SV40-Tauligen Proteins During G1/S Phase Transition

Abstract

Atrial tumor cardiomyocytes (AT-2) represent an immortalized cell line. AT-2 cells are derived from primary cultures of atrial cardiac muscle cells that originated from apha-MHC-SV40 T antigen transgenic mice. AT-2 cells grow as a monolayer and exhibit a doubling time of 24 hours. The T antigen oncoprotein is responsible for immortalizing AT-2 cells but the molecular details concerning how T antigen controls passage through the G1 restriction point and entry into S phase are not known. The primary objective of this project is to identify cell cycle dependent T antigen-protein complexes, specifically at the G1/S boundary, that may control AT-2 cell proliferation. The presence of T antigen associated proteins may explain why normal adult cardiomyocytes do not regenerate after injury and most notably do not develop tumors. To identify T antigen associated proteins. AT-2 cell growth is reversibly arrested at the G1/S boundary using the drug aphidicolin (7.5 ug/ml). Following release of a 16 hour exposure to aphidicolin, AT-2 cells typically exhibit a peak incorporation of 3H-thymidine within two hours (indicating entry into S phase). The T antigen associated protein complexes at the G1/S boundary are identified using the technique of immunoprecipitation. Immunoprecipitations are performed on three separate cultures, untreated, aphidicolin blocked and aphidicolin released. The cultures are radiolabeled with 35S-methionine and the entry of S phase is monitored by pulse labeling sister cultures with 3H-thymidine. Lysates are precleared with Sepharose protein-G beads. Standard techniques of immunoprecipitation are followed using commercially available monoclonal antibodies to T antigen (MAb 419), p53 (MAb 421) an ubiquitous tumor suppressor protein. Immunoprecipitated antigen-antibody complexes are analyzed on 7.5% denaturing polyacrylamide gels. Preliminary results have identified a unique protein of approximate molecular weight 48,000 Daltons. This protein (p48) is in the process of being purified and analyzed by microsequencing. Key Words: tissue culture, atrial heart cells, cell cycle, immunoprecipitation, T antigen, p53