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 obtain an insight into the mechanisms underlying escape of tumour cells to the specific cytotoxic T cell response. A primary tumour cell line and autologous tumour infiltrating lymphocytes were raised from a metastatic melanoma sample (ME15). Upon co-culture of tumour infiltrating lymphocytes with irradiated tumour cells, CTL specific for neoplastic cells were generated and cloned. Using a CTL clone, a cytotoxicity resistant tumour subline (ME15R) was 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 expression was exclusively detectable in ME15S cells were identified. Five of them matched with known expressed sequence tags encoding products of unidentified function. 2 showed high homology with a mitochondrial mRNA and with the gene encoding the S24 ribosomal protein. Most interestingly, genes coding for glutamine synthetase, TGF-beta-3 and PAX3, a well-characterised transcription factor, were only expressed in ME15S cells. The latter gene was found to be transcribed in all healthy tissues tested, but only in a subgroup 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 characteristics. 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.
Kocher
T
T
Department of Surgery, University of Basel. Thomas.Kocher@unibas.ch
Schraml
P
P
Spagnoli
G C
GC
Harder
F
F
Heberer
M
M
D001483:Base Sequence; D003602:Cytotoxicity, Immunologic; D015342:DNA Probes; D018432:Drug Resistance, Multiple; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000473:pathology; D008545:Melanoma / Q000235:genetics* / Q000276:immunology / Q000473:pathology / Q000601:surgery; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger