Characterization of mammary tumor cell lines from wild type and vitamin D3 receptor knockout mice☆
Introduction
1α,25 Dihydroxycholecalciferol (1,25D3), the biologically active form of vitamin D3, is a steroid hormone that mediates effects on calcium homeostasis through its nuclear receptor, the vitamin D3 receptor (VDR). The presence of the VDR in normal mammary gland (Zinser et al., 2002) and the demonstration that 1,25D3 inhibits the development of carcinogen induced pre-neoplastic lesions in mammary gland organ culture (Mehta et al., 1997) suggest that 1,25D3 and the VDR play a role in mammary gland development and may protect against breast tumor development. In addition, 1,25D3 exerts potent growth inhibitory effects on estrogen-dependent and estrogen-independent breast cancer cells both in vitro and in vivo (Chouvet et al., 1986, Abe et al., 1991, VanWeelden et al., 1998, Simboli-Campbell et al., 1996, Flanagan et al., 1999). In the widely studied estrogen receptor (ER) positive human breast cancer cell line MCF-7, 1,25D3 and its analogs induce growth arrest in G0/G1 (Simboli-Campbell et al., 1997) and apoptosis (Welsh, 1994, Simboli-Campbell et al., 1996, James et al., 1996). The VDR is found in over 80% of human breast cancers and does not necessarily co-localize with ER, making it an attractive target for treatment of early and late stage breast cancers (Colston et al., 1989, Berger et al., 1991). To this end, a number of synthetic vitamin D3 analogs that induce mammary tumor regression in animal models with minimal calcemic side effects have been developed, and some are in clinical trials (Binderup et al., 1991, Colston et al., 1992, Gulliford et al., 1998).
Despite intense research into the mechanism of action of 1,25D3 and its synthetic analogs, the exact role of the VDR in mediating cellular responsiveness to the growth regulatory effects of vitamin D3 compounds is unclear. Although some studies have reported correlation between VDR expression and sensitivity to vitamin D3 compounds, sublines of MCF-7 cells selected for resistance to 1,25D3 mediated growth inhibition continue to express the VDR, which in some cases remains transcriptionally active (Narvaez et al., 1996, Hansen et al., 2001). In addition, some vitamin D3 compounds which exert potent growth regulatory effects bind weakly to the VDR, and the existence of a plasma membrane 1,25D3 binding protein linked to intracellular signal transduction pathways raises the possibility that some effects of vitamin D3 compounds may be mediated through non-genomic mechanisms (Norman et al., 2002). Alternatively, 1,25D3 mediated apoptosis, which is induced by supraphysiologic concentrations of 1,25D3 (∼100 nM 1,25D3), is associated with generation of reactive oxygen species and mitochondrial disruption (Narvaez and Welsh, 2001), and previous studies have not ruled out the possibility that mitochondrial metabolism of high concentrations of 1,25D3 could induce oxidative stress and trigger apoptosis independently of the VDR.
To gain additional insight into the coupling of the VDR to growth regulatory pathways in breast cancer cells, cell lines were established from mammary tumors that developed in mice with targeted ablation of the VDR and their wild type (WT) counterparts. In this study, we utilize these cell lines to conclusively demonstrate that 1,25D3 mediated growth arrest and apoptosis in breast cancer cells requires the nuclear VDR.
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Animals, tumor formation and cell isolation
For generation of cell lines, mammary tumors were induced with 7,12-dimethylbenzanthracene (DMBA) in mice homozygous for a null allele of the VDR (VDRKO mice) and their wild type counterparts (WT mice). The VDRKO mice used in these studies were derived from animals originally obtained from Dr Marie Demay (Massachusetts General Hospital, Boston, MA), who generated the VDRKO mice on the C57BL/6J background by targeted ablation of the second zinc finger of the DNA binding domain (Li et al., 1997).
Characteristics of cell lines derived from mammary tumors generated in WT and VDRKO mice
Four transformed cell lines were established from DMBA induced mammary tumors which developed in VDRKO (KO240, KO288) mice and their WT (WT145, WT276) littermates. The morphology of these cell lines (Fig. 1, left panels) varied from small cuboidal cells to large, flat cells with extensive projections. To assess whether the cell lines were epithelial in origin, immunohistochemistry was used to detect the presence of cytokeratins, which are expressed in epithelial but not mesenchymal cell types.
Discussion
In these studies we have characterized a series of transformed mammary cell lines derived from mice with targeted ablation of the VDR and their WT littermates. Epithelial cells were isolated in parallel from DMBA induced mammary tumors, and established cell lines, which have been in culture for over 12 months, were obtained. Immunohistochemical staining indicated that the four cell lines are all epithelial in origin and display markers, such as vimentin, characteristic of epithelial cells which
Acknowledgements
This work was supported by National Institutes of Health (CA69700 to J.E. Welsh) and a Susan G. Komen Foundation pre-doctoral traineeship award to G.M. Zinser. The authors are grateful to Mark Suckow, DVM, for DMBA dosing, Jennifer Welsh for cell culture and Emily Tribble for tumor histology. We would also like to thank Paul MacDonald for the VDR expression construct and John Omdahl for the 24-OHASE luciferase construct.
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Portions of this work were presented at the 15th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Munich (Munchen), Germany, May 2002.