Characterization of a vitamin D₃-resistant MCF-7 cell line

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃], the active metabolite of vitamin D₃, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. We have previously demonstrated that 1,25-(OH)₂D₃ induces morphology (pyknotic nuclei, chromatin and cytoplasmic condensation, and nuclear matrix protein reorganization) consistent with the activation of apoptosis in MCF-7 cells. These morphological changes in 1,2,5-(OH)₂D₃- treated cells are associated with up-regulation of TRPM-2/clusterin and cathepsin B (genes associated with mammary gland apoptosis) and down regulation of bcl-2, an antiapoptotic gene. Thus, the inhibitory effects of 1,25-(OH)₂D₃ on MCF-7 cell growth involve activation of apoptosis. To investigate the mechanisms by which vitamin D₃ activates apoptosis, we have selected a vitamin D₃-resistant variant (MCF-7(D3Res) cells) by continuous culture of MCF-7 cells in 100 nM 1,25-(OH)₂D₃. The MCF-7(D3Res) cells represent a stably selected phenotype that grows equally well with or without 100 nM 1,25-(OH)₂D₃. In contrast to the MCF-7 cells from which they were derived (MCF-7(WT) cells), MCF-7(D3Res) cells do not exhibit apoptotic morphology, DNA fragmentation, or up-regulation of apoptosis-related proteins after treatment with 1,25-(OH)₂D₃. MCF-7(D3Res) cells exhibit cross resistance to severed vitamin D₃ analogs that are potent growth regulators of MCF-7(WT) cells. MCF-7(WT) and MCF-7(D3Res) cells exhibit comparable sensitivity to induction of apoptosis and up regulation of clusterin in response to the antiestrogen 4-hydroxytamoxifen. MCF-7(D3Res) cells express comparable levels of the vitamin D receptor (VDR), as assessed by Western blotting or ligand binding, as MCF-7(WT) cells. In both sensitive and resistant cell lines, 1,25-(OH)₂D₃ up-regulates whereas 4-hydroxytamoxifen down-regulates VDR protein expression, indicating appropriate homologous and heterologous VDR regulation in MCF-7(D3Res) cells. Gel shift analyses indicate that nuclear extracts from MCF-7(WT) and MCF-7(D3Res) cells bind equally well to the DR3 consensus vitamin D₃ response element. These data suggest that MCF-7(D3Res) cells have a functional VDR that is uncoupled from a functional apoptotic pathway. MCF-7(D3Res) cells offer a unique model system for identification of the mechanisms by which vitamin D₃ regulates the cell death pathway in breast cancer cells.