Adaptation to 5‐fluorouracil of the heterogeneous human colon tumor cell line ht‐29 results in the selection of cells committed to differentiation

Abstract

The HT-29 cell line contains a small proportion of differentiated polarized, enterocytic and mucus-secreting cell types (less than 95%) which can be selected under various pressure conditions, e.g., glucose deprivation or methotrexate. The purpose of the present work was to investigate whether this also applies to 5-fluorouracil (FUra). Stepwise adaptation of exponentially growing cells to 1, 5, 10 and 20 microM FUra results, after a phase of high mortality, in the emergence of adapted sub-populations with stable growth rates and curves, and IC50 6, 18, 37, and 110 times higher than in untreated cells respectively. FUra-adapted cells are all differentiated, according to 2 phenotypes: (I) polarized dome-forming cells which express carcinoembryonic antigen at their apical surface and (2) goblet cells which secrete a mucus of colonic immunoreactivity. These phenotypes are present in the parental population and are different from those selected e.g., by glucose deprivation or methotrexate. This differentiation pattern is maintained when the cells are subcultured in drug-free medium. Resistance to FUra is acquired through gene amplification as substantiated by a 4- to 6-fold increase of thymidylate synthase gene copies in cells stably adapted to the drug. Whether the same mechanism or others are responsible for the first steps of resistance to FUra remains to be elucidated. Altogether, these results support the hypothesis that some of the cells which are present in the parental line and are committed to differentiation possess advantages which allow them to immediately resist and secondarily adapt to FUra. Comparison of the differentiation characteristics of FUra-adapted cells with those from cells selected under other pressure conditions suggests that resistance and adaptation to either type of pressure may depend on the differentiated phenotype to which the cells are committed.

Schedule-dependent activity of 5-fluorouracil and irinotecan combination in the treatment of human colorectal cancer: in vitro evidence and a phase I dose-escalating clinical trial

“…In order to select HT-29 colon carcinoma cells resistant to FU (HT29 FUR) or to SN-38 (HT29 SN-38R), cells were continuously incubated in the presence of increasing concentrations of FU and SN-38 starting from 0.1 nM for both drugs (Lesuffleur et al, 1991). Cells were finally stabilised in the presence of 9 mM FU and 100 nM SN-38.…”

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

“…24,25 The TC7 clone was isolated from the colon cancer cell line Caco-2. 26 All cell lines were cultured as previously described 24,27 with modifications for TC7. 26 Experiments were done in 25 cm 2 or 75 cm 2 T-flasks (Corning Glass Works, Corning, NY) at 37°C in a 10%CO 2 /90% air atmosphere.…”

“…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.…”

“…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. Cells were grown on glass coverslips.…”

“…On the basis of our experience of drug resistance, 27 the IC 50 was determined after 6 days of continuous 5-FU exposure instead of 24 or 72 hr as done usually by other groups. The 8 cell lines displayed differences in 5-FU sensitivity as shown in Table I.…”