Expression of constitutively activated Ras (Q61L) prevents lovastatin from interrupting UCN-01–mediated ERK1/2 activation and significantly attenuates apoptosis induced by the regimen. (A) U266 cells were stably transfected with constructs encoding a constitutively activated mutant (Q61L) form of H-Ras or its empty vector control (neo). Cells were then treated with 150 nM UCN-01 (UCN) with or without 10 μM lovastatin (LV) for 24 hours (upper panels) or 48 hours (lower panels), after which cells were lysed and subjected to Western blot analysis to monitor expression of H-Ras and phosphorylated ERK1/2; 30 μg protein was loaded in each lane, and blots were subsequently stripped and reprobed for expression of β-actin to ensure equivalent loading and transfer of protein. Results are representative of 3 separate experiments. (B) U266/neo (neo) and U266/Q61L (Q61L) cells were incubated with 150 nM UCN-01 with or without 10 μM lovastatin for 24 hours, after which cell lysates (100 μg protein per condition) were subjected to a Ras activation assay (lower panels) as described in Figure 4D. In parallel, lysates from untreated cells were incubated with 100 μM GTPγS or 1 mM GDP for 30 minutes at 30°C for positive and negative controls, respectively (upper panels). Two additional studies yielded equivalent results. (C-D) U266/Q61L and neo cells were exposed to 150 nM UCN-01 in the presence or absence of either 10 μM lovastatin or 5 μM PD184352 (PD) for 24 hours (C) or 48 hours (D), after which the percentage of apoptotic cells was determined by annexin V–FITC/flow cytometry. The results represent the means ± SD for 3 separate experiments performed in triplicate. **Significantly lower than values for U266/neo cells (P < .01).