Identify werner protein molecular partners in S phase ALT cell.

摘要

The Werner protein (WRN) is encoded by the WRN gene. Mutations in this gene are the causal factor for the outset of an autosomal recessive progerid disorder known as Werner Syndrome. WRN has been proposed to function in ALT, a pathway that maintains telomere length independent of telomerase and involves high level of recombination processes observed in about 15% of cancer cells. These functions are probably accomplished by the interactions between WRN and many other proteins. Thus I studied the WRN complex formation in ALT cells to further investigate the potential role of WRN in ALT pathway. In this study, I used CCL75.1 cell as a representative for ALT cell lines and synchronized CCL75.1 cells to S phase using optimized double thymidine block. I found out that the optimal condition for harvesting S phase CCL75.1 cells was as follows: cells were blocked with media containing 4 mM thymidine for 18 hours, released in regular media for 9 hours, blocked again in 4 mM thymidine media for 19 hours and harvested after regular media incubation for 3 hours. Protein extracts from asynchronous and S phase CCL75.1 and Hela cells were subjected to immunoprecipitation of WRN protein followed by Western Blot analysis to determine the interaction between WRN and Ku70, PARP1 and TRF2. In both Hela and CCL75.1 cell lines, the interactions of WRN with Ku70 and PARP1 can be detected in asynchronous cells, but the association with TRF2 can only be detected during S phase. My results suggest that WRN complex in CCL75.1 cell is similar to that of Hela cell during S phase, indicating that the cell cycle related regulation of WRN complex is not different in ALT cells and that the difference between telomerase positive cell and ALT cell is probably not due to the variation of WRN complex composition in S phase. It is likely that WRN influences the activity of ALT cells in other ways.