Defects in apoptosis have been implicated in chemoresistance of colon cancer cells. We report here the ability to resist to 5-fluorouracil-induced apoptosis of 8 colon cancer cell lines differing in p53 and bax status: p53 ؊/0 bax ؉/؉ for TC7, SW480, HT-29; p53 ؉/؉ bax ؊/؊ for LS174T, LoVo; p53 ؉/؉ bax ؉/؊ for HCT116; p53 ؉/؉ or p53 ؉/0 bax ؉/؉ for LS513 or HCT-EB, respectively. To approximate to the in vivo therapy, the cell lines were exposed to a long-term treatment of 5-FU. The analysis of proteins implicated in the apoptotic pathway has shown that the independent analysis of p53 or bax status was not sufficient to predict the extent of drug-resistance of all cell lines. In p53 ؉/؉ cell lines but not in p53 ؊/0 cell lines, a low level of the pro-apoptotic Bax protein was correlated with a greater resistance of cells to 5-FU. In addition, we found that high levels of anti-apoptotic Bcl-2 and Bcl-x L proteins combined with a low level of Bax were correlated to high 5-FU resistance of wild-type p53 cell lines. The same correlation was obtained for 2 out of 3 mutated p53 cell lines. In conclusion, the relative levels of Bcl-2, Bcl-x L and Bax may altogether contribute to determine the resistance of a majority of colon tumor cells to long-term 5-FU treatment, whatever their p53 status.

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Adaptation of HT‐29 cells to increasing concentrations of methotrexate (MTX) results in the selection of differentiated populations which show sequential dose‐dependent changes of their differentiated phenotype with, at the highest concentrations (0.1 and 1 mM), a shift of differentiation from a mucus‐secreting to an enterocytic phenotype coinciding with an amplification of the DHFR gene. We show here that DHFR gene amplification itself does not play a role in the shift of differentiation. An alternative explanation is the presence, within the mucus‐secreting population, of an undetectable minor population of cells committed to enterocytic differentiation and able to develop resistance to higher concentrations of MTX. This was confirmed by cloning the population of cells resistant to 10 μM MTX. Out of 19 isolated clones, 17 were found to be mucus‐secreting and 2 enterocytic. We tested 9 of these clones for their ability to develop resistance to 0.1 mM MTX: only 1 of enterocytic phenotype, was found to develop resistance to this higher concentration and to amplify the DHFR gene. The ability of enterocytic cells to develop resistance to elevated MTX concentration through amplification of the DHFR gene was demonstrated in another enterocytic HT‐29 population selected by glucose deprivation. Enterocytic cells resistant to 10 μM MTX were also found, unlike mucus‐secreting cells, to be readily adaptable to 5‐fluorouracil, this occurring without amplification of the thymidylate synthase gene. Together these results highlight a previously uncharacterized relationship between commitment to enterocytic differentiation of colon‐cancer cells and their ability to develop resistance to MTX and 5‐fluorouracil. Int. J. Cancer 76:383–392, 1998.© 1998 Wiley‐Liss, Inc.