Telomeres have a G-rich repeated DNA sequence that maintains chromosome end stability. Telomerase is a reverse transcriptase-like ribonucleoprotein that maintains the telomere sequence. Telomerase is expressed in the majority of tumor cells but not in normal cells. Telomerase and telomeres are ideal tumor-specific targets for anti-cancer therapeutic strategies. Our preliminary studies show the discovery of the first telomerase inhibitors. Our studies also show that telomerase inhibitors induce telomere shortening, chromosome end fusion, and eventually cell death. Cells have shown the ability to adapt to telomerase inhibition. The goals of this proposal are to l) elucidate the molecular structure of telomerase and provide a more defined target for our anti-cancer strategies, and 2) determine the mechanism(s) of cellular adaptation to telomerase inhibitors. These goals will be accomplished by mapping and cloning the genes for the telomerase proteins and RNA template. Through our collaboration with Program l, purified telomerase will be used as a source of purified telomerase RNA for cloning. Somatic cell genetics will be used to map the telomerase activity. The new technique of Differential Display will be used to detect mRNAs expressed in posttelomerase-activated cells that are not expressed in pre-telomerase activated cells. The mapping will be coordinated with the isolation of candidate cDNA clones for the proteins and RNA to accelerate the identification of legitimate clones. The cDNA clones of the RNA will be screened for appropriate template sequence, secondary structure, and ability to direct in vitro and in vivo telomerase activity. The cDNAs for telomerase proteins will be expressed for production of antibodies. The antibodies and DNA clones will serve as probes for use in studies of telomerase expression in Program 3 and in tumor specimens from Program 4. The data from these studies will be used by Program 2 for structure modeling and specific drug design. Potential telomerase inhibitors, such as anti RNA oligonucleotides will be studied by Program l. The mechanism of adaptation to telomerase inhibitors will also be studied. Hypotheses concerning drug metabolism, telomerase up-regulation and telomerase specificity will be tested.
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