Bhagavathula Narasimharao, Hanosh Andrew W, Nerusu Kamalakar C, Appelman Henry, Chakrabarty Subhas, Varani James
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Int J Cancer. 2007 Oct 1;121(7):1455-62. doi: 10.1002/ijc.22858.
An siRNA directed against the extracellular calcium-sensing receptor (CaSR) was used to down-regulate this protein in CBS colon carcinoma cells. In additional studies, we utilized a variant of the parental CBS line that demonstrates CaSR expression but does not upregulate this protein in response to extracellular Ca(2+). In neither the siRNA-transfected cells nor the Ca(2+)-nonresponsive variant cells did inclusion of Ca(2+) in the culture medium inhibit proliferation or induce morphological alterations. Extracellular Ca(2+) also failed to induce E-cadherin production or a shift in beta-catenin from the cytoplasm to the cell membrane. In mock-transfected cells and in a Ca(2+)-responsive variant line derived from the same parental CBS cells, Ca(2+) treatment resulted in growth-reduction. This was accompanied by increased E-cadherin production and a shift in beta-catenin distribution from the cytoplasm to the cell membrane. Additionally, down-regulation of c-myc and cyclin D1 expression was observed in mock-transfected cells and in the Ca(2+)-responsive variant line (along with reduced T cell factor transcriptional activation). Neither c-myc nor cyclin D1 was significantly down-regulated in the siRNA-transfected cells or in the Ca(2+)-nonresponsive variant cells upon Ca(2+) stimulation. In histological sections of human colon carcinoma CaSR was significantly reduced as compared to the level in normal colonic crypt epithelial cells. Where CaSR expression was high, strong surface staining for E-cadherin and beta-catenin was observed. Where CaSR expression was reduced, beta-catenin surface expression was likewise reduced.
一种针对细胞外钙敏感受体（CaSR）的小干扰RNA（siRNA）被用于下调CBS结肠癌细胞中的这种蛋白质。在其他研究中，我们使用了亲代CBS细胞系的一个变体，该变体显示CaSR表达，但对细胞外Ca²⁺无反应，不会上调这种蛋白质。在siRNA转染的细胞和Ca²⁺无反应的变体细胞中，培养基中加入Ca²⁺均未抑制细胞增殖或诱导形态改变。细胞外Ca²⁺也未能诱导E-钙黏蛋白的产生，或使β-连环蛋白从细胞质转移到细胞膜。在模拟转染的细胞以及源自同一亲代CBS细胞的Ca²⁺反应性变体细胞系中，Ca²⁺处理导致细胞生长减少。这伴随着E-钙黏蛋白产生增加以及β-连环蛋白分布从细胞质转移到细胞膜。此外，在模拟转染的细胞和Ca²⁺反应性变体细胞系中观察到c-myc和细胞周期蛋白D1表达下调（同时T细胞因子转录激活减少）。在Ca²⁺刺激下，siRNA转染的细胞或Ca²⁺无反应的变体细胞中，c-myc和细胞周期蛋白D1均未显著下调。在人类结肠癌的组织学切片中，与正常结肠隐窝上皮细胞相比，CaSR显著减少。在CaSR表达高的部位，观察到E-钙黏蛋白和β-连环蛋白的强表面染色。在CaSR表达降低的部位，β-连环蛋白的表面表达同样降低。
