* Division of Nephrology, Department of Medicine;
Department of Anatomy and Cell Biology, Uniformed Services University, Bethesda, Maryland 20814
Received September 3, 1999; accepted November 10, 1999
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ABSTRACT |
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Key Words: gliotoxin; cytotoxicity; apoptosis; LLC-PK1 cells; TNF-; caspase; reactive oxygen species.
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INTRODUCTION |
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Apoptosis is a form of programmed cell death that accompanies a variety of diseases and disease models including renal diseases (Davis and Ryan, 1998). Gliotoxin has been shown to induce apoptosis in thymocytes, peripheral lymphocytes, macrophages, and L929 fibroblasts in vitro, and in thymus, spleen, and mesenteric nodes in vivo (Waring and Beaver, 1996
). Whether gliotoxin also induces apoptosis in renal cells or tissue has not been examined.
One of the major differences that distinguish apoptosis from necrosis is that the plasma membrane remains intact during apoptosis. Central to apoptosis is a group of well-conserved cysteine in active sites, aspartic acid-specific proteases, caspases. At least 13 members of the caspase family have been identified (Schwartz, 1998; Thornberry and Lazebnik, 1998
). Depending on their roles in apoptosis, this family of proteases can be divided into apoptosis initiators (including caspase-8, -9, -10), which participate in transduction of death signals, and apoptosis effectors (such as caspase-3, -6, -7, or caspase-3like proteases), which execute death signals. Among the members of the caspase family, caspase-3, -6, and -7 are the closest homologues of the Caenorhabditis elegans ced-3 gene product shown to be essential for apoptosis of 131 cells out of 1090 cells born during nematodal development. Apoptosis is a well-orchestrated operation, initiated by a diverse array of proapoptotic stimuli, and culminating in activation of caspase-3like proteases. These enzymes are either directly or indirectly involved in cutting off contacts with surrounding cells, rearranging the structure of cytoskeleton, shutting down DNA replication and repair, interrupting RNA splicing, destroying DNA, disrupting the nuclear structure, inducing the cell to display signals that mark it for phagocytosis, and disintegrating the cell into membrane-packaged apoptotic bodies (Schwartz, 1998
; Thornberry and Lazebnik, 1998
). Given the critical functions that caspases have, inhibition of caspases, especially caspase-3like proteases, has been shown to prevent the appearance of apoptosis in a wide variety of cells and tissues (Schwartz, 1998
; Thornberry and Lazebnik, 1998
).
One of the most important properties of epipolythiodioxopiperazines is their ability to go through a redox cycle in the presence of an appropriate reducing agent. Reduced gliotoxin has been specifically identified in cells following exposure to the toxin (Waring and Beaver, 1996). It has been proposed that the reduced gliotoxin undergoes a redox cycling that generates superoxide anion under aerobic conditions; during the conversion of the dithiol derivative to the disulfide form, the superoxide anion is then quickly dismutated to hydrogen peroxide (Waring and Beaver, 1996
). Hydrogen peroxide, as well as other reactive oxygen species produced by gliotoxin during the redox cycle in a cell-free system, directly damage plasmid and cellular DNA (Eichner et al., 1988
). Whether gliotoxin-induced reactive oxygen species account for its toxic effect on renal cells remains completely unknown. The present study examined the cytotoxicity induced by gliotoxin, the mode of cell death, and the roles of caspase-3like activity and reactive oxygen species in cell death in LLC-PK1 cells, a hog renal proximal tubular cell line.
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MATERIALS AND METHODS |
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Cell culture and treatment.
LLC-PK1 cells were purchased from American Tissue Culture Collection (Rockville, MD) and used between passage 1 to passage 15 after the original passage number 194. The cells were kept in Medium 199 plus 3% fetal bovine serum in a 37°C incubator supplied with 5% CO2. For assays of cell viability, the cells were plated down at 6 x 104 cells per well (confluent) in a 96-well plate and grew for 1822 h before treatment. The cells were preincubated with 100 ng/ml gliotoxin for 30 min prior to addition of TNF-.
Crystal violet assay.
Cells were stained with 0.5% crystal violet in methanol for 810 min at 22°C, then washed three times with 1X phosphate-buffered saline solution. The absorption measured at 550 nm was used as an index for cell viability (Wang et al., 1996).
DNA fragmentation assay.
The fragmented DNA in cytoplasma was detected according to Sei et al. (1998). Briefly, after treatment until 80% of the cells detached from dishes, the cells were lysed in 10 mM EDTA, 0.5% Triton X-100, and 5 mM TrisHCl (pH 8.0), and centrifuged at 10,000 x g for 30 min. The supernatant was digested with 0.1 mg/ml proteinase K at 50°C for 60 min, extracted with phenol/chloroform and precipitated with two volumes of 100% ethanol. The pellet was treated with 0.5 mg/ml RNase. The DNA was resolved in 1.8% agarose gel. The fragmented DNA in the cytosol was also quantified according to Burton (Burton, 1956). Briefly, the cell lysates were mixed with 1.5% diphenylamine reactive solution and incubated at 30°C for 24 h. The absorption was measured at A600.
Caspase activity assay.
The activity of caspase-3like proteases was measured as increases in hydrolysis of fluorogenic tetrapeptide substrate, Ac-DEVD-7-amino-4-methylcoumarin (Ac-DEVD-AMC), according to the manufacturer's instructions (BIOMOL, Plymouth Meeting, PA). Because caspase-7 also cleaves the substrate, the activity obtained here is referred as caspase-3like activity. Briefly, the cells were lysed in 25 mM HEPES Buffer (pH 7.5) containing 5 mM EDTA, 2 mM DTT, 0.1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and 0.1% Triton X-100 at 22°C for 10 min, then the supernatants were taken for measurement of hydrolysis of Ac-DEVD-AMC as a function of time at 22°C.
