title: Bifunctional alkylating agent-induced p53 and nonclassical nuclear factor kappaB responses and cell death are altered by caffeic acid phenethyl ester: a potential role for antioxidant/electrophilic response-element signaling.
abstract: Bifunctional alkylating agents (BFA) such as mechlorethamine (nitrogen mustard) and bis-(2-chloroethyl) sulfide (sulfur mustard; SM) covalently modify DNA and protein. The roles of nuclear factor kappaB (NF-kappaB) and p53, transcription factors involved in inflammatory and cell death signaling, were examined in normal human epidermal keratinocytes (NHEK) and immortalized HaCaT keratinocytes, a p53-mutated cell line, to delineate molecular mechanisms of action of BFA. NHEK and HaCaT cells exhibited classical NF-kappaB signaling as degradation of inhibitor protein of NF-kappaBalpha (IkappaBalpha) occurred within 5 min after exposure to tumor necrosis factor-alpha. However, exposure to BFA induced nonclassical NF-kappaB signaling as loss of IkappaBalpha was not observed until 2 or 6 h in NHEK or HaCaT cells, respectively. Exposure of an NF-kappaB reporter gene-expressing HaCaT cell line to 12.5, 50, or 100 muM SM activated the reporter gene within 9 h. Pretreatment with caffeic acid phenethyl ester (CAPE), a known inhibitor of NF-kappaB signaling, significantly decreased BFA-induced reporter gene activity. A 1.5-h pretreatment or 30-min postexposure treatment with CAPE prevented BFA-induced loss of membrane integrity by 24 h in HaCaT cells but not in NHEK. CAPE disrupted BFA-induced phosphorylation of p53 and p90 ribosomal S6 kinase (p90RSK) in both cell lines. CAPE also increased nuclear factor E2-related factor 2 and decreased aryl hydrocarbon receptor protein expression, both of which are involved in antioxidant/electrophilic response element (ARE/EpRE) signaling. Thus, disruption of p53/p90RSK-mediated NF-kappaB signaling and activation of ARE/EpRE pathways may be effective strategies to delineate mechanisms of action of BFA-induced inflammation and cell death signaling in immortalized versus normal skin systems.
authors.affiliation: Cell and Molecular Biology Branch, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5400, USA.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Minsavage
authors.forename: James F
authors.initials: JF
authors.lastname: Dillman
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D015153:Blotting, Western; D002109:Caffeic Acids / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter / Q000235:genetics; D016158:Genes, p53 / Q000187:drug effects*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D008466:Mechlorethamine / Q000494:pharmacology*; D009151:Mustard Gas / Q000494:pharmacology*; D016328:NF-kappa B / Q000187:drug effects*; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives* / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon / Q000096:biosynthesis / Q000235:genetics; D020218:Response Elements / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection
chemical_list: D000477:Alkylating Agents; D000975:Antioxidants; D002109:Caffeic Acids; D016328:NF-kappa B; D018336:Receptors, Aryl Hydrocarbon; D008466:Mechlorethamine; D007770:L-Lactate Dehydrogenase; D008156:Luciferases; C055494:caffeic acid phenethyl ester; D010626:Phenylethyl Alcohol; D009151:Mustard Gas