Abstract 1232: The loss of p16INK4a function is essential for Werner Syndrome mouse embryo fibroblasts to escape from senescence

Abstract

Abstract p16INK4a is a tumor suppressor gene contributing to cell cycle arrest as a cyclin dependent kinase inhibitor. It has been shown that the expression of p16INK4a protein is up-regulated along with the cellular senescence and the loss of p16INK4a function could elongate the cellular life span. These data suggest that p16INK4a promote cellular senescence. However, it is still debated that the expression regulation of p16INK4a in mouse cells is due to cell culture condition. Previous study has showed that mouse embryo fibroblasts (MEFs) from late generation of Werner Syndrome (WS) mice (G5 mTR−/− Wrn−/−) senesced very rapidly, and the senescent MEF cells are prone to escape senescence and spontaneously immortalize. To understand the molecular basis for WS MEFs escaping from senescence, we compared the expression level of proteins involved in regulating cellular senescence. Western blotting showed that all immortalized WS MEFs lost their expression of p16INK4a and re-expression of p16INK4a could induced cellular senescence, suggested the loss of p16INK4a function is essential for maintaining senescence status of WS MEFs. To further evaluate the role of p16INK4a in senescence induced by telomere dysfunction, p16INK4a-/− mice were crossed with Wrn−/− mice to generate MEFs with genotypes of p16INK4a-/−, Wrn−/− and p16INK4a-/−Wrn−/−. Growth curve of these three cell lines revealed that Wrn−/− MEFs grew slower than p16INK4a-/− MEFs, the p16INK4a-/−Wrn−/− MEFs grew about the same as p16INK4a-/− MEFs. This data suggested that p16INK4a-/− rescued the growth barriers occurred in Wrn−/− MEFs. In consistent with this, Western blotting showed that loss of p16INK4a in Wrn−/− MEFs (p16INK4a-/−Wrn−/−) could partially eliminate the DNA damage responses caused by Wrn−/−, e.g., the expression of H2AX, p19ARF. Furthermore, telomere dysfunction was initiated by introducing TRF2ΔBΔM into these cells. In response to this stimulation, Wrn−/− MEFs senesced in several passages. In contrast to this, p16INK4a-/− and p16INK4a-/−Wrn−/− MEFs could continue grow and “kicked out” the exogenous TRF2ΔBΔM in their late passages. In summary, our data suggested that in the context of telomere dysfunction, loss of p16INK4a function could prevent the cells from senescence. This work was supported by The New Century Excellent Scholarship to Y.L, NSFC grant to Y.L. (30771194, 30970598) and to W.T. (30960152). We thank Dr. Sandy Chang from Yale University and Dr. Ronald A Depinho from Harvard University for providing transgenic mice used in this study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1232. doi:10.1158/1538-7445.AM2011-1232