Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase
Mouad Edderkaoui,1
Peggy Hong,1
Eva C. Vaquero,1
Jong K. Lee,1
Lars Fischer,1,3
Helmut Friess,3
Markus W. Buchler,3
Markus M. Lerch,2
Stephen J. Pandol,1 and
Anna S. Gukovskaya1
1Department of Medicine, Veterans Affairs Greater Los Angeles Healthcare System and University of California, Los Angeles, California; 2Department of Gastroenterology, Endocrinology and Nutrition, Ernst-Moritz-Arndt Universität Greifswald, Germany; and 3Department of General Surgery, University of Heidelberg, Heidelberg, Germany
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ABSTRACT
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The extracellular matrix (ECM) facilitates pancreatic cancer cells survival, which is of central importance for pancreatic adenocarcinoma that is highly fibrotic. Here, we show that reactive oxygen species (ROS) mediate the prosurvival effect of ECM in human pancreatic cancer cells. Fibronectin and laminin stimulated ROS production and NADPH oxidase activation in pancreatic cancer cells. Both pharmacological and molecular approaches show that fibronectin stimulated ROS production through activation of NADPH oxidase and NADPH oxidase-independent pathways and that 5-lipoxygenase (5-LO) mediates both these pathways. Analyses of the mechanisms of ROS production by ECM proteins and growth factors indicate that activation of NADPH oxidase (Nox4) is a common mechanism employed both by ECM proteins and growth factors to increase ROS in pancreatic cancer cells. We also found that Nox4 is present in human pancreatic adenocarcinoma tissues and that these tissues display membrane NADPH oxidase activity. ECM proteins and growth factors activate NADPH oxidase through different mechanisms; in contrast to ECM proteins, growth factors activate NADPH oxidase through 5-LO-independent mechanisms. Inhibition of 5-LO or NADPH oxidase with pharmacological inhibitors of these enzymes and with Nox4 or 5-LO antisense oligonucleotides markedly stimulated apoptosis in cancer cells cultured on fibronectin. Our results indicate that ROS generation via 5-LO and downstream NADPH oxidase mediates the prosurvival effect of ECM in pancreatic cancer cells. These mechanisms may play an important role in pancreatic cancer resistance to treatments and thus represent novel therapeutic targets.
pancreas
REACTIVE OXYGEN SPECIES (ROS) are highly reactive O2 metabolites that include superoxide radical (O2·), hydrogen peroxide (H2O2), and hydroxyl radical (HO·) (44). Whereas ROS are classically thought of as cytotoxic and mutagenic (23, 33), recent evidence suggests that ROS such as superoxide and H2O2 serve as regulators in signal transduction pathways (38). ROS are involved in proliferation, cell death, and adhesion pathways (7, 9, 39, 42). The targets of ROS include such key signaling molecules as transcription factor NF-kB, MAPKs, and tyrosine phosphatases (10, 15, 46, 55).
In phagocytes, large amounts of ROS are produced during the oxidative burst by plasma membrane NAD(P)H oxidase, a well-characterized multicomponent enzyme with gp91phox and p22phox catalytic subunits that together form an integral complex called flavocytochrome b558 (3, 4). Nonphagocytic cells also produce significant amounts of ROS, albeit much smaller than in phagocytes (4, 5, 25). In nonphagocytic cells, ROS are produced by mitochondria as well as by a number of ROS-generating plasma membrane and cytosolic enzymes (44). Over the past several years, members of the family of nonphagocytic NADPH oxidases (NOXes) homologous to gp91phox were shown to mediate ROS production in nonphagocytic cells (4, 5, 25, 26). On the basis of our present state of knowledge, the family consists of seven members. NOXes are classified into three main groups according to their domain structure. In particular, Nox1, -3, and -4 are very similar in size and structure to gp91phox (now termed Nox2), and they also contain the domains required to transfer electrons from NADPH to molecular oxygen to form superoxide. NOXes are expressed (at different levels) in various tissues and are thought to be a major mechanism of ROS production in nonphagocytic cells.
The products of the phospholipase A2 (PLA2)/lipoxygenase (LO) pathway such as leukotrienes and HETEs are also involved in ROS generation. They may participate in activation of NADPH oxidase (30, 5153) and also produce ROS as byproducts during oxidation of arachidonic acid by lipoxygenases (22).
We recently showed that different Nox isoforms are expressed in pancreatic cancer cells (48). In particular, Nox4 is present in these cells at both mRNA and protein levels. Growth factors activate NADPH oxidase resulting in an increase in cellular ROS. We also showed that ROS produced via NADPH oxidase mediate the prosurvival effect of growth factors in pancreatic cancer cells (48).
The important characteristic of pancreatic adenocarcinoma is high expression of extracellular matrix (ECM) protein (16, 29, 49, 54). When deprived of ECM, normal cells undergo apoptosis. This process, termed anoikis, controls cell proliferation by deleting misplaced cells. Cancer cells are usually resistant to anoikis. We recently showed that detachment from ECM stimulates pancreatic cancer cell death and that fibronectin and laminin markedly protect pancreatic cancer cells from death (47). The mechanisms through which the ECM inhibits pancreatic cancer cell death are not well understood. In particular, the role of ROS in the prosurvival effects of ECM proteins has not been studied. Information on the effects of ECM proteins on cellular ROS, in general, are limited (20, 32, 50). Nothing is known on the effects of ECM on ROS production in pancreatic cancer cells.
