Resistance to high concentrations of methotrexate and 5-fluorouracil of differentiated HT-29 colon-cancer cells is restricted to cells of enterocytic phenotype

Abstract

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.

Requirement of both mucins and proteoglycans in cell‐cell dissociation and invasiveness of colon carcinoma HT‐29 cells

“…25 Furthermore, this clone highly expresses 2 MUC genes, which were previously studied in colon carcinoma: MUC5AC, which is not expressed in normal colon but found in colon carcinomas, and MUC1, which is upregulated in colon carcinomas and associated to poor outcome. 18 -21,23 We showed a significant invasive behavior for HT-29 5M21 cloned cells, whereas parental HT-29 cells were not invasive, in in vitro assay on collagen type I.…”

“…The parental HT-29 cell-line, 29 referred to as HT-29 STD, and HT-29 5M21 cells, cloned from a HT-29 cell subpopulation resistant to 10 Ϫ5 M methotrexate, 25 were a gift from Dr. T. Lesuffleur (Unité 505 INSERM). Cells were grown in DMEM (Life Technology Gibco), supplemented with 10 % fetal calf serum and antibiotics (50 i.u./ml penicillin and 50 g/ml streptomycin).…”

“…37 However, cloning of HT-29 MTX subpopulation has shown a great diversity in the phenotypic properties expressed by the cells of this subpopulation, particularly in terms of MUC1 and/or MUC5AC expression. 25 On the other hand, the type of extracellular matrix is also known to influence the behavior of cells via the activation of different integrins. Interestingly, the present experiments allowed characterization of an HT-29 clone that displayed a spontaneous invasive behavior in collagen type I in comparison to the parental cell population, and this first observation provided a cellular model allowing determination of the molecular partners of such behavior.…”

“…A homogenous subpopulation of mucin-secreting cells was selected by culture of the HT-29 cell line in the presence of increasing concentrations of the anticancer-drug methotrexate (MTX), and cell cloning of this subpopulation yielded a highly mucin-secreting clone (5M21). 24,25 In contrast to the parental HT-29 cells, which display a round morphology and develop as a cell multilayer, HT-29 5M21 clone grows as a monolayer of polarized cells which secrete an abundant mucus layer, with MUC5AC being the major gel-forming mucin.…”

Resistance of colon cancer cells to long‐term 5‐fluorouracil exposure is correlated to the relative level of Bcl‐2 and Bcl‐XL in addition to Bax and p53 status

“…For continuous drug treatment, 5-fluorouracil (Sigma Chemical Co., St. Louis, MO) at the indicated concentrations was added 24 hr after seeding and then every day during several weeks as previously described. 27,28 Determination of IC 50 of 5-FU Exponentially growing cells were seeded, at the same density as reported above, in 96-well microtiter plates (Corning) in the presence of increasing concentrations of 5-FU and analyzed after 6 days as previously described. 27 Determination of apoptosis Nuclear fragmentation.…”