Abstract
Recently, apoptosis has been implicated as one of the end points of cells exposed to chemotherapeutic agents. The p53 and Bcl-2 family of proteins are involved in chemotherapy-induced apoptosis, but in a cell type-dependent manner. We sought to determine the roles played by the p53 and Bcl-2 family of proteins in 5-fluorouracil (5-FU)-induced apoptosis of human colon cancer cell lines. We first studied the p53 genetic and functional status, and then 5-FU, at inhibitory concentration of 50% (IC50) doses, was used to induce apoptosis, which was confirmed by morphological analysis and enzyme-linked immunosorbent assay (ELISA). Bcl-2, Bcl-X(L), Bax, Bad, Bak and p53 protein expression was analysed by Western blotting. Using five human colon cancer cell lines, we found that equitoxic (IC50) doses of 5-FU induced apoptosis in both wild-type p53 and mutant p53 cells. Analysis of the steady-state levels of Bcl-2 family proteins showed high expression of Bcl-X(L) in all of the cell lines except Colo320. Bcl-2 was expressed in two of them. Bax presented with the lowest basal expression and Bad showed homogeneous expression. On the other hand, Bak expression varied more than fivefold among these cells. In cells containing wild-type p53 (e.g. LoVo), 5-FU-induced apoptosis was accompanied by increased expression of Bax and Bak without consistent modulation of other bcl-2 family proteins. In contrast in cells containing mutant p53 (e.g. DLD1), Bak expression was remarkably increased. There was a significant correlation between chemosensitivity and Bcl-X(L) to Bax ratio, rather than Bcl-2 to Bax. In conclusion, these results suggest that some members of the Bcl-2 family of proteins, in human colon cancer cell lines, are modulated by 5-FU and that the ratio of Bcl-X(L) to Bax may be related to chemosensitivity to 5-FU.
Highlights
P53 genetic and functional status, and 5-FU, at inhibitory concentration of 50% (ICj) doses, was used to induce apoptosis, which was confirmed by morphological analysis and enzyme-linked immunosorbent assay (ELISA)
Anti--actin mouse monoclonal antibody (m_Ab) w-as purchased from Siama
Apoptotic cells are indicated by arrowheads doses (IC ) of 5-FU induced apoptosis in all cancer cell lines (CCCLs) as determined by morphological analy sis after acridine orange (AO) staining, (Figure 1)
Summary
5-FU wvas provided by Kyoxx a Hakko Kog-vo Co. Tokyo. Different cell numbers and incubation times wvith a chemotherapeutic agent were used to determine the optimal assay conditions for all experiments. Untreated cells w-ere used as the control. The determinations of significant differences amona the cell lines w-ere made w-ith the Mann-Whitney test. I.e. a DNA-directed effect is observed w-hen cells are continuously exposed for a relatixely long time (Inaba et al 1990: Aschele et al 1992). We confirmed this to be the case in our preliminarys experiments. All the experiments w-ere performed using the floating and attached cells. DNA sequences were determined by the dideoxy nucleotide-termination method w ith sequence primers synthesized in the amplified region.
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