In the present study, we provide evidence that in pancreatic cancer cells, ECM proteins, fibronectin, and laminin increase intracellular ROS and stimulate NADPH oxidase activity. Both pharmacological and molecular approaches show that fibronectin stimulated ROS production through activation of NADPH oxidase and NADPH oxidase-independent pathways. 5-LO is a critical regulator of ECM-induced ROS generation; it mediates both activation of NADPH oxidase and ROS generation through an NADPH oxidase-independent mechanism. Inhibiting ROS with NADPH oxidase and 5-LO inhibitors, Nox4, or 5-LO antisense oligonucleotide stimulated apoptosis in pancreatic cancer cells. Thus NADPH oxidase and 5-LO provide mechanisms through which ECM proteins protect pancreatic cancer cells from death. The prosurvival effect of ROS may be an important mechanism of pancreatic cancer cell resistance to therapy.
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MATERIALS AND METHODS
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Reagents.
The polyclonal Nox4 antibody generated from a His-tagged recombinant fragment of human Nox4 encoding the COOH-terminal amino acids 494513 (described in Ref. 32) was a gift from Dr. Barry Goldstein (Thomas Jefferson University, Philadelphia, PA). Antibody against 5-LO was from Cayman Chemical and anti-
5 antibody was from Santa Cruz Biotechnology; 2'7'-dichlorofluorescein diacetate (DCFH-DA) was from Molecular Probes (Eugene, OR); DEVD-AMC (Ac-Asp-Glu-Val-Asp-aminomethylcoumarin) was from Peptide Institute (Osaka, Japan); zVAD-fmk [z-Val-Ala-Asp(OMe)-CH2F] was from Enzyme Systems Products (Livermore, CA). AACOCF3, nordihydroguaiaretic acid (NDGA), baicalein, and REV-5901 were from Calbiochem (San Diego, CA). All other reagents were from Sigma (St. Louis, MO).
Cell culture.
Human pancreatic adenocarcinoma cell lines, the poorly differentiated MIA PaCa-2, slightly differentiated PANC-1, and moderately to well-differentiated CAPAN-1, were obtained from the American Type Culture Collection (Manassas, VA). MIA PaCa-2 and PANC-1 cells were grown in 1:1 DMEM-F-12 medium (GIBCO Invitrogen, Grand Island, NY) supplemented with 15% FBS, 4 mM L-glutamine, and antibiotic/antimicotic solution (Omega Scientific, Tarzana, CA). CAPAN-1 cells were grown in RPMI 1640 medium (GIBCO) supplemented with 10% FBS, 4 mM L-glutamine, and antibiotic/antimicotic solution (Omega Scientific). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and were used between passages 4 and 12.
MIA PaCa-2, CAPAN-1, and PANC-1 cells were plated at a density of 5 x 105 cells/ml on 100-mm culture dishes, coated with either poly(2-hydroxyethyl metacrylate) (polyHEMA) or with an ECM protein, cultured for up to 72 h in serum-free DMEM-F-12 or RPMI 1640 media, collected, and processed for the specified analyses. PolyHEMA prevents both cell attachment on plastic and endogenous cell matrix deposition (14). Plates were coated twice with 3 ml polyHEMA dissolved in 95% ethanol (10 mg/ml), allowed to dry, and washed twice with PBS before being used. Compared with polyHEMA, we observed similar but less-pronounced responses in cells grown on plastic or on plastic coated with polylysine (24). Plates coated with human laminin, fibronectin (Sigma), or bovine collagen I (Chemicon, Temecula, CA) were prepared by applying 2 µg/cm2 of the specified protein for 1 h at room temperature. Cells were cultured for 48 h or indicated time in the absence of FBS. Inhibitors or vehicle were added to the culture medium just before plating the cells.
Tissue samples.
Pancreatic cancer tissues were obtained from surgical specimens from four different patients with pancreatic ductal cell adenocarcinoma. Tissue samples were frozen in liquid nitrogen immediately on surgical removal and maintained at 80°C until use. All studies were approved by the ethics committees of the University of Heidelberg, Germany, and by the human subjects committee of the West Los Angeles Veterans Affairs Greater Los Angeles Healthcare System.
Generation of Rho0 cells.
MIA PaCa-2 Rho0 cells were depleted of mitochondrial DNA (mtDNA) as we described previously (48) by incubating wild-type cells (Rho+) for 68 wk with 100 ng/ml ethidium bromide. The medium was supplemented with 4.5 mg/ml glucose, 50 µg/ml uridine, and 100 µg/ml pyruvate to compensate for the respiratory metabolism deficit as previously described. After selection, the MIA PaCa-2 Rho0 cells were cultured in the above-specified medium without ethidium bromide. mtDNA depletion was verified with PCR using human mtDNA-specific primers and with Western blot analysis for mitochondrial DNA coded cytochrome-c oxidase, subunit IV, as we discussed previously (48).
