Regulation of gene expression by 1 alpha,25-dihydroxyvitamin D-3 and its analog EB1089 under growth-inhibitory conditions in squamous carcinoma cells

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. Her e, we examined the effects of 1 alpha ,25(OH)(2)D-3 and its analog EB1089 o n proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15, and SCC25. A range of sensiti vities 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 prote in, and no significant variation was observed in 1 alpha ,25(OH)(2)D-3-depe ndent induction of the endogenous 24-hydroxylase gene, or of a transiently transfected 1 alpha ,25(OH)(2)D-3- sensitive reporter gene. The antiprolife rative effects of 1 alpha ,25(OH)(2)D-3 and EB1089 in SCC25 cells were anal yzed by screening more than 4,500 genes on two cDNA microarrays, yielding 3 8 up-regulated targets, including adhesion molecules, growth factors, kinas es, and transcription factors. Genes encoding factors implicated in cell cy cle regulation were induced, including the growth arrest and DNA damage gen e, gadd45 alpha, and the serum- and glucocorticoid-inducible kinase gene, s gk, Induction of GADD45 alpha protein in EB1089-treated cells was confirmed by Western blotting. Moreover, while expression of proliferating cell nucl ear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitati on studies revealed increased association between GADD45 alpha and PCNA in treated cells, consistent with the capacity of GADD45 alpha to stimulate DN A repair. While 1 alpha ,25(OH)(2)D-3 and EB1089 modestly induced transcrip ts encoding the cyclin-dependent kinase inhibitor p21(waf1)/(cip1), no chan ges in protein levels were observed, indicating that p21(waf1)/(cip1) induc tion 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 funct ional VDRs.