One of the key steps in human carcinogenesis is the activation of a telomere maintenance system that allows the continued proliferation of transformed cells. The telomeres of primary human cells shorten with each cell division and this attrition eventually induces cell cycle arrest, thus limiting the proliferative capacity of emerging tumorigenic clones. Malignant cells are known to overcome this barrier through the activation of telomerase, the RNP reverse transcriptase that synthesizes telomeric DNA onto chromosome ends. In the preceding funding period, we have described a regulatory pathway in cancer cells that controls the action of telomerase at individual chromosome ends. This telomere length regulation depends on the telomere specific protein complex, shelterin, which acts in cis to inhibit telomerase. Shelterin's accumulation at telomeres is proportional to the length of the telomeric repeat array, resulting in a greater inhibition of telomerase at longer telomeres. In the next funding period, we will further define the mechanism of telomerase inhibition by shelterin (AIM 1). The work in AIM 2 will focus on the positive regulation of telomerase-mediated telomere extension in human tumor cell lines. Whereas the expression of telomerase components and the biogenesis of the enzyme has been analyzed in detail, little is known about how telomerase is recruited to chromosome ends, how this recruitment is regulated, and whether telomerase requires additional activation steps. Our preliminary data has revealed putative positive regulators of telomerase-mediated telomere elongation and we propose to define these pathways and others like it further. In a second new direction, we will focus on the mechanism and regulation of telomere shortening (AIM 3), which is poorly understood, despite its importance as a presumed tumor suppressor pathway. The attrition of human telomeres is much faster than expected from the `end-replication problem' but the process underlying this accelerated terminal sequence loss is not known. Our preliminary data suggests that telomere attrition involves shelterin regulated degradation of the C-rich telomeric DNA strand. We propose to identify the genes responsible for telomere shortening in (pre-) malignant cells and study the regulation of this processing step. It is anticipated that insights into the molecular basis of telomere length control will provide opportunities for therapeutic intervention in a wide variety of human cancers, including leukemias, lymphomas, and most carcinomas.