Endothelial cell dysfunction and cytoskeletal changes associated with repression of p16INK4a during immortalization

摘要

The immortalization process is a fundamental step in the development of most (if not all) human cancers, including the aggressive endothelial cell (EC)-derived malignancy angiosarcoma. Inactivation of the tumor suppressor p16^INK4a and the development of multiple chromosomal abnormalities are features of angiosarcoma that are recapitulated during telomerase-mediated immortalization of human ECs in vitro. The present study used a panel of telomerase-immortalized bone marrow EC (BMEC) lines to define the consequences of inactivation of p16^INK4a on EC function and to identify molecular changes associated with repression of p16^INK4a. In a comparison of two immortalized BMEC mass cultures and six clones, the cell lines that repressed p16^INK4a showed a higher rate of proliferation and an impaired ability to undergo morphogenic differentiation and form vessel-like structures in vitro. Proteomic comparison of a p16^INK4a-negative and a p16^INK4a-positive BMEC mass culture at early- and late-passage time points following transduction with telomerase reverse transcriptase (hTERT) revealed altered expression of cytoskeletal proteins, including vimentin and α-tropomyosin (αTm), in the immortal cells. Immunoblot analyses of a panel of 11 immortal clones showed that cells that lacked p16^INK4a expression tended to accumulate more dramatic changes in these cytoskeletal proteins than cells that retained p16^INK4a expression. This corresponded with aberrant cytoskeletal architectures among p16^INK4a-negative clones, which featured thicker actin stress fibers and less fluid membrane ruffles than p16^INK4a-positive cells. A direct link between p16^INK4a repression and defective EC function was confirmed by analysis of normal cells transfected with small interfering RNA (siRNA) targeting p16^INK4a. siRNA-mediated repression of p16^INK4a significantly impaired random motility and vessel formation in vitro. This report is the first to demonstrate that ECs that repress the expression of p16^INK4a are prone to defects in motility, morphogenesis and cytoskeletal organization. These defects are likely to reflect alterations that occur during the development of EC-derived malignancies.