Intracellular ROS levels were measured by flow cytometry in cells loaded with the redox-sensitive dye DCFH-DA (as described in Ref. 48). The nonfluorescent DCFH-DA readily diffuses into the cells, where it is hydrolyzed to the polar derivative DCFH, which is oxidized in the presence of H2O2 to highly fluorescent DCF. DCFH in addition to H2O2 is also oxidized by HOCl and nitric oxide but not by superoxide (19). Approximately 1 x 106 cells were incubated in the dark for 30 min at 37°C with 10 µM DCFH-DA, harvested, and resuspended in the medium without DCFH-DA. Fluorescence was recorded on the FL-1 channel of FACScan (Becton-Dickinson) flow cytometer, and data were analyzed with the Cell Quest program.
Measurement of NADPH oxidase activity.
Superoxide production was measured in total cell or tissue homogenates by using lucigenin-derived chemiluminescence according to the protocols described previously (37, 40, 48); cells or tissue samples from human pancreatic adenocarcinomas were homogenized in a Dounce homogenizer in the medium containing 50 mM phosphate buffer, 1 mM EGTA, and 150 mM sucrose. Homogenates were centrifuged, and pellets (membrane fractions) were stored at 80°C. For superoxide measurements, 50 µg protein were diluted in 500 µl of the same lysis buffer. Dark-adapted lucigenin was added to the sample, and chemiluminescence measurement was immediately started. Chemiluminescence (in arbitrary units) was measured at 15-s intervals for 1 min in a Turner 20/20 luminometer (Turner Designs, Sunnyvale, CA). NADPH was used as a substrate. The specificity of the measurement was confirmed by adding either the nonenzymatic superoxide scavenger tiron (10 mM) or superoxide dismutase (Sigma; 200 U/ml). In some experiments, the homogenate was preincubated for 10 min with DPI as described in the figures. In this and other assays, protein concentration was measured by the Bradford assay (Bio-Rad Laboratories, Hercules, CA).
It has been shown that at high doses, lucigenin can by itself stimulate additional superoxide production, which is especially pronounced with NADH as a substrate (21, 28). As we described previously (48), we measured chemiluminescence in membrane fractions at 5, 25, 50, 100, and 200 µM lucigenin in the presence and absence of NADPH or NADH. By using both NADH and NADPH as substrates, we confirmed that at 550 µM, lucigenin does not induce an artificial O2· production in pancreatic cancer cells. The data presented were obtained with 15 µM lucigenin.
Transfections.
Transfection of MIA PaCa-2 cells with Nox4 antisense oligonucleotides was done as we described previously (48). The Nox4 antisense (AS) phosphorothioate oligonucleotides Nox4 AS1 (1.5 nmol; 5'-AGC TCC TCC AGG ACA CAG CC-3') or Nox4 AS2 (5'-GGA CAC AGC CAT GCC GCC-3') (1) was applied for a 60-mm dish. The Nox4 scrambled oligonucleotides (5'-TCG AGG AGG TCC TGT GTC GG-3') was used as a control. Six hours posttransfection, fresh medium was supplied and cells were cultured for an additional 72 h before specified analyses. Transfection with 5-LO AS oligonucleotides was performed using the same technique with 5-LO AS phosphorothioate oligonucleotides 5-LO AS1 (5'-CAC AGT CAC GTC GTC GTA TGA ATC CAC C-3') (7) or 5-LO AS2 (5'-GTG ACC GTG TAG GAG GGC AT-3') and 5-LO scrambled phosphorothioate oligonucleotides (5'-CGT CCA GAC CAA GGA CGA GCT TGC A-3') as a control.
The efficiency of transfection assessed by expression of the green fluorescent protein containing plasmid was
5060% in both cell lines MIA PaCa-2 and PANC-1. We did not use CAPAN-1 cells in transfection experiments because of their low transfection efficiency. The specificity of Nox4 and 5-LO AS oligonucleotides was checked by BLASTing them against the human genome.
The transfection efficiency for CAPAN-1 cells was low; therefore, transfection experiments were performed with MIA PaCa-2 and PANC-1 cells only.
Measurements of apoptosis.
Internucleosomal DNA fragmentation was measured by using the Cell Death Detection ELISAPlus kit (Roche Molecular Biochemicals, Manheim, Germany) according to the manufacturer's instructions.
Phosphatidylserine externalization was analyzed with the Annexin-V-FLUOS Staining Kit from Roche Biochemicals (Indianapolis, IN) as we described previously (47, 48). Cells were collected and resuspended at a density of 1 x 106 cells in 500 µl of binding buffer containing 2 µl annexin V (AnV) and 1 µl propidium iodide (PI), incubated in the dark for 30 min at room temperature, and analyzed by flow cytometry.
Effector caspase (DEVDase) activity was measured by a fluorogenic assay in whole cell lysates using DEVD-AMC as a substrate, as we described (47, 48). The lysate (50100 µg protein) was incubated with 10 µM substrate in a reaction buffer [25 mM HEPES (pH 7.5), 10% sucrose, 0.1% CHAPS, and 10 mM DTT] at 37°C. Caspase substrate cleavage releases AMC, which emits a fluorescent signal with 380-nm excitation and 440-nm emission. Fluorescence was calibrated using a standard curve for AMC.
