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¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
Interleukin-8 (IL-8), originally identified as a neufrophil chemotactic factor, exhibits a potent angiogenic activity, Here, we demonstrated the IL-8 mRNA is constitutively expressed in human hepatocellular cartinoma cells (HCCs) and also abundantly expressed in the hepatoma cell line, HepG2. However, IL-8 mRNA was not detected in uninvolved liver tissue surrounding HCCs.
In order to investigate the promoter regions essential for constitutive IL-8 gene expression in HCCs, we performed transient transfection experiments utilizing CAT reporter constructs containing deletions of the human IL-8 promoter. We found that the sequence 5'-AGGAAG-3' at -137 bp to -132bp of the IL-8 promoter was shown to be a polyomavirus enhancer A binding protein 3(PEAS) site which can cooperate with the activator protein 1(AP-1). The introduction of a mutation in either the PEA3 or AP-1 site abolished the constitutive promoter activity, and mutation at both the PEA3 and AP-1 sites showed the lowest IL-8 promoter activity in HepG2 cells. Moreover, electrophoretic mobility shift assay demonstrated constitutive formation of PEA3 and AP-1 complexes in HepG2 cells. In immunohistochemistiy analysis, PEA8 and IL-8 proteins coexisted in HCC tumors with marked angiogenesis.
Further, we applied cis-element double strand oligodeoxynucleotides (ODNs), "decoy" ODNs against PEA3 binding sites in order to trap PEA3 molecules in the nuclei of HCCs. Transfection of PEA3 decoy ODNs showed rapid regression of transplanted HepG2 cells in nude mice, which significantly inhibited angiogenesis with decreased IL-8 mRNA expression.
This is the first report that in HCCs PEA3 cooperates with AP-1 to play crucial a role in promoting constitutive IL-8 expression, which can induce tumor angiogenesis in hepatomas. Therefore, it is possible that transcription factor PEA3 is a new target of anti-angiogenic cancer therapy for hepatoma.