Interferon-γ-Dependent Stimulation of Fas Antigen in SV40-Transformed Human Keratinocytes: Modulation of the Apoptotic Process by Protein Kinase C

• Published in: Journal of investigative dermatology Vol. 105; no. 6; pp. 810 - 815
• Main Authors: Takahashi, Hidetoshi, Kobayashi, Hiroya, Hashimoto, Yoshio, Matsuo, Shinobu, Iizuka, Hajime
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-12-1995
• Subjects: Apoptosis; Cell Differentiation; Cell Transformation, Viral; Cells, Cultured; fas Receptor - genetics; Humans; Interferon-gamma - pharmacology; Keratinocytes - cytology; Protein Kinase C - physiology; RNA, Messenger - analysis; Simian virus 40 - genetics; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0022-202X; 1523-1747
• DOI: 10.1111/1523-1747.ep12326577

Fas antigen is a cell membrane protein that has been suggested to mediate apoptosis. Using SV 40-transformed human keratinocytes, we investigated the Fas-antigen-dependent apoptotic process. The expression of Fas antigen m RNA was markedly induced by interferon-γ (IFN-γ) treatment (500 U/ml). After IFN-γ treatment in the presence of anti-Fas monoclonal antibody, apoptosis was induced, as detected by the formation of nuclesome-sized fragments of DNA and morphologically by apoptotic cells with round homogeneous nuclear beads detected by acridine orange staining. The apoptotic SV 40-transformed keratinocytes were analyzed quantitatively by enzyme-linked immunosorbent assay using anti histone and peroxidase-conjugated anti-DNA antibodies and peroxiadase-conjugated anti-DNA antibodies to detect cell death. The IFN-γ-and anti-Fas antibody-dependent apoptosis was observed by 3 h, and the maximal response was observed by 12 h. The induction of apoptosis was significantly augmented by treatment with 10 ng/ml 12-o-tetradecanoyl-phor-bol-13-acetate (TPA). TPA alone had no effect on either Fas antigen expression or on the apoptotic process. Other protein kinase C activators (1-olecyl-2-acetylglycerol and mezerein) also stimulated IFN-γ-dependent apoptosis, whereas 4-o-methyl phorbol myristate acetate, a very weak protein kinase C activator, had only a slight effect. The TPA-induced augmentation of apoptosis was inhibited by the protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2- methyl piperazine dihydrochloride (H-7). However, H-7 inhibited only the TPA-induced augmentation of apoptosis; the basal IFN-γ- and anti-Fas-dependent apoptosis remained in the presence of H-7. Northern blot analysis revealed that c-jun mRNA was induced by IFN-γ plus anti-Fas antibody treatment as well as by TPA treatment; the addition of IFN-γ alone to the incubation medium had no effect on the expression of c-jun mRNA. These results indicate that IFN-γ induces a Fas-antigen-dependent apoptotic process in SV40-transformed keratinocytes and that TPA augments the process through the activation of protein kinase C.