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Regulation of Gene Expression by 1,25-Dihydroxyvitamin D₃ and Its Analog EB1089 under Growth-Inhibitory Conditions in Squamous Carcinoma Cells

Departments of Physiology and Medicine (N.A., R.L., Y.B., A.B., J.H.W.) McGill University Montreal, Quebec H3G 1Y6, Canada
Department of Otolaryngology-Head and Neck Surgery (D.J.E., M.J.B.), and Montreal Center for Experimental Therapeutics in Cancer (M.J.B., J.H.W.) Jewish General Hospital and McGill University Montreal, Quebec, H3T 1E2, Canada

Analogs of 1,25-dihydroxyvitamin D₃ (1, 25(OH)₂D₃) inhibit growth in vitro and in vivo of cells derived from a variety of tumors. Here, we examined the effects of 1,25(OH)₂D₃ and its analog EB1089 on proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15, and SCC25. A range of sensitivities to 1,25(OH)₂D₃ and EB1089 was observed, from complete G₀/G₁ arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. All lines expressed similar levels of vitamin D₃ receptor (VDR) mRNA and protein, and no significant variation was observed in 1,25(OH)₂D₃-dependent induction of the endogenous 24-hydroxylase gene, or of a transiently transfected 1,25(OH)₂D₃-sensitive reporter gene. The antiproliferative effects of 1,25(OH)₂D₃ and EB1089 in SCC25 cells were analyzed by screening more than 4,500 genes on two cDNA microarrays, yielding 38 up-regulated targets, including adhesion molecules, growth factors, kinases, and transcription factors. Genes encoding factors implicated in cell cycle regulation were induced, including the growth arrest and DNA damage gene, gadd45, and the serum- and glucocorticoid-inducible kinase gene, sgk. Induction of GADD45 protein in EB1089-treated cells was confirmed by Western blotting. Moreover, while expression of proliferating cell nuclear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitation studies revealed increased association between GADD45 and PCNA in treated cells, consistent with the capacity of GADD45 to stimulate DNA repair. While 1,25(OH)₂D₃ and EB1089 modestly induced transcripts encoding the cyclin-dependent kinase inhibitor p21^waf1/cip1, no changes in protein levels were observed, indicating that p21^waf1/cip1 induction does not contribute to the antiproliferative effects of 1,25(OH)₂D₃ and EB1089 in SCC cells. Finally, in partially resistant SCC9 cells, there was extensive loss of target gene regulation (10 of 10 genes tested), indicating that resistance arises from widespread loss of 1,25(OH)₂D₃-dependent gene regulation in the presence of normal levels of functional VDRs.

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