1,25-Dihydroxyvitamin D(3) (1,25D(3)), the active metabolite of
vitamin D(3), inhibits
breast cancer cell growth in vivo and in vitro. To examine mechanisms of 1,25D(3) induced growth arrest and apoptosis, cell lines were established from DMBA induced mammary
tumors derived from
vitamin D(3) receptor knockout (VDRKO) and wild type (WT) mice. Two VDRKO (KO240, KO288) and two WT (WT145, WT276) cell lines were selected and characterized. All four cell lines express cytokeratins indicative of an epithelial origin, as well as
vimentin, which is expressed in many transformed cell lines. The tumorigenicity of the cells was confirmed in vivo as all four cell lines form
estrogen responsive
tumors in nude mice. Both WT cell lines express the VDR
protein and are sensitive to growth inhibition by 1,25D(3) at doses as low as 1 nM. Flow cytometric analysis indicated that 1,25D(3) induces G(0)/G(1) arrest and apoptosis in the WT cell lines. In contrast, both cell lines established from
tumors that developed in VDRKO mice lack VDR
mRNA and
protein. Cells from WT mice exhibit 1,25D(3) inducible transcriptional activity, as measured by reporter gene assays, but cells from VDRKO mice do not. Cells from VDRKO mice are also completely resistant to 1,25D(3) mediated growth arrest and apoptosis over the range of 0.01-100 nM 1,25D(3). VDRKO cells are also resistant to the synthetic
vitamin D(3) analogs
EB1089 and CB1093 that are more potent
growth inhibitors than 1,25D(3) in WT cells. This data conclusively demonstrate that the induction of cell cycle arrest and apoptosis in
breast cancer cells by 1,25D(3),
EB1089 and CB1093 is dependent on the nuclear VDR. Cells lacking VDR remain sensitive to growth arrest mediated by 9-cis
retinoic acid, a
ligand for the
retinoid x receptor which can heterodimerize with the VDR. Sensitivity to apoptosis induced by the
DNA damaging agent
etoposide is not altered in VDRKO cells, indicating that VDR ablation does not impair apoptotic pathways in general. All four cell lines display equal sensitivity to
tamoxifen induced growth arrest. These
estrogen responsive, transformed cell lines which differentially express the VDR provide a novel model system for identification of the mechanisms by which 1,25D(3) regulates proliferation and apoptosis in
breast cancer cells.