CELLULAR RAS ACTIVITY IS REQUIRED FOR PASSAGE THROUGH MULTIPLE POINTS OF THE G(0)/G(1) PHASE IN BALB/C 3T3 CELLS

Microinjection experiments demonstrated a requirement for cellular ras activity late in G(1). In this study, we used two separate methods to identify an additional requirement for cellular ras activity early in the G(0)/G(1), phase of the cell cycle. Quiescent BALB/c cells were injected with anti-ras antibody prior to stimulation with serum. The cells would therefore be inhibited in progression through the cell cycle at the earliest point requiring ras function. Alternatively, cells were inhibited in late G(1) as in previous studies by injecting anti-ras several hours after serum addition to quiescent cells. The injected cultures were then treated with chemical cell cycle inhibitors known to function in mid-G(1). Cells injected with anti-ras prior to serum stimulation were retained at a point of ras requirement prior to the execution point of the chemical inhibitor, while cells injected 3 to 5 h after serum stimulation were retained at a point of ras requirement downstream of the execution point of the chemical inhibitor. To confirm these results, quiescent BALB/c cells were injected with anti-ras antibody prior to or several hours following serum addition. In this case, however, second injections of oncogenic ras or adenoviral E1A protein were performed to overcome the inhibitory effects of the anti-ras antibody. Cells injected prior to serum addition were clearly inhibited at an early point of Ras requirement since they required 5 or 6 h longer to enter S phase than cells injected with anti-ras antibody after serum addition.