Bifunctional Alkylating Agent-Induced p53 and Nonclassical Nuclear Factor κB Responses and Cell Death Are Altered by Caffeic Acid Phenethyl Ester: A Potential Role for Antioxidant/Electrophilic Response-Element Signaling
- Cell and Molecular Biology Branch, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland
- Address correspondence to:
James F. Dillman III, Cell and Molecular Biology Branch, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5400. E-mail: james.dillman{at}us.army.mil
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 κB (NF-κB) 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-κB signaling as degradation of inhibitor protein of NF-κBα (IκBα) occurred within 5 min after exposure to tumor necrosis factor-α. However, exposure to BFA induced nonclassical NF-κB signaling as loss of IκBα was not observed until 2 or 6 h in NHEK or HaCaT cells, respectively. Exposure of an NF-κB reporter gene-expressing HaCaT cell line to 12.5, 50, or 100 μM SM activated the reporter gene within 9 h. Pretreatment with caffeic acid phenethyl ester (CAPE), a known inhibitor of NF-κB 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-κB 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.
Footnotes
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The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.116145.
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ABBREVIATIONS: BFA, bifunctional alkylating agent(s); NM, nitrogen mustard (bis-(2-chloroethyl) methylamine or mechlorethamine); SM, sulfur mustard (bis-(2-chloroethyl) sulfide); NF-κB, nuclear factor κB; TNFα, tumor necrosis factor-α; IκB, inhibitor protein of nuclear factor κB; IKK, IκB kinase(s); IL, interleukin; ATM, ataxia-telangiectasia-mutated; ATR, ataxia-telangiectasia-related; ARE/EpRE, antioxidant response element/electrophilic response element; CAPE, caffeic acid phenethyl ester; NHEK, normal human epidermal keratinocytes(s); CEES, 2-chloroethyl ethylsulfide; TDG, thiodiglycol (2,2-thiodiethanol); DMSO, dimethyl sulfoxide; HU, hydroxyurea; LDH, lactate dehydrogenase; AhR, aryl hydrocarbon receptor; p90RSK, p90 ribosomal S6 kinase; Nrf2, nuclear factor E2-related factor 2; MEK, mitogen activated protein kinase kinase; ERK, extracellular signal-activated kinase; XRE, xenobiotic response element(s); IB, immunoblotted; PAGE, polyacrylamide gel electrophoresis.
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↵1 Current affiliation: Toxicology and Environmental Sciences, ExxonMobil Biomedical Sciences, Inc., Annandale, New Jersey.
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- Received October 25, 2006.
- Accepted January 2, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