Western blot analysis.
Cells were incubated in a lysis buffer A (0.5 mM EDTA, 150 mM NaCl, 50 mM Tris, and 0.5% Nonidet P-40, pH 7.5) for 30 min at 4°C. Tissue samples from human pancreatic adenocarcinomas were homogenized in the lysis buffer B (150 mM NaCl, 50 mM Tris, 1% deoxycholic acid, 1% Triton X-100, and 0.1% SDS). Both buffers A and B were supplemented with 1 mM PMSF and 5 µg/ml each of protease inhibitors pepstatin, leupeptin, chymostatin, antipain, and aprotinin. Cell and tissue lysates were centrifuged for 10 min at 13,000 g. Supernatants were collected, and proteins were separated by SDS-PAGE (Invitrogen) and electrophoretically transferred to nitrocellulose membranes. Nonspecific binding was blocked with 5% milk in Tris-buffered saline (and 4 mM Tris base TBS; 100 mM NaCl, pH 7.5). Membranes were washed in TBS containing 0.05% Tween 20 (TTBS) and incubated for 2 h with the indicated primary antibodies and then for 1 h with horseradish peroxidase-conjugated secondary antibody. Blots were developed with the Supersignal chemiluminescent substrate (Pierce).
Immunohistochemisrty.
Immunostaining of frozen tissue sections from human pancreatic adenocarcinomas was performed as we described previously (17). The sections were incubated in a blocking medium containing PBS-glycine, 1% goat serum, 1% BSA, and 1% gelatin for 30 min. Then slides were incubated for 10 min in a working medium (PBS supplemented with 0.05 M glycine, 0.1% goat serum, 0.1% BSA, and 0.1% gelatin) followed by a 2-h incubation with anti- Nox4 antibody dissolved in a working medium. Bound antibody was detected with FITC-conjugated secondary antibody.
Statistical analysis.
Results are expressed as means ± SE from at least three independent experiments. Statistical analysis was done using unpaired Student's t-test. P < 0.05 was considered statistically significant.
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RESULTS
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Fibronectin, laminin, and collagen I stimulate intracellular ROS production in MIA PaCa-2, CAPAN-1, and PANC-1 pancreatic carcinoma cells.
Using the redox-sensitive fluorescence probe DCFH-DA, we determined that ECM proteins stimulated ROS production in pancreatic cancer cell lines (Fig. 1). In MIA PaCa-2 (Fig. 1A), CAPAN-1 (Fig. 1B), and PANC-1 cells (Fig. 1C), levels of intracellular ROS were higher in cells attached to ECM proteins than in detached cells. Fibronectin and laminin increased intracellular ROS to the same extent in all three cell lines tested (Fig. 1D). Thus fibronectin produces the same increase in ROS in poorly differentiated (MIA PaCa-2), slightly differentiated (PANC-1), and well-differentiated (CAPAN-1) cells. Collagen I only stimulated ROS in PANC-1 cells (Fig. 1, AC). Increases in cellular ROS by fibronectin and laminin were of the same magnitude as effects of exogenous 10 µM H2O2.

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Fig. 1. Extracellular matrix (ECM) proteins increase intracellular reactive oxygen species (ROS) in pancreatic cancer cells. MIA PaCa-2 (A), CAPAN-1 (B), and PANC-1 (C) cells were cultured without serum for 48 h on polyHEMA or ECM proteins: fibronectin (FN), laminin (LN), and collagen I. Changes in intracellular ROS were measured by FACS analysis in cells labeled with the redox-sensitive dye 2'7'-dichlorofluorescein diacetate (DCFH-DA). Histograms in A-C are representative of at least 3 independent experiments. For quantitative analysis (D), DCF fluorescence was normalized on that in cells cultured on polyHEMA. Values are means ± SE (n = 3). *P < 0.05 vs. cells cultured on polyHEMA. In E, MIA PaCa-2 cells were preincubated for 1 h at 4°C with or without monoclonal anti- 5-integrin antibody (10 µg/ml). DCF fluorescence was normalized on that in cells preincubated without antibody. Values are means ± SE (n = 3).*P < 0.05 vs. cells without pretreatment with antibody.
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The effect of ECM proteins on cell signaling is mediated through their receptors, integrins. Figure 1E shows that blockade of fibronectin receptor
5
1-integrin (2, 18) with anti-
5 integrin antibody greatly inhibited the fibronectin-induced increase in ROS. Figure 2 shows that detachment from ECM causes time-dependent decrease in ROS, whereas in attached cells, intracellular ROS levels are maintained for up to 72 h.

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Fig. 2. Cells detached from ECM lose the ability to maintain intracellular ROS. MIA PaCa-2 cells were cultured for the indicated times without serum on polyHEMA or FN. DCF fluorescence was normalized on that in cells cultured for 3 h. At 3 h, there was no difference in DCF fluorescence in cells cultured on polyHEMA and FN. Values are means ± SE from at least 3 independent experiments for each time point.
