Abstract
Human HeLa cells and murine L(S) cells are highly sensitive to the cytocidal activity of tumor necrosis factor (TNF) when simultaneously treated with the inhibitor of protein synthesis cycloheximide. This cytocidal activity of TNF was inhibited up to 90% in both cell lines after a 15-60-min pretreatment with 3-10 ng/ml of phorbol 12-myristate 13-acetate (PMA). This inhibition was long lasting for HeLa cells but transient for L(S) cells. The protection afforded by PMA was most effective when the cells were pretreated with this phorbol ester, but it decreased when PMA was added together with TNF or after TNF addition. This finding suggested that PMA interfered with one of the early steps in the mechanism of action of TNF. A pretreatment with the calcium ionophore A23187 also reduced the cytocidal activity of TNF in both HeLa and L(S) cells to about the same extent. Treatment of these cells with either PMA or A23187 significantly decreased the binding of 125I-TNF to cell surface receptors. This decrease paralleled the time course and dose-response of the inhibition of cytocidal activity. In addition, treatment of HeLa cells with 1-oleyl-2-acetyl-glycerol (OAG) also induced a rapid loss of TNF binding capacity. Since OAG, PMA, and A23187 are all activators of protein kinase C (Ca2+/phospholipid-dependent enzyme), these results suggest that this kinase is involved in modulation of TNF sensitivity. Furthermore, depletion or inhibition of protein kinase C antagonized PMA-induced effects on TNF cytotoxicity and binding to receptors. Internalization of bound TNF was not significantly affected by PMA treatment, and Scatchard analysis of binding data indicated that PMA decreased TNF receptor binding affinity rather than the number of TNF-binding sites. These findings suggest that protein kinase C may have a physiological role in mediating TNF sensitivity.
Highlights
Human HeLa cells and murine L(S) cells are highly mediated by its binding to specific receptors
Tumor necrosis factor (TNF)’ is a protein originally identified as amediator of antitumor activity present in the serum of mice sequentially injected with Bacillus Calmette-Guerin
The increase in cytotoxicity in L(S) cells treated with 30 ng/ml of PMA for more than 1 h was much greater than the increase in TNF binding. This observation may be explained by the previous observation that a relatively small number of receptors is sufficient for the cytocidal activity of TNF in some cell lines [8].These results suggest that the inhibition of cytotoxicity by PMA is correlated with a reduced binding of TNF to specific receptors
Summary
Inhibition of the Cytocidal Activityof TNF by PMA-Human HeLa S2cells and murine L(S) cells are sensitive to the cytocidal activity of TNF in the presence of the inhibitor of protein synthesis CHX[21]. This activity of TNF is measured by cytotoxicity assays. HeLa and L(S)cells were treated with CHX and different doses of TNF in short-term cytotoxicity assays. Correlation between the Inhibition of Cytotoxicity by PMA and Decreased Binding of TNF to Cellular Receptors-A 30min treatment with different doses of PMA reduced the binding of lZ5I-TNF to HeLa and L(S) cells (Fig. 2).
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