Analogs of 1 alpha ,25-dihydroxyvitamin D-3 (1 alpha, 25(OH)(2)D-3) inhibit growth in vitro and in vivo of cells derived from a variety of tumors. Here, we examined the effects of 1 alpha ,25(OH)(2)D-3 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 alpha ,25(OH)(2)D-3 and EB1089 was observed, from complete G(0)/G(1) arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. All lines expressed similar levels of vitamin D, receptor (VDR) mRNA and protein, and no significant variation was observed in 1 alpha ,25(OH)(2)D-3-dependent induction of the endogenous 24-hydroxylase gene, or of a transiently transfected 1 alpha ,25(OH)(2)D-3- sensitive reporter gene. The antiproliferative effects of 1 alpha ,25(OH)(2)D-3 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 alpha, and the serum- and glucocorticoid-inducible kinase gene, sgk, Induction of GADD45 alpha 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 alpha and PCNA in treated cells, consistent with the capacity of GADD45 alpha to stimulate DNA repair. While 1 alpha ,25(OH)(2)D-3 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 alpha ,25(OH)(2)D-3 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 alpha ,25(OH)(2)D-3-dependent gene regulation in the presence of normal levels of functional VDRs.
