Abstract
Abstract: :
Purpose: Uveal melanoma is the most common primary intraocular tumor in adults with an annual incidence of six to eight new cases per million. This cancer is characterized by its propensity to metastasize to liver. The identification of up– and down–regulated genes are important determinants of tumor development, growth and progression. A subtractive library was thus prepared to identify genes which are expressed in the primary tumor when compared to the normal tissue. Methods: The Suppressive Subtractive Hybridization (SSH) technique was used to isolate genes that are differentially expressed in a cell line (TP31) derived from primary uveal melanoma compared to uveal melanocytes. A library was thus constructed from the resulting subtracted cDNA and 484 clones were selected randomly and screened for differential expression. Dot blot arrays of clones from the subtracted library were hybridized with labeled probes from either TP31 cell line or uveal melanocyte populations. Clones that were recognized by the TP31 cell line probe and not by the uveal melanocyte probe were confirmed to be differentially expressed. Results: From the differentially expressed library, 123 candidate clones were selected, sequenced and compared to the human genome, cDNA, and EST databases. Among these clones, 36 have previously been characterized whereas 52 corresponded to EST and 35 are unknown. Primers were designed to specifically amplify by RT–PCR the 87 clones corresponding to EST or which are unidentified using cDNA from normal tissues as well as cell lines. As many as 10 clones were shown to be only expressed either by uveal melanoma or by both uveal melanoma and other types of cancer. The expression of these clones was further corroborated by Northern blot analysis. Conclusion: The analysis of the subtraction library revealed genes that are specifically expressed by primary uveal melanoma. Some of these genes are novel and thus represent candidate genes for the development of diagnostic tools for early screening of uveal melanoma.
Keywords: gene screening • melanocytes • tumors