5547

Hepatocyte growth factor activator inhibitor type 2 (HAI-2)-related small peptide (H2RSP) is a small nuclear peptide recently identified in our laboratory. In this study, we examined the expression and cellular localization of H2RSP in normal, hyperplastic, adenomatous and carcinomatous colorectal tissues, and investigated the possible roles of H2RSP on cellular proliferation. Anti-H2RSP polyclonal antibody was raised against the recombinant human H2RSP. Immunohistochemistry and in situ hybridization were performed using surgical specimens of the patients with various gastrointestinal diseases. In normal gastrointestinal mucosae, H2RSP was immunohistochemically localized in the cytoplasm of the epithelial cells at the crypt and translocated into the nucleus of the surface epithelial cells. Its mRNA was detected mainly in the cells at the crypt by in situ hybridization. The hyperplastic epithelial cells showed almost same staining pattern as normal epithelium, while adenoma cells showed decreased or mixed nuclear and cytoplasmic stainig patterns. In adenocarcinomas, H2RSP was predominantly located in the cytoplasm of almost all cancer cells with various intensities, and nuclear localization was hardly visible. The level of H2RSP mRNA was apparently decreased in the cancer cells compared with corresponding normal epithelial cells by real-time RT-PCR. When cellular localization of H2RSP was examined in fractionated samples of cultured human colorectal carcinoma cells (DLD-1), H2RSP was gradually translocated from the cytoplasm into the nucleus along with the increased cellular density of DLD-1 cells in vitro. In addition, Chinese hamster ovary (CHO) cells stably transfected with H2RSP cDNA revealed the reduced cell growth in vitro. H2RSP was bound to poly(rG)-coated beads, and did not interact with any nuclear protein, single-stranded or double-stranded DNA in pull-down assay using GST-H2RSP fusion protein. The results suggest that H2RSP might be an RNA-binding protein, and have a potentially important role in the proliferation and/or differentiation of the gastrointestinal epithelial cells by translocating from the cytoplasm into the nucleus. Therefore, the impaired nuclear translocation of H2RSP may contribute to colorectal carcinogenesis.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]