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ECM proteins increase cellular ROS through NADPH oxidase and 5-LO, and 5-LO mediates both NADPH oxidase-dependent and independent pathway(s).
To determine the sources of intracellular ROS, we first used a pharmacological approach (Fig. 3). We applied inhibitors of various ROS-producing systems at concentrations reported to completely inhibit their target enzymes. The superoxide scavenger tiron decreased ROS levels in cells cultured on both polyHEMA and ECM proteins, indicating the contribution of superoxide in ROS generation in pancreatic cancer cells (Fig. 3A).

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Fig. 3. Fibronectin and laminin induce ROS production through 5-lipoxygenase (5-LO)- and NAD(P)H oxidase-mediated pathways. MIA PaCa-2 (A and C) and CAPAN-1 (B and D) cells were cultured without serum for 48 h on polyHEMA, FN or LN, and in the presence or absence of the inhibitors of ROS-generating systems: NADPH oxidase inhibitor DPI (15 µM); phospholipase A2 inhibitor AACOCF3 (10 µM); 5-LO inhibitors nordihydroguaiaretic acid (NDGA; 10 µM) and REV-5901 (REV; 15 µM); and superoxide scavenger tiron (10 mM). In A and B, changes in intracellular ROS were measured by FACS analysis in cells labeled with DCFH-DA. In C and D, superoxide production in the absence and presence of 100 µM NADPH was measured by lucigenin-derived chemiluminescence in total cell homogenate. Both DCF fluorescence (A and B) and luminescence (C and D) were normalized on that in cells cultured on polyHEMA without inhibitors. Values are means ± SE from at least 3 independent experiments. *P < 0.05 vs. cells on polyHEMA without inhibitors; #P < 0.05 vs. cells on the same ECM protein without inhibitors.
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In MIA PaCa-2, CAPAN-1 (Fig. 3A) cells cultured on polyHEMA (the substrate that prevents both cell attachment on plastic and endogenous ECM deposition) (47), ROS generation was partially but significantly inhibited by DPI, an inhibitor of NADPH-dependent oxidases (4). Inhibitors of the PLA2/5-LO pathway, in particular, the inhibitor of cytosolic PLA2 AACOCF3 (24), a broad-spectrum LO inhibitor [NDGA (31, 41)], and the selective inhibitor of 5-LO REV-5901 (43), did not significantly affect intracellular ROS levels in cells cultured on polyHEMA (Fig. 3, A and B). Moreover, the inhibitory effect of DPI plus REV-5901 on the basal ROS level was the same as with DPI alone (Fig. 3A). These results suggest that NADPH-oxidase but not PLA2 /5-LO contributes to the ROS production in cells cultured on polyHEMA.
By contrast, ROS production in pancreatic cancer cells cultured on fibronectin was inhibited by both DPI and inhibitors of the PLA2/5-LO pathway (Fig. 3, A and B). These data (Fig. 3, A and B) indicate that in cells cultured on ECM proteins, ROS production is mediated through 5-LO and NADPH oxidase. In cells cultured on either polyHEMA or ECM proteins, inhibitors of 12-LO, xanthine oxidase, and nitric oxide synthase (6, 35, 36) did not affect intracellular ROS (Table 1), indicating no involvement of these enzymes.
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Table 1. Inhibitors of xanthine oxidase, 12-LO, and nitric oxide synthase did not affect cellular ROS in MIA PaCa-2 cells
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The experiments on MIA PaCa-2 cells show that the effects of laminin on cellular ROS are also inhibited by DPI and REV-5901 (Fig. 3A). They suggest that laminin employs the same mechanisms as fibronectin to increase cellular ROS.
DPI and REV-5901 inhibited the fibronectin-induced increase in cellular ROS in both poorly differentiated MIA PaCa-2 (Fig. 3A) and well-differentiated CAPAN-1 (Fig. 3B) cells.
To further elucidate the role of NADPH oxidase in the effects of ECM proteins on cellular ROS, we measured the effects of ECM proteins on NADPH oxidase activity. Both fibronectin and laminin stimulated NADPH oxidase activity in membrane fractions from MIA PaCa-2 cells (Fig. 3C). NADPH oxidase activity in cells cultured on fibronectin (as well as on polyHEMA) was completely prevented by DPI (Fig. 3C). On the other hand, the 5-LO inhibitor REV-5901 did not inhibit NADPH oxidase activity in cells cultured on polyHEMA but abrogated NADPH oxidase activation by fibronectin (Fig. 3C). These data suggest that 5-LO mediates fibronectin-induced activation of NADPH oxidase but not the basal NADPH oxidase activity. Fibronectin stimulated NADPH ox-idase activity in both poorly differentiated MIA PaCa-2 (Fig. 3C) and well-differentiated CAPAN-1 (Fig. 3D) cells.