Cytofluorometric analyses of propidium iodide staining.
The cells were plated at 1 x 106/well in a 6-well plate. Cytofluorometric analyses of propidium iodide staining were performed according to Nicoletti et al. (1991). Briefly, both detached and attached cells were collected and incubated in a hypotonic fluorochrome solution (propidium iodide 0.5 mg/ml in 0.1% sodium citrate plus 0.1% Triton X-100) overnight at 4°C. The propidium iodide fluorescence of each individual nucleus was measured with excitation of 488 nm and emission of 620 nm. The cell debris was excluded from analysis by appropriately raising the forward scatter threshold. Hypodiploid nuclei due to the condensation of nuclear chromatin appeared at sub G0/G1 position.
Mitochondrial membrane potential measurement.
Briefly, the cells were loaded with 3 µg/ml JC-1 at 37°C for 30 min. After gating out small-sized debris, the red and green emitted fluorescence were collected through 575/40 nm (FL2) and 525/40 nm (FL1) bandpass filters, respectively (Salvioliet et al., 1997).
Reactive oxygen species measurement.
The cells were placed at 1 x 106/well in a 6-well plate. The cells were preloaded with 20 µM freshly prepared DCFH-DA in the buffer containing (in mM) 145 NaCl, 5 KCl, 1 Na2HPO4, 1 CaCl2, 0.5 MgSO4, 5 glucose, and 10 HEPES, pH 7.4 at 37°C for 15 min, then treated for 40 min. The cells were analyzed by flow cytometry (Robinson et al., 1997).
All numerical data are expressed as means of three experiments +/- standard errors, unless indicated otherwise. Statistical analyses were performed by unpaired t test or analyses of variance (ANOVA) as appropriate. Multiple post comparisons were made by Dunnett analyses.
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RESULTS |
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DISCUSSION |
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Despite the fact that TNF- is a well-known potent inducer of apoptosis, LLC-PK1 cells are insensitive to the cytokine, being in accordance with the insensitivity of kidney in vivo (Bohlinger et al., 1996
). The mechanism underlying the potentiating effect of gliotoxin appears to be due to its inhibitory effect on the activation of a transcription factor, NF-
B (Pahl et al., 1996
; Ward et al., 1999
; Zhou et al., 1998
). The NF-
B is a survival signal demonstrated in a variety of cells and tissues (Magnusson and Vaux, 1999
; Ward et al., 1999
; Wang et al., 1996
; Zhou et al., 1998
).
The mechanisms that underlie gliotoxin-induced apoptosis remain largely unclear. A recent study on thymocytes from Waring's group has shed light on this issue (Waring et al., 1997). Gliotoxin induced phosphorylation of histone H3, thus increasing sensitivity of chromatin to nuclease digestion. The effect of gliotoxin is mediated by protein kinase A, as gliotoxin raised cyclic AMP levels and the activity of protein kinase A, and its effects on H3 phosphorylation and apoptosis were inhibited by a number of specific inhibitors of protein kinase A (Waring et al., 1997
). The identity of the nuclease responsible for gliotoxin-induced apoptosis remains unknown. An endonuclease identified to be responsible for internucleosomal DNA degradation is caspase dependent (Sakahira et al., 1998
). Based on the inhibitory effect of z-VAD, a general inhibitor of caspases, Ward et al (1999) have suggested that gliotoxin-induced apoptosis in the human granulocytes is mediated by caspases. It is noteworthy that the complexity of the mechanisms underlying caspase-3, apoptosis, and cell death has not been well understood. A caspase inhibitor may inhibit apoptosis but may not block cell death (Deas et al., 1998
; Trapani et al., 1998
). The present study has provided direct evidence that caspase-3 was involved in cell death potentiated or induced by gliotoxin. Moreover, inhibition of caspases by BAF significantly reduced cell death (Figs. 3, 4, and 8
), although the cytoprotective effect diminished as the cells were treated with gliotoxin for a long period of time or by a high dose of insult (Figs. 3 and 8C
).
This study found that TNF- alone also increased hydrolysis of DEVD-AMC, even though the cells appeared perfectly healthy. The activation of caspase-3 without apoptosis is not a unique phenomenon observed only here. Wilhelm et al. (1998) have shown that T lymphocytes acquired high intracellular caspase-3like activity upon activation by mitogens and IL-2 without evidence of apoptosis. Whether a checkpoint exists further downstream in the apoptotic pathway or the caspases have a role outside cell death remains to be further investigated.
A variety of toxins and chemicals induce cytotoxicity via reactive oxygen species (Savolainen et al., 1998). Gliotoxin is not exceptional (Figs. 9 and 10
). Although the cell death was accompanied by a rise in reactive oxygen species levels, it is difficult to prove that such a rise was the cause rather than a consequence of cell death. However, in view of the ability of gliotoxin to go through redox cycle and the completely inhibitory effect of N-acetylcysteine, we believe that the increases in the levels of reactive oxygen species entirely mediated the cell death. N-acetylcysteine also inhibited the caspase-3like activity, suggesting that reactive oxygen species activate caspase-3like proteases, then apoptosis and cell death follow. However, the effect of reactive oxygen species is not solely mediated via caspases, as the cytoprotection promoted by N-acetylcysteine was more potent than that of BAF.
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ACKNOWLEDGMENTS |
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NOTES |
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