Identification of a novel gene product: Resveratrol-Induced Nuclear Protein (RINP)

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Background and Aim: Resveratrol is an antioxidant found in grape skins and other natural food products. Many studies have shown that resveratrol has anti-cancer properties. We have reported that Resveratrol significantly inhibits pancreatic cancer cell proliferation and induces apoptosis. The current study is aimed to To elucidate underlying molecular mechanisms for anti-cancer properties of resveratrol. Methods and results: An oligonucleotide microarray analysis was used to identify genes modified by resveratrol in CD18 pancreatic cancer cell line. Among multiple genes that are up- or down-regulated, we found an uncharacterized novel gene is significantly upregulated by Resveratrol, which is further confirmed by real time RT-PCR. With 5-RACE and 3-RACE (Rapid Amplification of cDNA End) PCR based on the EST sequence of the novel gene, a full length cDNA of the novel gene is cloned out. The cDNA is 1879 bp long containing a 615 bp open reading frame, which encodes a protein with 205 amino acid residues. Computational analysis using PSORT II online software suggests that the novel gene product contains a putative single strand DNA binding domain. Therefore, we named this novel gene product Resveratrol-induced DNA binding protein (RINP). By fusing ORF of the novel gene with V5 epitope sequence in a pLenti 6/V5 expression vector, we studied the localization of RINP by immunofluoresence staining and confocal microscope. We found that full length RINP is localized in the nucleus. Since both the N and C terminals of RINP are heavily basic and thus are possible sites for a nuclear localization signal, therefore, we established two truncated RINP expression vectors containing only either N-terminal or C-terminal RINP alone. We found that either N-terminal or C-terminal RINP alone is enough to signal the novel protein to nucleus. We also examined distribution of RINP in multiple normal human tissues using real time RT-PCR. We found that RINP is expressed in every tissue tested with highest expression in liver and placenta and the lowest in brain. Conclusion: We have characterized a novel gene induced by Resveratrol in pancreatic cancer cells, CD18 cell line, which contains a putative single strand DNA binding site and is localized to the nucleus. Further studies are undergoing to identify the function of this gene with a focus on its role in resveratrol-induced growth inhibition and apoptosis in cancer cells.