Resistance of HBL100 human breast epithelial cells to vitamin D action
Resistance of HBL100 human breast epithelial cells to vitamin D action
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DOI：
10.1093/carcin/20.4.577
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发表时间：
1999-04-01
期刊：
影响因子：
4.7
通讯作者：
Mehta, R
中科院分区：
文献类型：
--
作者：
Agadir, A;Lazzaro, G;Mehta, R
维生素D类似物是乳腺癌细胞生长的有效抑制剂，但许多乳腺癌细胞系对维生素D的生长抑制作用表现出不同程度的抗性。在这项研究中，我们研究了人乳腺上皮细胞系HBL 100对1，25-二羟基维生素D-3 [1，25（OH）（2）D-3]的抗性机制，用猴病毒40（SV 40）大T抗原永生化的人乳腺癌细胞系HBL 100和维生素D敏感的乳腺癌细胞系ZR 75 -1，检测了维生素D-3受体（VDR）的表达、DNA结合和转录激活活性。蛋白质印迹分析显示，VDR基因在两种细胞系中的表达相当，然而，凝胶阻滞试验表明，核蛋白来自ZR 75 -1细胞，而不是来自HBL 100;细胞表达的维生素D反应元件（VDRE）的结合活性增加了9倍。利用瞬时转染实验，我们发现在ZR 75 -1中VDRE被激活了8倍，然而在HBL 100细胞中没有观察到响应于1，25（OH）（2）D-3的激活。另一方面，VDR表达载体的共转染可以恢复HBL 100细胞中1，25（OH）（2）D-3诱导的VDRE转录。此外，VDR在HBL 100细胞中的稳定表达导致细胞对1，25（OH）（2）D-3的生长抑制作用的敏感性增强。由于CV-1细胞表达很少的内源性VDR，VDR和大T抗原的相互作用在这些细胞中进行。通过瞬时共转染，我们观察到大T抗原的表达强烈抑制1，25（OH）（2）D-3诱导的CV-1细胞中的VDRE转录活性，并呈剂量依赖性。当VDR浓度为120 ng时，这种抑制作用被完全逆转。因此，维生素D-3对HBL 100细胞生长抑制作用的丧失可能是由于细胞内大T抗原的表达所致，进一步证明维生素D-3有效抑制细胞生长需要VDR。
Vitamin D analogs are effective inhibitors of breast cancer cell growth, but many breast cancer cell lines show various degrees of resistance to the growth inhibitory effect of vitamin D, In this study, we investigated the mechanism of 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] resistance of the human breast epithelial cell line HBL100, which had been immortalized by Simian virus 40 (SV40) large T antigen, We determined the expression, DNA binding and transactivation activity of vitamin D-3 receptor (VDR) in HBL100 and a vitamin D-sensitive ZR75-1 breast cancer cell line. Western blot analysis revealed a comparable expression of VDR gene in both cell lines, However, gel retardation assays demonstrated nuclear proteins from ZR75-1 cells but not from HBL100; cells expressed a 9-fold increase in the binding activity with a vitamin D response element (VDRE). Using a transient transfection assay, we showed that the VDRE was activated by 8-fold in ZR75-1, However, in HBL100 cells there was no activation observed in response to 1,25(OH)(2)D-3. On the other hand, co-transfection of a VDR expression vector could restore 1,25(OH)(2)D-3-induced VDRE transcription in HBL100 cells. Moreover, stable expression of VDR in HBL100 cells resulted in enhanced sensitivity of the cells to the growth inhibitory effect of 1,25(OH)(2)D-3. Since CV-1 cells express very little endogenous VDR, the interactions of VDR and large T antigen were carried out in these cells, By transient cotransfection, we observed that expression of the large T antigen strongly inhibited 1,25(OH)(2)D-3-induced VDRE transcriptional activity in a dose-dependent fashion in CV-1 cells. At 120 ng VDR concentration, the inhibition was completely reversed, Thus the loss of the growth inhibitory effect of vitamin D-3 in HBL100 cells may be caused by the expression of the large T antigen in the cells, and provide further evidence that VDR is required for efficient growth inhibition by vitamin D-3.