The treatment of human HL-60 promyelocytic leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of tumor necrosis factor (TNF) transcripts. The study reported here has examined TPA-induced signaling mechanisms responsible for the regulation of TNF gene expression in these cells. Run-on assays demonstrated that TPA increases TNF mRNA levels by transcriptional activation of this gene. The induction of TNF transcripts by TPA was inhibited by the isoquinolinesulfonamide derivative H7 but not by HA1004, suggesting that this effect of TPA is mediated by activation of protein kinase C. TPA treatment also resulted in increased arachidonic acid release. Moreover, inhibitors of phospholipase A2 blocked both the increase in arachidonic acid release and the induction of TNF transcripts. These findings suggest that TPA induces TNF gene expression through the formation of arachidonic acid metabolites. Although indomethacin had no detectable effect on this induction of TNF transcripts, ketoconazole, an inhibitor of 5-lipoxygenase, blocked TPA-induced increases in TNF mRNA levels. Moreover, TNF mRNA levels were increased by the 5-lipoxygenase metabolite leukotriene B4. In contrast, the cyclooxygenase metabolite prostaglandin E2 inhibited the induction of TNF transcripts by TPA. Taken together, these results suggest that TPA induces TNF gene expression through the arachidonic acid cascade and that the level of TNF transcripts is regulated by metabolites of the pathway, leukotriene B4 and prostaglandin E2.
Tumor necrosis factor (TNF) is a polypeptide cytokine that is cytotoxic to some but not all tumor cells. The basis for resistance to the cytotoxic effects of this agent remains unclear. We have studied the development of TNF resistance in human ZR-75-1 breast carcinoma cells. ZR-75-1 cells have undetectable levels of TNF RNA and protein. However, TNF transcripts are transiently induced in these cells by exposure to recombinant human TNF. This induction of TNF RNA is associated with production of TNF-like protein in cell lysates and culture supernatants. Stable resistance to TNF-induced cytotoxicity develops when ZR-75-1 cells are exposed to increased concentrations of TNF. The TNF-resistant cells, designated ZR-75-1R, continuously express TNF transcripts and a TNF-like protein. Furthermore, ZR-75-1R cell supernatants contain cytotoxic activity that is abrogated by polyclonal antibody against TNF. The ZR-75-1R cells also possess TNF receptors that are occupied or down-regulated by the TNF-like protein. These findings thus suggest that (') TNF induces TNF transcripts and production of a TNF-like protein in ZR-75-1 cells and (it) resistance to TNF-induced cytotoxicity is associated with stable TNF expression.Tumor necrosis factor (TNF) is a polypeptide cytokine that exerts a wide variety of biological effects (1). TNF was originally identified by its ability to cause hemorrhagic necrosis in subcutaneous murine tumors (2). Subsequent studies showed that TNF is cytotoxic to certain murine and human tumors, both in vitro and in vivo (1-3).The cytotoxic effects of TNF appear to be cell cycledependent. TNF causes accumulation ofcells in G2 phase and cytolysis in late stages of mitosis (4). This cytolytic effect is enhanced by inhibitors of RNA and protein synthesis (5, 6). Tumor cells sensitive and resistant to TNF-induced cytotoxicity have similar numbers of cell surface receptors. Furthermore, both sensitive and resistant cells internalize and degrade TNF after receptor binding (7). Thus, cell surface receptors appear to be necessary but not sufficient for TNF cytotoxicity (8)(9)(10). The basis for the sensitivity or resistance of transformed cells to TNF remains unknown.Exposure of TNF-sensitive L-929 mouse fibroblasts to TNF results in the development of stable TNF resistance (11). Paradoxically, this TNF-resistant subline produces a TNF-like protein that is cytotoxic to the parent L929 cell line. We recently demonstrated that certain human epithelial tumor cell lines inherently resistant to TNF also produce both TNF mRNA and protein (unpublished work). These findings suggested that production of TNF may be associated with TNF resistance. The present studies investigated this relationship in human breast carcinoma cells sensitive to the cytotoxic effects of TNF in vitro. The results show that TNF treatment induces both TNF expression and resistance to TNF cytotoxicity. METHODSCell Culture. The ZR-75-1 human breast carcinoma cells were obtained from the American Type Culture Collection and grown in RPMI 1640 ...
Macrophage colony-stimulating factor (CSF-1; M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocytes. The effects of CSF-1 are mediated through binding to specific, high-affinity surface receptors encoded by the c-fms gene. CSF-1 and c-fms gene expression was investigated in fresh human acute myeloblastic leukemic cells by Northern blot hybridization using cDNA probes. 4.0-kb CSF-1 transcripts were detected in 10 of 17 cases of acute myeloblastic leukemia (AML), while c-fms transcripts were detected in 7 of 15. Coexpression of CSF-1 and c-fms was observed in five cases, and in five other cases neither gene was expressed. In situ hybridization demonstrated that transcripts for CSF-1 were present in 70-90% of cells in each of three cases studied while c-fms mRNA was detected in 40-70% of cells. The constitutive expression of CSF-1 transcripts was associated with production of CSF-1 protein, although detectable amounts of CSF-1 were not secreted unless the cells were exposed to phorbol ester. These results demonstrate that leukemic myeloblasts from a subset of patients with AML express transcripts for both the CSF-1 and CSF-1 receptor genes, often in the same leukemic cells in vitro.
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