A Down's syndrome associated gene, Single Minded 2 gene short form (SIM2-s), is specifically expressed in colon tumors but not in the normal colon. Antisense inhibition of SIM2-s in a RKO-derived colon carcinoma cell line causes growth inhibition, apoptosis, and inhibition of tumor growth in a nude mouse tumoriginicity model. The mechanism of cell death in tumor cells is unclear. In the present study, we investigated the pathways underlying apoptosis. Apoptosis was seen in a tumor cell-specific manner in RKO cells but not in normal renal epithelial cells, despite inhibition of SIM2-s expression in both of these cells by the antisense. Apoptosis was depended on WT p53 status and was caspase-dependent; it was inhibited by a pharmacological inhibitor of mitogen-activated protein kinase activity. Expression of a key stress response gene, growth arrest and DNA damage gene (GADD)45alpha, was up-regulated in antisense-treated tumor cells but not in normal cells. In an isogenic RKO cell line expressing stable antisense RNA to GADD45alpha, a significant protection of the antisense-induced apoptosis was seen. Whereas antisense-treated RKO cells did not undergo cell cycle arrest, several markers of differentiation were deregulated, including alkaline phosphatase activity, a marker of terminal differentiation. Protection of apoptosis and block of differentiation showed a correlation in the RKO model. Our results support the tumor cell-selective nature of SIM2-s gene function, provide a direct link between SIM2-s and differentiation, and may provide a model to identify SIM2-s targets.