Taken together, the results in Fig. 3 show that inhibition of 5-LO prevented both effects of fibronectin, namely, the activation of NADPH oxidase and the increase in cellular ROS. These data indicate that 5-LO mediates an increase in cellular ROS by fibronectin. They further suggest that activation of NADPH oxidase is one mechanism through which 5-LO increases cellular ROS. DPI, which completely inhibited NADPH oxidase activity (Fig. 3C), only partially decreased fibronectin-induced increase in ROS in MIA PaCa-2 cells (Fig. 3A). Furthermore, the inhibition of 5-LO with REV-5901 caused additional inhibition of ROS response to fibronectin in cells treated with DPI, abrogating the effect of fibronectin on ROS (Fig. 3A). These results suggest that ECM-induced ROS production is mediated through both NADPH oxidase and NADPH oxidase-independent mechanisms. They also suggest that 5-LO mediates both of these mechanisms.
Lack of the effects of REV-5901 on cellular ROS and NADPH oxidase activity indicates that 5-LO is not involved in ROS generation in cells cultured on polyHEMA (Fig. 3, AC). DPI only partially decreased cellular ROS in MIA PaCa-2 cells cultured on polyHEMA, suggesting the contribution of mechanisms other than NADPH oxidase.
Inhibiting 5-LO or NADPH oxidase with 5-LO or Nox4 AS oligonucleotides decreases ROS production in pancreatic cancer cells cultured on fibronectin.
We showed previously that Nox4 is expressed in pancreatic cancer cells at both mRNA and protein levels (48). To determine the role of Nox4 in the generation of ROS by fibronectin, we transfected MIA PaCa-2 and PANC-1 cells with Nox4 AS (as well as scrambled) oligonucleotides. We used two different AS oligonucleotides targeting human Nox4 (Fig. 4). Both AS1 (Fig. 4, AC) and AS2 (Fig. 4, D and E) were effective at reducing levels of endogenous Nox4 protein when transfected into MIA PaCa-2 and PANC-1 cells. Both AS oligonucleotides showed inhibition of ROS (Fig. 4). In cells cultured on fibronectin, Nox4 AS oligonucleotides decreased both intracellular ROS levels (Fig. 4, B and E) and NADPH oxidase activity (Fig. 4C) compared with cells transfected with the scrambled oligonucleotides.

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Fig. 4. Transfection with NADPH oxidase Nox4 antisense oligonucleotides inhibits cellular ROS and NADPH oxidase activity in cells cultured on FN. MIA PaCa-2 (AC) or PANC-1 (D and E) were transfected with antisense oligonucleotide 1 (AS1), antisense oligonucleotide 2 (AS2), or scrambled (S) Nox4 oligonucleotides and cultured for 72 h without serum on FN. In A and D, Nox4 protein levels were measured in transfected cells by immunoblotting. Blots were stripped and reprobed for actin to confirm equal loading. The densitometric ratios of the intensity of the Nox4 band (normalized to actin) between cells transfected with antisense and scrambled oligonucleotides were 0.3 in MIA PaCa-2 cells transfected with AS1 (A) and 0.5 and 0.6 in PANC-1 cells transfected, respectively, with AS1 and AS2 (D). Changes in intracellular ROS (B and E) were measured by FACS analysis in cells labeled with DCFH-DA. Histograms are representative of 3 independent experiments. Superoxide production (C) was measured by lucigenin-derived chemiluminescence in membrane fractions in the presence of 100 µM NADPH. The chemiluminescence values were normalized on those in cells transfected with scrambled oligonucleotides. Values are means ± SE from 3 independent experiments. *P < 0.05 vs. cells transfected with scrambled oligonucleotides.
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Similarly, to confirm our results obtained with the pharmacological inhibitors on the involvement of 5-LO, we transfected MIA PaCa-2 and PANC-1 cells with 5-LO AS oligonucleotides. The results show a decreased level of intracellular ROS (Fig. 5, B and E) and NADPH oxidase activity (Fig. 5C) in cells transfected with 5-LO AS oligonucleotides and cultured on fibronectin.

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Fig. 5. Transfection with 5-LO antisense oligonucleotides inhibits cellular ROS and NADPH oxidase activity in cells cultured on fibronectin. MIA PaCa-2 (AC) or PANC-1 (D and E) was transfected with AS1, AS2, or S 5-LO oligonucleotides and cultured for 72 h without serum on fibronectin. Protein levels of 5-LO (A and D) were measured in transfected cells by immunoblotting. Blots were stripped and reprobed for actin to confirm equal loading. The densitometric ratios of the intensity of 5-LO band (normalized to actin) between cells transfected with antisense and scrambled oligonucleotides were 0.3 in MIA PaCa-2 cells transfected with AS1 (A) and 0.4 in PANC-1 cells transfected with either AS1 or AS2 (D). Changes in intracellular ROS (B and E) were measured by FACS analysis in cells labeled with DCFH-DA. Histograms are representative of 3 independent experiments. Superoxide production (C) was measured by lucigenin-derived chemiluminescence in membrane fractions in the presence of 100 µM NADPH. The chemiluminescence values were normalized on those in cells transfected with scrambled oligonucleotides. Values are means ± SE from 3 independent experiments. *P < 0.05 vs. cells transfected with scrambled oligonucleotides.
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The results of transfection experiments confirm the involvement of 5-LO and Nox4 in fibronectin-induced ROS production.
Involvement of mitochondrial NADH dehydrogenase to the ECM-induced increase in cellular ROS.
