Tumor necrosis factor (TNF)-induced protein phosphorylation in a human rhabdomyosarcoma cell line is mediated by 60-kD TNF receptors (TR60).
In the present study, we used a cloned derivative, KYM-1D4, of the human rhabdomyosarcoma cell line, KYM-1, known to express high numbers of the two tumor necrosis factor (TNF) receptors, TR60 and TR80, and to be highly sensitive to TNF alpha-mediated cytotoxicity/antiproliferation, to investigate the role of TR60 and TR80 in protein phosphorylation. Using permeabilized KYM-1D4 cells, it was found that TNF alpha strongly induced phosphorylation of proteins of molecular weight 80, 65, 58, 42, and 30 kD. Addition of a monoclonal antibody (MoAb) against TR60 was shown to induce cytotoxicity/antiproliferation in KYM-1D4 cells and the same pattern of protein phosphorylation as TNF alpha, whereas addition of an MoAb against TR80 was both noncytotoxic and ineffective in inducing protein phosphorylation. In contrast, in a highly TNF alpha-resistant KYM-1-derived cell line, 37B8R, no protein phosphorylation was induced with either TNF alpha or the agonistic anti-TR60 MoAb. However, when 37B8R was allowed to revert to partial TNF sensitivity by culture in the absence of TNF alpha, the resultant cell line, 37B8S, was found to regain inducibility of protein phosphorylation by TNF alpha. These results indicate that expression of functional TR60 in KYM-1-related cell lines is principally involved in TNF-mediated cytotoxicity/antiproliferation and is necessary for the induction of protein phosphorylation. Nevertheless, the latter, although apparently strongly associated with cytotoxicity, was probably involved in protective mechanisms because protein kinase C inhibitors that inhibited TNF alpha and anti-TR60-induced phosphorylation increased the cytotoxic/antiproliferative response to these mediators.
Mire-Sluis
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Division of Immunobiology, National Institute for Biological Standards and Control, Potters Bar, Herts, UK.
Meager
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D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D012208:Rhabdomyosarcoma / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha