Statins, administered alone or in combination with UCN-01, induce perturbations in protein prenylation. (A) U937 cells (left panels) were exposed for 18 hours to 100 nM UCN-01 (UCN) with or without 20 μM lovastatin (LV), 40 μM fluvastatin (FV), or 30 μM simvastatin (SV), respectively; alternatively, Jurkat cells (right panels) were treated with 150 nM UCN-01 with or without 20 μM lovastatin for 18 hours. (B) RPMI8226 cells (8226) were exposed to 100 nM UCN-01 with or without 10 μM lovastatin for 24 hours and U266 to 150 nM UCN-01 with or without 10 μM lovastatin for 48 hours. (C) U937 cells were incubated with low concentrations of UCN-01 (50 to 75 nM) with or without 2.5 to 5 μM lovastatin for 48 hours or 72 hours, respectively. For panels A-C, after treatment, cells were lysed and prenylation status of H-Ras, RhoB, and Rap1A was determined by Western blot analysis. Each lane was loaded with 30 μg protein; blots were subsequently stripped and reprobed for expression of β-actin to ensure equivalent loading and transfer of protein. The results of a representative experiment are shown; an additional study yielded equivalent results. UP indicates unprenylated (corresponding to slower-migrating bands); P, prenylated. (D) U937 cells were exposed to 100 nM UCN-01 with or without 20 μM lovastatin for 18 hours and U266 cells to 150 nM UCN-01 with or without 10 μM lovastatin for 24 hours. At the end of the incubation period, cells were lysed and subjected (400 μg protein per condition) to a Ras activation assay as described in “Ras activation assay” (right panels). In parallel, untreated U937 and U266 cells were lysed, and cell extracts incubated/loaded with 100 μM GTPγS or 1 mM GDP for 30 minutes at 30°C for positive and negative controls, respectively (left panels). Ras activity is reflected by the amount of Ras (Ras-GTP) pulled down by Raf-1 RBD agarose beads. IgG (H) indicates IgG heavy chain. The results are representative of 3 separate experiments.