promotes cell proliferation by mechanism(s) that do not require telomere elongation.
Specific Aim #4 tests the specific hypothesis that telomerase has such functions, in telomere protection and promoting cell proliferation, that are separable its synthesis of telomeric DNA in vivo. The goal is a fuller understanding of the role(s) in cancer and aging played by telomerase activation.
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