Human colon cell line HT-29: Characterisation of 1,25-dihydroxyvitamin D 3 receptor and induction of differentiation by the hormone

Abstract

The human colon carcinoma cell line HT-29 differentiates into functional enterocytes upon replacement of glucose by galactose in the culture medium. Since the differentiation of other types of cells is associated with the modulation of 1,25-dihydroxycholecalciferol (1,25(OH) 2D 3) receptor concentrations and since enterocytes are classical target cells for 1,25(OH) 2D 3 we have examined the HT-29 cells to determine whether the differentiated and undifferentiated stages could be directly linked to the presence of 1,25(OH) 2D 3 receptors. HT-29 cells were grown in Dulbecco's modified medium containing 10% fetal calf serum (FCS) and glucose or galactose. Cell differentiation was assessed by measuring the brush border hydrolase, maltase. 1,25(OH) 2D 3 receptors were studied in the cells after 48 h without FCS. Nuclear uptake was measured in intact dispersed cells and the receptor protein was further characterized by vitamin D metabolite binding specificity, sucrose density gradient analysis and binding to DNA-cellulose. Maltase activity was 5-fold greater in differentiated HT-29 cells than in undifferentiated cells. Scatchard analysis showed a highly specific saturable (9500 sites per cell) high affinity (2 × 10 −10 M), binding of 1,25(OH) 2D 3 in undifferentiated cells. This receptor-like protein sedimented at 3.3S, bound to and eluted from DNA-cellulose and had all the characteristics of a 1,25(OH) 2D 3 receptor. No specific binding was detected in differentiated HT-29 cells. The presence of 1,25(OH) 2D 3 receptors in undifferentiated HT-29 cells implies that these cells are targets for vitamin D. The maltase activity increased significantly when undifferentiated cells were exposed to 1,25(OH) 2D 3 for 5–6 days, indicating that the hormone can promote differentiation of HT-29 cells. These results demonstrate that HT-29 cells can provide a new model for studying steroid receptor regulation and cell differentiation.