Figure 3.

β-Lapachone selectively induced elevation of E2F1 protein in human pancreatic cancer cells (A) and colon cancer cells (B), but not in normal colonic epithelial cells (C). (A) Human pancreatic cancer cells (Paca-2) were exposed to β-lapachone at 0 μM (lane 1), 0.5 μM (lane 2), 2 μM (lane 3), and 4 μM (lane 4) for 0.5 h and were harvested for determination of E2F1 level by Western blot. Monoclonal antibody against E2F1 was obtained from Santa Cruz Biotechnology. (B) SW480 and NCM460 were treated with β-lapachone at 2 μM and were harvested after 20 min (lane 2), 1 h (lane 3), 2 h (lane 4), or 4 h (lane 5). Control cells were treated with an equal volume of DMSO (lane 1). Whole-cell extracts were prepared, and the E2F1 level was determined by Western blotting with a monoclonal antibody against E2F1. (C) Effects of β-lapachone on E2F2, -3, -4, and -5 were similarly determined from the same set of cell lysates of B with monoclonal antibodies from Santa Cruz Biotechnology. β-Actin was used as a loading control. (D) SW 480 cells (lanes 1–5) and NCM 460 cells (lanes 6–8) were treated with β-lapachone at 4 μM for 0.5 h (lanes 2 and 7), 1 h (lanes 3 and 8), 2 h (lane 4), or 4 h (lane 5). Control cells were treated with an equal volume of DMSO (lanes 1 and 6). Nuclear extracts were prepared, and E2F1 activity was determined by the electromobility shift assay by using a ³²P-labeled, 100-bp, double-stranded DNA fragment containing three E2F consensus sequences (18). Arrow denotes the location of different forms of E2F protein–DNA complexes. Results represent one of three independent experiments. (E) Expression of pRB and phosphorylation status in cell lines used. Lane 1, DLD1; lane 2, SW480; lane 3, NCM 460; lane 4, MCF7; lane 5, MCF10A. Whole-cell lysates were prepared, and Western blotting was used to analyze pRB by using a monoclonal antibody from Santa Cruz Biotechnology.