INDUCTION OF GROWTH-INHIBITION OF 293 CELLS BY DOWN-REGULATION OF THECYCLIN-E AND CYCLIN-DEPENDENT-KINASE-4 PROTEINS DUE TO OVEREXPRESSIONOF TIS21

We earlier reported that TIS21 mRNA expression was markedly decreased in A549 and NCIH69 human lung cancer cells and in thymic carcinoma tis sues obtained from transgenic mice containing simian virus 40 large T antigen (J Cancer Res Clin Oncol 121:279-284, 1995). To determine how TIS21 inhibits growth, we made 293 cells that constitutively expressed TIS21 protein. The constitutive TIS21 expresser lines C9 and C11 grew to a lower saturation density than did those in th vector-transfected clones (V7 and V10) and antisense-transfected clones (AS1 and AS4), a nd the size of the C9 and C11 cells increased significantly after tran sfection with TIS21 cDNA. The serum-stimulated cell cycle was analyzed by fluorescence-activated cell sorting after double thymidine treatme nt; V10 progressed normally through the cell division cycle, but C9 an d C11 cells accumulated continuously in G(1) phase until 36 h after tr eatment. On the other hand, the progression of cells that had already entered to 5 or G(2)/M phase was not inhibited. When cell-cycle regula tory proteins were measured, C9 and C11 cells showed significantly red uced synthesis of cyclin E and cyclin-dependent kinase (cdk) 4 as well as a decrease in cyclin E-associated cdk activity. These observations led us to conclude that TIS21 overexpression in G(1) phase decreased the amounts of cyclin E and cdk4, thereby decreasing the activity of c dks at the G(1)-S transition. (C) 1998 Wiley-Liss, Inc.