Essential contribution of Ets-1 to constitutive Pim-3 expression in human pancreatic cancer cells.
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.
Li
Ying-Yi
YY
Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan.
Wu
Yu
Y
Tsuneyama
Koichi
K
Baba
Tomohisa
T
Mukaida
Naofumi
N
D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics; D000090063:Proto-Oncogene Mas; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C494041:ETS1 protein, human; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; C499249:PIM3 protein, human; D017346:Protein Serine-Threonine Kinases