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S. Landreville, C. Lupien, C. Salesse; Identification of Tumor Markers Potentially Important for Uveal Melanoma Development . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3412.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Uveal melanoma is the most prevalent primary cancer of the eye and the most common noncutaneous site of melanoma with an annual frequency of 6–8 cases per million of population in North America. Uveal melanoma is derived from malignant transformation of normal uveal melanocytes and this cancer is characterized by its propensity to metastasize to liver. Carcinogenesis occurs as a stepwise accumulation of molecular genetic events involving activation of oncogenes, inactivation of tumor–suppressor genes and dysfunction of proteins encoded by genes involved in DNA repair and maintenance of genomic stability. It is thus important to find genes that are involved in uveal melanoma development. Methods: The Suppressive Subtractive Hybridization (SSH) method was used to isolate genes that are differentially expressed in a cell line (TP31) derived from primary uveal melanoma compared to normal uveal melanocytes. Libraries were thus constructed from the resulting subtracted cDNAs. Clones were selected randomly and were confirmed to be overexpressed or downexpressed in the TP31 cell line using differential screening. Primers were designed to specifically amplify by RT–PCR some of these clones using cDNA from normal tissues as well as cell lines. Semi–quantitative RT–PCR analyses with uveal melanoma cells demonstrated that many genes are specifically expressed by uveal melanoma cells when compared to normal uveal melanocytes. Western and Northern blot analyses were used to corroborate the expression of these uveal melanoma specific genes. Results: We found a specific expression of calcineurin A alpha (CALNA), topoisomerase II alpha subunit (TOP2A) and colonic and hepatic tumor overexpressed gene (ch–TOG) in uveal melanoma by semi–quantitative RT–PCR analyses and identified a new splice variant of CALNA. The expression of this new variant of CALNA was confirmed by Western blot using a TP31 cell line extract. Semi–quantitative RT–PCR analyses also demonstrated that clones UVM1–35, UVM3–49 and UVM1–109, corresponding respectively to an EST, a hypothetical protein and an unknown were specifically expressed by uveal melanoma. Northern blot analyses using the TP31 cell line mRNA were used to determine the length of the complete transcript and the presence of splice variants of these 3 clones which are not yet characterized. Conclusions: This study resulted in a valuable collection of novel cDNA fragments. These libraries form the basis for more detailed expression analysis of specific genes in uveal melanoma and investigation of their putative roles.
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