Transfection of the H-2Kb and neor genes into BL6-8 (H-2Kb-, H-2Db+) melanoma clone resulted in various phenotypic changes with appearance of soybean agglutinin (SBA) and Grifonia Simplicifolia 1-B4 (GS1B4) lectin binding carbohydrates and loss of melanoma-associated antigen (MAA). In parallel H-2Kb gene-transfected melanoma cells showed increased sensitivity to TNF lysis. To further delineate the ability of H-2Kb gene to induce the phenotypic changes and TNF sensitivity, BL6-8 melanoma clone was transfected with the H-2Kb gene alone without cotransfection with neor gene and transfected cells were selected for adherence to SBA lectin-conjugated agarose beads. Analysis of isolated clones revealed that 38 of 47 tested clones have been found to be expressing the H-2Kb Ag, SBA, and GS1B4 lectin binding carbohydrates but lost MAA, e.g., H-2Kb+, lectin+, MAA-, and in parallel these cells became sensitive to TNF lysis. Although all clones with high expression of H-2Kb Ag were sensitive to TNF lysis, it seems unlikely that H-2K molecules are directly required for or involved in TNF-induced melanoma cell lysis. This conclusion is based on findings that four H-2Kb-transfected clones selected on SBA-agarose beads did not expressed H-2Kb Ag but manifested increase in SBA and GS1B4 lectin binding and loss of MAA and also became sensitive to TNF lysis. It seems that increase in TNF sensitivity is a part of the broad phenotypic changes induced by the H-2K gene that remained stable even in the clones in which the transfected H-2Kb gene was lost or down-regulated. We believe that the effects of the H-2Kb gene on melanoma cell phenotype and TNF sensitivity are indirect and are probably mediated via its inhibition of the melanoma-associated ecotropic retrovirus production and activation of some repressed cellular genes. Study of the mechanisms responsible for TNF sensitivity of BL6 melanoma cells revealed that the H-2Kb gene transfection resulted in an increase in p55 TNF receptor expression. TNF-induced activation of phospholipase A2 and release of arachidonic acid metabolites was observed only in the H-2Kb transfected, but not in BL6-8 melanoma cells transfected with neor or class II H-21Ak genes. TNF resistance of BL6 melanoma cells appeared to be due to a block in transduction of the lytic signal that was reversed after transfection with H-2Kb gene.
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