In addition to membrane NADPH oxidase, DPI can also inhibit mitochondrial NADH dehydrogenase (27). To evaluate the contribution of mitochondrial NADH dehydrogenase, we used MIA PaCa-2 Rho0 cells depleted of mtDNA that codes for this enzyme. Using PCR and Western blot analysis, we showed the absence of mtDNA in Rho0 cells (48).
In Rho0 cells, fibronectin and laminin increased intracellular ROS levels similar to parental (wild type) Rho+ MIA PaCa-2 cells (Fig. 6A).

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Fig. 6. FN and LN stimulate ROS generation in MIA PaCa-2 Rho0 cells depleted of mitochondrial DNA (mtDNA). MIA PaCa-2 Rho0 cells depleted of mtDNA were generated by incubating wild-type cells for >6 wk with 100 ng/ml of ethidium bromide. The absence of mtDNA was confirmed by PCR and Western blot analysis as described in MATERIALS AND METHODS and in Ref. 48. MIA PaCa-2 Rho0 (A) or parental MIA PaCa-2 (B) cells were cultured for 48 h without serum on polyHEMA, FN, or LN in the presence or absence of the mitochondrial complex inhibitor rotenone (1 µM; B). Changes in intracellular ROS were measured by FACS analysis in cells labeled with DCFH-DA. Histogram is representative of at least 3 independent experiments. Values were normalized on those in cells cultured in the same conditions on polyHEMA. Values are means ± SE from 3 independent experiments.
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To evaluate contribution of mitochondria in fibronectin-induced ROS production, we also applied mitochondrial complex I inhibitor rotenone to the wild-type MIA PaCa-2 Rho+ cells. In cells cultured with 1 µM rotenone for 48 h, fibronectin stimulated intracellular ROS similar to that in the absence of rotenone (Fig. 6B). These results indicate that the mitochondrial NADH dehydrogenase is not a major contributor to the ECM-induced ROS generation in MIA PaCa-2 cells.
Antioxidants as well as Nox4 and 5-LO AS oligonucleotides stimulate apoptosis in pancreatic cancer cells cultured on ECM proteins.
It has been published previously (47) that ECM proteins inhibit death responses in pancreatic cancer cells. In particular, we (47) found that attachment to fibronectin or laminin significantly increased the survival of MIA PaCa-2 cells cultured on polyHEMA without growth factors. We also found that the prosurvival effect of ECM proteins was similar to that of growth factors (48).
In the following experiments, we measured the role of ROS in the prosurvival effects of ECM proteins. Inhibition of intracellular ROS with antioxidants (DPI, REV-5901, and tiron) stimulated internucleosomal DNA fragmentation, a hallmark of apoptosis, in the MIA PaCa-2 cells cultured on fibronectin or laminin (Fig. 7A). In contrast, there was no effect on DNA fragmentation of the inhibitors of enzymes that we found not involved in ROS production in pancreatic cancer cells, allopurinol (xanthine oxidase), baicalein (12-LO), and N-nitro-L-arginine methyl ester (nitric oxide synthase) (not shown). Antioxidants increased DNA fragmentation both in poorly differentiated MIA PaCa-2 and in well-differentiated CAPAN-1 cells (Fig. 7, A and B).

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Fig. 7. Inhibition of ROS stimulates internucleosomal DNA fragmentation and decreases survival in pancreatic cancer cells. MIA PaCa-2 (A, C, and D) and CAPAN-1 (B) cells were cultured without serum for 48 h on polyHEMA, FN, or LN in the presence or absence of NADPH oxidase inhibitor DPI (15 µM), 5-LO inhibitor REV (15 µM), superoxide scavenger tiron (10 mM), or the broad-spectrum caspase inhibitor zVAD-fmk (100 µM). Internucleosomal DNA fragmentation (A and B) was measured using cell death detection ELISA kit. Phosphatidylserine externalization (C) was measured in cells labeled with annexin V (An V)/propidium iodide (PI) and analyzed by FACS. An/PI cells were considered as live cells; An+/PI and An+/PI+ cells were considered as cells dying through apoptosis and apoptosis associated with secondary necrosis (dead cells). Caspase-3 activity was measured in cell lysates by fluorogenic assay (D). Values were normalized to those in cells cultured on polyHEMA without inhibitors. Values are means ± SE from at least 4 independent experiments. In AC, *P < 0.05 vs. cells cultured on the same condition without inhibitors. In D, *P < 0.05 vs. cells cultured on polyHEMA; #P < 0.05 vs. cells cultured on FN protein without inhibitors.
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Cell survival was also analyzed by flow cytometry in cells stained with AnV and PI. AnV was applied to detect phosphatidylserine externalization, a marker of apoptosis; PI was applied to identify loss of plasma membrane integrity, a hallmark of necrosis. We showed previously (47) that pancreatic cancer cells with apoptotic nuclear morphology are either in the An+/PI or An+/PI+ group. An+/PI is a group of cells with early apoptosis, whereas the An+/PI+ group includes cells with late apoptosis associated with the secondary necrosis (47). Figure 7C shows that DPI increased the percentage of dead cells (An+/PI together with An+/PI+) from 35% to 55%. It also decreased survival of MIA PaCa-2 cells from 62% down to 38%.
