321 A Novel in Vitro Model of Conditionally Immortalized Human Vascular Smooth Muscle Cells A Tool for Aging Studies HARRIS PRATSINIS, a CATHERINE DEMOLIOU-MASON, b ALUN HUGHES, b AND DIMITRIS KLETSAS a,c a Institute of Biology, National Centre for Scientific Research “Demokritos,” 153 10, Athens, Greece b Department of Clinical Pharmacology, National Heart & Lung Institute, Imperial College of Science Technology and Medicine, St.Mary’s Hospital, London W2 1NY, UK INTRODUCTION It is well established that serial in vitro propagation of normal human cells even- tually leads to replicative senescence. 1 However, certain viral and cellular oncogenes have the ability to immortalize normal cells. Conditionally immortalized rodent 2 or human 3 fibroblast lines have proved very useful for the study of cellular senescence. We have developed a conditionally immortalized human vascular smooth muscle cell (HVSMC) line, designated SM1, after transfection of primary vein-derived SMCs with a nonreplicative retroviral vector containing a temperature-sensitive (tsA58) mutant of simian virus 40 (SV40) large T-antigen (LT-antigen). 4 In contrast to normal HVSMCs, which stop dividing after 10–15 passages, SM1 cells have been in culture at the permissive temperature (36°C) for over 200 population doublings. Their growth rate at the permissive temperature is much higher than that of normal HVSMCs; furthermore, they achieve a higher cell density. At the nonpermissive temperature (39°C), however, LT-antigen expression is downregulated, a phenome- non accompanied by growth arrest and acquisition of a senescent cell-phenotype: di- minished BrdU incorporation, increase in cell size, appearance of perinuclear vacuoles, increased SA-β-Gal staining, and expression of the cell cycle inhibitor p21 WAF1/SDI1 at high levels. 4 AUTOCRINE REGULATION OF SM1 PROLIFERATION We previously showed that normal human fibroblasts control their proliferation by secreting autocrine growth factors and that this mechanism persists during in vitro aging. 5 Because serum withdrawal under permissive conditions does not lead SM1 cells to growth arrest, we tested whether their transformation was accompanied by c Corresponding author. Phone: + 30 1 6503565; fax: + 30 1 6511767. dkletsas@mail.demokritos.gr