Abstract
Tumour necrosis factor (TNF)-alpha induces apoptosis in a human acute myeloid leukaemia cell line, Kasumi-1. To examine the role of protein phosphorylation in signal transduction of TNF-alpha-induced apoptosis, a variant cell line resistant to TNF-alpha was established by an intermittent challenge of Kasumi-1 cells with increasing concentrations of TNF-alpha for 6 months. The mechanism of resistance to TNF-alpha appears to be in the post-receptor pathway because expression of p55 TNF receptor in the variant cells is increased compared with that of the parental Kasumi-1 cells. In renaturation assays, TNF-alpha induced a rapid activation of different protein kinases of different molecular weights, including the 50 kDa protein kinase (PK50) followed by the 35 kDa protein kinase (PK35), in the parental Kasumi-1 cells. The dose-response of TNF-alpha required to activate PK50 and PK35 was closely related to concentrations of TNF-alpha that induced apoptosis. Treatment of Kasumi-1 cells with ceramide also activated PK35. In TNF-resistant variant cells, activation of PK35 in response to TNF-alpha or ceramide was practically nil. These findings suggest that activation of PK35 through the ceramide pathway may play an important role in signal transduction of TNF-alpha in the Kasumi-1 cell line, while the decreased activation of PK35 may explain the insensitivity of the variant cells towards TNF-alpha.
Highlights
We identified a rapid activation by Tumour necrosis factor (TNF)-o of the 50 kDa protein kinase (PK50) followed by activation of the 35 kDa protein
TNF-a failed to activate PK35 in the TNF-resistant variant cells selected from Kasumi-1 cells. These findings suggest that PK50 and PK35 play important roles in the signal transduction of TNF-a-induced apoptosis in the human myeloid leukaemia cell line, Kasumi-1
Cytological studies under light microscopy revealed that TNF-a-treated parental cells exhibited morphological features consistent with apoptosis, including marked condensation and fragmentation of nuclei, whereas untreated parental cells and both treated and untreated variant cells rarely exhibited apoptosis
Summary
Reagents [y-32P]ATP (3000 Ci mmol-h) was purchased from ICN Biomedicals (Costa Mesa, CA, USA). 4-[3-(4-Iodophenyl)2- (4-nitrophenyl) - 2H -5- tetrazolio]-1,3 - benzene disulphonate (WST-1) and 1-methoxy-5-methylphenazinium methylsulphate (1-methoxy PMS) were purchased from Dojindo (Kumamoto, Japan). Reagents [y-32P]ATP (3000 Ci mmol-h) was purchased from ICN Biomedicals (Costa Mesa, CA, USA). 4-[3-(4-Iodophenyl)2- (4-nitrophenyl) - 2H -5- tetrazolio]-1,3 - benzene disulphonate (WST-1) and 1-methoxy-5-methylphenazinium methylsulphate (1-methoxy PMS) were purchased from Dojindo (Kumamoto, Japan). Myelin basic protein (MBP), 0phosphoserine, O-phosphothreonine, O-phosphotyrosine, ceramide and ATP were purchased from Sigma (St Louis, MO, USA). Human TNF-a and monoclonal antibodies against P55 and P75 TNF receptors were purchased from Genzyme (Cambridge, MA, USA). Granulocyte macrophage colony-stimulating factor (GM-CSF) was a gift from Sandoz. Monoclonal anti-mitogen-activated protein (MAP) kinase was purchased from Zymed (San Francisco, CA, USA). Sepharose-conjugated goat anti-mouse IgG was purchased from Organon Teknika (West Chester, PA, USA)
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