Identification of genes differentially expressed in melanoma sublines derived from a single surgical specimen characterised by different sensitivity to cytotoxic T-lymphocyte activity

In this study we used differential display technology in an attempt to obta in an insight into the mechanisms underlying escape of tumour cells to the specific cytotoxic T cell response. A primary tumour cell line and autologo us tumour infiltrating lymphocytes were raised from a metastatic melanoma s ample (ME15). Upon co-culture of tumour infiltrating lymphocytes with irrad iated tumour cells, CTL specific for neoplastic cells were generated and cl oned. Using a CTL clone, a cytotoxicity resistant tumour subline (ME15R) wa s immunoselected. We applied a]PCR-based differential display technique to amplify DNA sequences differentially expressed in ME15 sublines sensitive ( S) or resistant (R) to specific CTL killing. 10 different sequences whose e xpression was exclusively detectable in ME15S cells were identified. Five o f them matched with known expressed sequence tags encoding products of unid entified function. 2 showed high homology with a mitochondrial mRNA and wit h the gene encoding the S24 ribosomal protein. Most interestingly, genes co ding for glutamine synthetase, TGF-beta-3 and PAX3, a well-characterised tr anscription factor, were only expressed in ME15S cells. The latter gene was found to be transcribed in all healthy tissues tested, but only in a subgr oup of established melanoma cell lines. Taken together, our data underline the relevant potential of differential display technology in the molecular analysis of paired tumour lines endowed with different phenotypic character istics. Cloning of entire open reading frames and transfection studies are warranted to clarify the role of individual differentially displayed genes in the escape of tumour cells from cellular immune response.