This Application proposes to identify genes that may be up or downregulated in breast cancer cells after treatment with vitamin D. The Investigator, based on the fact that vitamin D induces apoptosis of breast cancer cells and that apoptosis is an active process that requires gene transcription and protein synthesis, hypothesizes that vitamin D induces a specific pattern of gene expression in breast cancer cells which triggers and/or facilitates apoptosis. Furthermore, as result of that gene induction by vitamin D, any alterations in the regulation and or expression of any of these genes could confer resistance to apoptosis. The Applicant proposes to identify and characterize genes induced by vitamin D that are involved in the apoptotic process in breast cancer cells as well as to determine their expression in relation to apoptosis and survival. Finally, she proposes to identify genes which are up- regulated in a breast cancer cell line selected for being resistant to the growth inhibitory effects of vitamin D. The breast cancer cell line MCF-7 as well as a vitamin D-resistant cell line (MCF-7 Dres) will be grown in the presence and absence of vitamin D, and cells will be harvested at four different time points. Total RNA will be extracted using conventional methods, and will be used to identify the genes mentioned in Aims 1 and 3 by differential display RT- PCR as well as for preparing CDNA libraries for isolating full length CDNAS that may represent novel genes. The expression of those genes will be confirmed, in vitro, by northern blotting of RNA derived from MCF-7 and MCF-Dres cells treated with vitamin D or a vitamin D analog (EB1089), as well as in other human breast cancer cell lines, in additional cancer cell lines and as in normal cell lines. Distribution and specificity of the expression of the vitamin D-regulated genes will be examined in vivo in the nude mouse in which breast cancer cells will be implanted into mammary fat pads. Different treatments will be applied to different groups of mice before excising the tumors. Tumors will be analyzed morphologically (EM, apoptotic and mitotic index), biochemically and at molecular level (temporal expression of vitamin D-regulated genes by northern blotting or RT-PCR, as well by in situ hybridization). Vitamin D-signalling will be characterized in MCF-7Dres cells, to confirm the functionality of the VDR in this cell line, by analyzing the induction of two known vitamin D regulated genes on western blots. Identification and characterization of genes related with survival in MCF-7Dres cells will be also assessed using the same methodology previously described in Specific Aim 1, and in vitro and in vivo studies (se stated in aim 2) will be performed in order to explore the expression of those genes. The results of this application may provide markers for assessing the efficacy of vitamin D and analogs in induction of tumor regression in human patients; and the characterization of genes involved in the effect of vitamin D on tumor cell growth will provide a framework for design of novel therapeutics which target the growth regulatory pathways.
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