Vitamin D3 is believed to reduce the risk of colon cancer, and serum levels inversely correlate with colorectal cancer incidence. The active metabolite, 1alpha,25-dihydroxyvitamin D3, has previously been shown to inhibit growth and promote differentiation of colon cancer cells. The vitamin D analogue, EB1089, is currently under clinical trial in a variety of cancers because of its growth-inhibitory effects in vitro and reduced hypercalcemic effects in vivo. The mechanism of growth inhibition by EB1089, however, remained to be determined. In this study we examined the effects of alpha,25-dihydroxyvitamin D3 and EB1089 on five colorectal tumor cell lines (two adenoma and three carcinoma) to determine the mechanism of growth inhibition and to ascertain whether premalignant adenoma cells were responsive to these agents. 1alpha,25-Dihydroxyvitamin D3 and EB1089 induced p53-independent apoptosis in adenoma and carcinoma cell lines in a dose-dependent manner between 10(-10) and 10(-6) M. EB1089, as well as inducing apoptosis, increased the proportion of cells in the G1 phase, particularly in the adenoma cell lines. In two of the three carcinoma cell lines (SW620 and PC/JW), levels of apoptosis induced by EB1089 were similar or greater than those induced by 1alpha,25-dihydroxyvitamin D3. Although the carcinoma cell line HT29 was relatively resistant to apoptosis induced by EB1089 compared with 1alpha,25-dihydroxyvitamin D3, EB1089 markedly inhibited cell yields. These observations offer promise for the clinical use of EB1089. To determine whether the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 was potentially via a differentiation pathway, alkaline phosphatase activity was measured as a marker of differentiation. Induction of alkaline phosphatase was observed in the floating apoptotic cells as well as in the adherent population. A link between the induction of differentiation and apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 is suggested by the occurrence of apoptosis subsequent to the induction of differentiation. To investigate the molecular pathway to apoptosis induction, members of the Bcl-2 family of proteins were examined (Bcl-2, Bcl-x, Bax, and Bak). Decreased Bcl-2 was observed in some cell lines, particularly in response to EB1089, but was not essential for apoptosis. Levels of the proapoptotic protein Bak, however, were consistently increased in all of the five cell lines in association with apoptosis induced by either agent. The results implicate Bak protein in the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 or its analogue EB1089. The ability of EB1089 to induce apoptosis in colorectal carcinoma cells suggests that this or other vitamin D analogues may prove clinically effective for the treatment of colorectal cancer. Furthermore, the fact that it induces cell cycle arrest and apoptosis in the premalignant adenoma cells may suggest an application in colorectal cancer chemoprevention.