Essential contribution of Ets-1 to constitutive Pim-3 expression in human pancreatic cancer cells
Essential contribution of Ets-1 to constitutive Pim-3 expression in human pancreatic cancer cells
复制标题
DOI：
10.1111/j.1349-7006.2008.01059.x
复制
发表时间：
2009-03-01
期刊：
影响因子：
5.7
通讯作者：
Mukaida, Naofumi
中科院分区：
文献类型：
--
作者：
Li, Ying-Yi;Wu, Yu;Mukaida, Naofumi
我们之前证明了具有丝氨酸/苏氨酸激酶活性的原癌基因Pim-3在癌细胞中异常表达，而在胰腺的正常细胞中不表达。为了阐明Pim-3在胰腺癌细胞中异常表达的分子机制，我们构建了与人Pim-3基因5'侧缺失突变体连接的荧光素酶表达载体，并用该载体转染人胰腺癌细胞。高达-264 bp的区域是构成Pim-3基因表达所必需的，而Ets-1结合位点（-216和-211 bp之间）的突变降低了荧光素酶的活性。此外，Ets-1 mRNA和蛋白在人胰腺癌细胞系中与Pim-3共同组成表达。染色质免疫沉淀实验表明，Ets-1与人类Pim-3基因5'侧区-249 ~ -183 bp之间存在组成性结合。转染野生型Ets-1可诱导Pim-3启动子活性及其蛋白表达，转染优势型Ets-1或Ets-1小干扰RNA （siRNA）可降低Pim-3启动子活性及其蛋白表达。此外，显性负的Ets-1和Ets-1 siRNA转染人胰腺癌细胞后，可减少Bad丝氨酸磷酸化的数量（112）并诱导凋亡。最后，Pim-3 cDNA转染逆转了Ets-1 sirna诱导的细胞凋亡增加和Bad丝氨酸磷酸化降低（112）。这些观察结果表明，转录因子Ets-1可以诱导Pim-3的异常表达，从而阻止人胰腺癌细胞的凋亡。[j] .癌症科学2009；100:396-404。
We previously demonstrated that the proto-oncogene Pim-3 with serine/threonine kinase activity was aberrantly expressed in cancer cells but not in the normal cells of the pancreas. In order to elucidate the molecular mechanism underlying aberrant Pim-3 expression in pancreatic cancer cells, we constructed luciferase expression vectors linked to 5'-flanking deletion mutants of the human Pim-3 gene and transfected human pancreatic cancer cells with the resultant vectors. The region up to -264 bp was essential for constitutive Pim-3 gene expression, and the mutation in the Ets-1 binding site (between -216 and -211 bp) reduced luciferase activities. Moreover, Ets-1 mRNA and protein were constitutively expressed together with Pim-3 in human pancreatic cancer cell lines. Chromatin immunoprecipitation assay demonstrated constitutive binding of Ets-1 to the 5'-flanking region of human Pim-3 gene between -249 and -183 bp. Pim-3 promoter activity and its protein expression were induced by transfection with wild type-Ets-1 and were reduced by transfection with dominant negative-Ets-1 or Ets-1 small-interfering RNA (siRNA). Furthermore, dominant negative-Ets-1 and Ets-1 siRNA reduced the amount of Bad phosphorylated at its Ser(112) and induced apoptosis, when they were transfected into human pancreatic cancer cells. Finally, Pim-3 cDNA transfection reversed Ets-1 siRNA-induced increase in apoptosis and decrease in Bad phosphorylation at its Ser(112). These observations would indicate that the transcription factor Ets-1 can induce aberrant Pim-3 expression and subsequently prevent apoptosis in human pancreatic cancer cells. (Cancer Sci 2009; 100: 396-404).