Li Ying-Yi, Wu Yu, Tsuneyama Koichi, Baba Tomohisa, Mukaida Naofumi
Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan.
Cancer Sci. 2009 Mar;100(3):396-404. doi: 10.1111/j.1349-7006.2008.01059.x. Epub 2008 Dec 16.
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.
我们先前证明，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，但在胰腺正常细胞中不表达。为了阐明胰腺癌细胞中Pim-3异常表达的分子机制，我们构建了与人类Pim-3基因5'-侧翼缺失突变体相连的荧光素酶表达载体，并用所得载体转染人类胰腺癌细胞。至-264 bp的区域对于Pim-3基因的组成型表达至关重要，Ets-1结合位点（-216至-211 bp之间）的突变降低了荧光素酶活性。此外，Ets-1 mRNA和蛋白在人类胰腺癌细胞系中与Pim-3一起组成型表达。染色质免疫沉淀分析表明Ets-1与人类Pim-3基因5'-侧翼区域（-249至-183 bp之间）组成型结合。用野生型Ets-1转染可诱导Pim-3启动子活性及其蛋白表达，而用显性负性Ets-1或Ets-1小干扰RNA（siRNA）转染则使其降低。此外，当将显性负性Ets-1和Ets-1 siRNA转染到人类胰腺癌细胞中时，它们会降低Bad在其Ser(112)位点的磷酸化水平并诱导细胞凋亡。最后，Pim-3 cDNA转染逆转了Ets-1 siRNA诱导的细胞凋亡增加以及Bad在其Ser(112)位点磷酸化水平的降低。这些观察结果表明，转录因子Ets-1可诱导人类胰腺癌细胞中Pim-3异常表达，并随后阻止细胞凋亡。
