Regulation of gene expression by 1α,25-dihydroxyvitamin D3 and its analog EB1089 under growth-inhibitory conditions in squamous carcinoma cells
Regulation of gene expression by 1α,25-dihydroxyvitamin D3 and its analog EB1089 under growth-inhibitory conditions in squamous carcinoma cells
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DOI：
10.1210/me.15.7.1127
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发表时间：
2001-07-01
影响因子：
--
通讯作者：
White, JH
中科院分区：
文献类型：
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作者：
Akutsu, N;Lin, R;White, JH
1 α，25-二羟基维生素D-3（1 α，25（OH）（2）D-3）的类似物在体外和体内抑制源自多种肿瘤的细胞的生长。在此，我们研究了1 α，25（OH）（2）D-3及其类似物EB 1089对人头颈部鳞状细胞癌（SCC）细胞系SCC 4、SCC 9、SCC 15和SCC 25增殖和靶基因调控的影响。观察到对1 α，25（OH）（2）D-3和EB 1089的一系列敏感性，从SCC 25细胞的完全G（0）/G（1）停滞到SCC 9细胞生长的仅50%抑制。所有细胞系都表达了相似水平的维生素D受体（VDR）mRNA和蛋白质，并且在内源性24-羟化酶基因或瞬时转染的1 α，25（OH）（2）D-3敏感报告基因的1 α，25（OH）（2）D-3依赖性诱导中未观察到显著变化。1 α，25（OH）（2）D-3和EB 1089在SCC 25细胞中的抗增殖作用通过在两个cDNA微阵列上筛选超过4，500个基因来分析，产生38个上调的靶点，包括粘附分子、生长因子、激酶和转录因子。诱导编码与细胞周期调控有关的因子的基因，包括生长停滞和DNA损伤基因gadd 45 α，以及血清和糖皮质激素诱导的激酶基因sgk。通过Western印迹证实EB 1089处理的细胞中GADD 45 α蛋白的诱导。此外，虽然在EB 1089处理的细胞中增殖细胞核抗原（PCNA）的表达减少，但免疫共沉淀研究显示，在处理的细胞中GADD 45 α和PCNA之间的关联增加，这与GADD 45 α刺激DNA修复的能力一致。虽然1 α，25（OH）（2）D-3和EB 1089适度诱导编码细胞周期蛋白依赖性激酶抑制剂p21（waf 1）/（cip 1）的转录物，但未观察到蛋白水平的变化，表明p21（waf 1）/（cip 1）诱导对1 α，25（OH）（2）D-3和EB 1089在SCC细胞中的抗增殖作用没有贡献。最后，在部分耐药的SCC 9细胞中，靶基因调控大量丢失（检测的10个基因中有10个），表明在正常水平的功能性VDR存在下，1 α，25（OH）（2）D-3依赖性基因调控的广泛丢失引起耐药。
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.