STABLE DIFFERENTIATION OF A HUMAN COLON ADENOCARCINOMA CELL-LINE BY SODIUM-BUTYRATE IS ASSOCIATED WITH MULTIDRUG-RESISTANCE

Colorectal cancers are often com posed of cell types representing vari ous differentiated cell lineages, however little is known concerning t he relationship of differentiation and drug resistance in these cancer s. The present study was performed to develop and characterize a stabl e, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, t hen recultured in standard medium. Foci of flat-appearing cells appear ed and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differ entiated phenotype in standard culture conditions in the absence of so dium butyrate. It is characterized by the formation of a polarized mon olayer with dome formation and the presence of prominent apical microv illi and tight junctions. This cell line demonstrated reduced growth i n soft agar and nude mice compared with the parental cell line. LS174T -D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold an d 3.4-fold, respectively, in LS174T-D compared with parental cells. Pr oliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cis platin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-f old), and methotrexate (12.5-fold). Intracellular uptake of (H-3)-5-fl uorodeoxyuridine did not differ significantly in the differentiated an d undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione -S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion o f glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium buty rate induces or selects for colon cancer cells with features of entero cytic differentiation. This stably differentiated cell line is associa ted with glutathione-mediated multidrug resistance, and provides a mod el for further studies of differentiation in normal and cancerous colo n. (C) 1994 Wiley-Liss, Inc.*