One of the general characteristics of cancer cells is genomic instability. Though it is still unclear what causes this instability, a hypothesis gaining increasing attention is that free chromosome ends, either from chromosome breakage or from loss of the telomere sequences which cap the ends, are prone to illegitimate recombination events. Thus, telomeres provide stability to the chromosomes. However, there appears to be a gradual loss of telomere sequences with each cell division, perhaps because of the end-replication problem. Tumor cells do have shortened telomeres, but they also possess greatly elevated levels of the enzyme telomerase to overcome the end-replication problem, while normal cells do not. Thus, telomerase is an attractive target for new anti-cancer agents because of the expected selectivity for neoplastic cells. Furthermore, we already have preliminary evidence that inhibiting telomerase can kill cancer cells. We therefore propose to characterize telomerase and its inhibitors in much more detail, in order to select the most effective agents and define their biological effects. Specifically, we propose: l) To isolate, purify, and characterize telomerase(s) from several human tumor types. Telomerase from selected breast, lung, and colon cancer specimens will be isolated and characterized biochemically, and telomerase from the well-characterized HeLa human tumor cell line will be purified. In addition, we will design new, faster, and more sensitive telomerase activity assays. 2) To elucidate the mechanism and specificity of human tumor telomerase inhibition by selected agents. Agents will include: (a) nucleoside/nucleotide analogs, (b) nonnucleoside reverse transcriptase inhibitors, and (c) antisense molecules. Their ability to inhibit isolated telomerase will be determined. Comparison with other nucleotide processing enzymes such as reverse transcriptase, terminal transferase, and DNA polymerases will help to delineate structural requirements for specific inhibition of telomerase. 3) To compare the formation and repair of drug- induced lesions in telomeres versus bulk DNA. In particular, we will examine the actions of DNA-reactive agents on the structure and function of telomeric DNA. Agents will include: (a) an AT-specific alkylating minor groove binder, (b) a GC-specific DNA intercalating agent, and (c) an enediyne strand scission agent. From these studies we hope to learn the biochemical and biological consequences of telomerase inhibition, which prototype structures offer the best promise of specific inhibition, and how tumor cells cope with lesions in the telomeres. Overall, this work will shed new light on how interference with the telomere/telomerase system could provide a new and selective therapeutic strategy for human cancer.
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