Abstract
UV irradiation triggers apoptosis through both the membrane death receptor and the intrinsic apoptotic signaling pathways. Bax, a member of the Bcl-2 family of proteins, translocates from the cytosol to the mitochondrial membrane during UV-induced apoptosis, but the regulation of Bax translocation by UV irradiation remains elusive. In this study, we show that Bax translocation, caspase-3 activation and cell death by UV irradiation are not affected by Z-IETD-fmk (caspase-8 inhibitor), but delayed by Pifithrin-alpha (p53 inhibitor), although Bid cleavage could be completely abolished by Z-IETD-fmk. Co-transfecting YFP-Bax and Bid-CFP into human lung adenocarcinoma cells, we demonstrate that translocation of YFP-Bax precedes that of Bid-CFP, there is no significant FRET (fluorescence resonance energy transfer) between them. Similar results are obtained in COS-7 cells expressing YFP-Bax and Bid-CFP. Furthermore, using acceptor photobleaching technique, we observe that there is no interaction between YFP-Bax and Bid-CFP in both healthy and apoptotic cells. Additionally, during UV-induced apoptosis there is downregulation of Bcl-x(L), an anti-apoptotic protein. Overexpression of Bcl-x(L) in cells susceptible to UV-induced apoptosis prevents Bax translocation and cell death, repression of Bid protein with siRNA (small interfering RNA) do not inhibit cell death by UV irradiation. Taken together, these data strongly suggest that Bax translocation by UV irradiation is a Bid-independent event and inhibited by overexpression of Bcl-x(L).
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenocarcinoma / enzymology
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Adenocarcinoma / metabolism
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Adenocarcinoma / pathology*
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Animals
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Apoptosis / drug effects
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Apoptosis / radiation effects*
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BH3 Interacting Domain Death Agonist Protein / genetics
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BH3 Interacting Domain Death Agonist Protein / metabolism*
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Benzothiazoles / pharmacology
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COS Cells
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Caspase 3 / metabolism
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Caspase 8 / metabolism
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Caspase Inhibitors
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Cell Line, Tumor
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Chlorocebus aethiops
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Cysteine Proteinase Inhibitors / pharmacology
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Enzyme Activation
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Fluorescence Resonance Energy Transfer
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Humans
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Lung Neoplasms / enzymology
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Lung Neoplasms / metabolism
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Lung Neoplasms / pathology*
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Oligopeptides / pharmacology
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Protein Transport
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RNA Interference
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RNA, Small Interfering / metabolism
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Recombinant Fusion Proteins / metabolism
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Time Factors
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Toluene / analogs & derivatives
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Toluene / pharmacology
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Transfection
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Tumor Suppressor Protein p53 / antagonists & inhibitors
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Tumor Suppressor Protein p53 / metabolism
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Ultraviolet Rays*
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bcl-2-Associated X Protein / genetics
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bcl-2-Associated X Protein / metabolism*
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bcl-X Protein / genetics
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bcl-X Protein / metabolism
Substances
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BAX protein, human
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BCL2L1 protein, human
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BH3 Interacting Domain Death Agonist Protein
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BID protein, human
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Benzothiazoles
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Caspase Inhibitors
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Cysteine Proteinase Inhibitors
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Oligopeptides
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RNA, Small Interfering
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Recombinant Fusion Proteins
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TP53 protein, human
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Tumor Suppressor Protein p53
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bcl-2-Associated X Protein
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bcl-X Protein
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benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone
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Toluene
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pifithrin
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CASP8 protein, human
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Caspase 3
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Caspase 8