Most cancers ( approximately 85%) are telomerase-positive cancer cells (TA) and maintain telomere length by telomerase. Some cancers that are telomerase-negative, and maintain the length of their telomeres by one or more mechanisms referred to as alternative lengthening of telomeres (ALT). The existence of ALT cells in a substantial minority of human tumors indicates that telomerase inhibitors will be ineffective in this tumor subset. It may also be useful to develop inhibitors of ALT. However, the determinants and the early detection marker of ALT are incompletely understood. There are no early detection markers for the ALT phenotype. Two duplex telomere binding proteins, TRF1 and TRF2, have been identified in humans. TRF1 is a negative regulator of telomere length. TRF2 has a protective role in telomere. Another human protein, TIN2, forms a complex with TRF1 and TRF2 and thus appears to be a crucial organizer of a complex containing TRFl-associated proteins (TANK2, TANK1), hPOT1 and TRF2-associated proteins (hRapl). TIN2 interacts with TRF1 and TRF2, via different domains in vivo and in yeast. Furthermore, overexpression of TIN2 deletion mutants that binds only TRF2 or TIN2 si-RNA induces apoptosis, suggesting that the TIN2-organized telomere complex has a critical role in modulating telomere structure and protecting chromosome ends. The TIN2-complex may be a basal telomere complex that recruits unknown factors that function in telomeric DNA damages or recombination mechanism proposed to be characteristic of ALT. I hypothesize that TA cells or ALT cells have different components in their TIN2 telomere complexes. Here, I propose to find the determinants of ALT: 1) I will compare telomere complexes from both TA and ALT cells using one or two dimensional SDS-PAGE and mass spectrometry to identify the determinants of ALT phenotypes, and confirm results using molecular biochemical analysis. 2) I will compare posttranslational modification in the telomere complexes of TA and ALT cells which may be responsible for ALT phenotypes, also using the assays in aim 1. These experiments will provide a first glimpse of differences between TA and ALT cells, and crucial preliminary data for a larger proposal to identify important targets for inhibitors and early detection markers.
