ESTIMATION OF PCNA MESSENGER-RNA STABILITY IN CELL-CYCLE BY A SERUM-DEPRIVATION METHOD

A simple scheme was developed to study the mRNA stability of the proli ferating cell nuclear antigen (PCNA) gene during cellular transition f rom the G(1)/S boundary to a quiescent state. By this scheme, CHO.K1 c ells were grown to about 80% confluence and then serum-starved for 40 h for synchronization in a quiescent state. The quiescent cells were s erum-stimulated for a period of time (between 8 h and 12 h) and then g rown in serum-free medium until being harvested for further analyses. The cellular PCNA mRNA level was analyzed by Northern blotting. As com pared with that in cells which were continuously incubated in serum-co ntaining medium, the decline of the mRNA level, after reaching the pea k, in these serum-deprived cells was virtually devoid of mRNA synthesi s. Thus, this mRNA decay was taken for the measurement of mRNA stabili ty. The advantage of the scheme is that, unlike the treatment of trans cription inhibitors, it does not prevent the cells from completing the rest of the cell cycle before returning to the resting state, and so the mRNA stability observed is fell cycle dependent. In contrast with the previous report that the stability of PCNA mRNA in quiescent cells is less by severalfold than that in S phase cells, our study shows th at the mRNA stability of PCNA remained constant during the cellular tr ansition from G(1)/S boundary to quiescent state. (C) 1995 Wiley-Liss, Inc.