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ET-18-OCH₃ Inhibits Nuclear Factor-B Activation by 12-O-Tetradecanoylphorbol-13-acetate but not by Tumor Necrosis Factor- or Interleukin 1¹

Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157-1016 [L. W. D., F. C., M. A. R., P. K. S.], and Department of Genetics, Stanford University, Stanford, California 94305 [P. A. R., M. R.]

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) is a synthetic diether phospholipid that is competitive with phosphatidylserine binding to the regulatory domain of protein kinase C (PKC). Our previous studies indicate that the selective inhibition of tumor cell growth by ET-18-OCH₃ may be due to altered signal transduction mechanisms, including the inhibition of PKC. To further define the mechanism of action of ET-18-OCH₃, we have used it to study the role of PKC in regulation of the transcription factor NF-B, which is activated by diverse stimuli. In the 293.27.2 human kidney cell line, as in hematopoietic cells of all lineages, NF-B is stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), tumor necrosis factor- (TNF-), and interleukin-1 (IL-1). The response to either TNF- or IL-1 is synergistically enhanced by TPA. However, the regulatory mechanisms and signal transduction systems responsible for NF-B activation in response to these different stimuli have not been determined in detail. We have used ET-18-OCH₃ and auranofin, which inhibit PKC by different mechanisms, to assess the role of PKC in NF-B activation. ET-18-OCH₃ markedly inhibits TPA-induced NF-B activation, as measured by HIV long terminal repeat-directed expression of ß-galactosidase. The IC₅₀ for inhibition by ET-18-OCH₃ is approximately 2 µM, a noncytotoxic concentration. Inhibition of TPA-induced NF-B activation was dependent upon preincubation with ET-18-OCH₃, and the drug was active at approximately 2 mol% of total cellular phospholipid. ET-18-OCH₃ did not inhibit NF-B activation by either TNF- or IL-1, indicating that there are multiple distinct signal transduction pathways leading to activation of NF-B. We have confirmed these results using auranofin, an antirheumatic drug that is a specific PKC inhibitor interacting with the catalytic domain. Like ET-18-OCH₃, auranofin blocked NF-B activation by TPA but not by TNF- or IL-1. Also like the ether lipid, auranofin only partially blocked the synergy exhibited by TPA and TNF-. To confirm the role of NF-B in this response, we measured NF-B by electrophoretic mobility shift assay. Both ET-18-OCH₃ and auranofin inhibited cellular induction of the active NF-B complex in response to TPA but not in response to TNF-. Together, these data define distinct pathways leading to NF-B activation. One pathway, induced by TPA, is PKC dependent; the other, induced by TNF- or IL-1, is pKC independent. ET-18-OCH₃ inhibits only the PKC-dependent pathway and may be useful as a biological response modifier in combination with other chemotherapeutic agents.

¹ This work was supported in part by Grants CA-48995 from NIH and from the North Carolina Biotechnology Center. This study used the facilities of the Membrane Lipid and Tissue Culture core laboratories of the Comprehensive Cancer Center of Wake Forest University supported by CA-12197. F. C. was supported by a fellowship from L. Berti, Champion Industries, Winston-Salem, NC. M. A. R. was supported by a fellowship from the National Cancer Institute (CA-09045). M. R. is a Senior Fellow of the Leukemia Society of America.

² To whom requests for reprints should be addressed, at Department of Biochemistry, Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016.

³ Present address: Department of Developmental Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

⁴ Present address: German Cancer Research Center, D6900 Heidelberg, Germany.

Received 5/15/95. Accepted 8/23/95.

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