Inhibition of ROS with antioxidants also activates effector caspases (Fig. 7D). Both caspase activation (Fig. 7D) and DNA fragmentation (not illustrated) induced by antioxidants were inhibited by zVAD-fmk, a broad-spectrum caspase inhibitor.
To further determine the role of NADPH oxidase and 5-LO in the inhibition of apoptosis by fibronectin, we used a molecular approach. Transfection of MIA PaCa-2 and PANC-1 cells with Nox4 or 5-LO AS oligonucleotides increased both DNA fragmentation and the percentage of AnV+-positive cells (Fig. 8, AD). Inhibition of Nox4 or 5-LO by AS oligonucleotides also activated effector caspases (Fig. 8, E and F). Of note, both AS1 (Fig. 8) and AS2 (not shown) Nox4 oligonucleotides and both AS1 (Fig. 8) and AS2 (not shown) 5-LO oligonucleotides demonstrated similar stimulation of apoptosis.

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Fig. 8. Transfection with antisense Nox4 or 5-LO stimulates apoptosis in pancreatic cancer cells. MIA PaCa-2 (A and CE) and PANC-1 (B) were transfected with AS1 or scrambled Nox4 and 5-LO oligonucleotides and cultured for 72 h without serum on FN. Internucleosomal DNA fragmentation (AC) was measured using cell death detection ELISA kit. Phosphatidylserine externalization (D) was measured in cells labeled with AnV/PI and analyzed by FACS. An+/PI and An+/PI+ cells were considered as dead cells. Caspase-3 activity (E and F) was measured in cell lysates by fluorogenic assay. Values are means ± SE from at least 3 independent experiments. In AC, E, and F, values were normalized to those in cells transfected with scrambled oligonucleotides. *P < 0.05 vs. cells transfected with scrambled oligonucleotides.
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These data (Figs. 7 and 8) indicate that inhibition of ROS with antioxidants or transfection with Nox4 or 5-LO AS oligonucleotides stimulates apoptosis in MIA PaCa-2 and PANC-1 cells. Apoptosis induced by ROS inhibition is medifound ated by caspases.
Effect of the combination of fibronectin and growth factors on cellular ROS and apoptosis is greater than that of fibronectin or growth factors alone.
We (48) showed previously that growth factors increased cellular ROS in pancreatic cancer cells through activation of NADPH oxidase. Thus NADPH oxidase activation mediates a common pathway for both growth factors and ECM proteins to generate ROS in pancreatic cancer cells. As distinct from fibronectin, inhibition of 5-LO (Fig. 9, A and B) or PLA2 (48) had no effect on growth factor-induced cellular ROS or NADPH oxidase activity. Together with the data in Fig. 3, these results indicate that 5-LO is involved in ROS production by ECM proteins but not by growth factors.

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Fig. 9. Mechanisms of ROS production and inhibition of apoptosis by growth factors and ECM proteins partially overlap. MIA PaCa-2 cells were cultured for 48 h on polyHEMA (AD) or FN (C and D) with or without 15% FBS or 100 ng/ml IGF-I and in the presence or absence of the inhibitors of ROS-generating systems: NADPH oxidase inhibitor DPI (15 µM) and 5-LO inhibitors NDGA (10 µM), and REV (15 µM). Changes in intracellular ROS (A and C) were measured by FACS analysis in cells labeled with DCFH-DA. Superoxide production (B) was measured in the presence of 100 µM NADPH by lucigenin-derived chemiluminescence in total cell homogenate. Internucleosomal DNA fragmentation (D) was measured using cell death detection ELISA kit. DCF fluorescence (A and C), luminescence (B), and DNA fragmentation (D) were normalized on that in cells cultured on polyHEMA without inhibitors and without growth factors. Values are means ± SE from at least 3 independent experiments. *P < 0.05 vs. cells cultured on polyHEMA with (A) or without (BD) growth factors) and without inhibitors; #P < 0.05 vs. cells cultured in the same conditions on polyHEMA.
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We further measured the effects of combined action of fibronectin and growth factors (FBS or IGF-I) on the cellular ROS and death responses in MIA PaCa-2 cells. In these experiments, we used concentrations of FBS and IGF-1 that provided maximal ROS production as we showed previously (48). Figure 9C shows that the combination of fibronectin and FBS (or IGF-I) produces a greater increase in cellular ROS than growth factors or fibronectin alone. Similarly, growth factor-induced inhibition of apoptosis was greater in cells cultured on fibronectin than in those cultured on polyHEMA (Fig. 9D). The effects of fibronectin and growth factors on the cellular ROS and DNA fragmentation were, however, not additive, indicating that growth factors and ECM proteins employ overlapping mechanisms to increase ROS and inhibit DNA fragmentation.
Nox4 is present and active in human pancreatic adenocarcinomas.
Western blot analysis showed that both Nox4 and fibronectin are present in tissue samples from human pancreatic adenocarcinomas (Fig. 10A). Furthermore, NADPH oxidase activity was present in the membranes fractions of pancreatic cancer (Fig. 10B). Strong Nox4 immunoreactivity was present in the cancer cells (Fig. 10C). There was no staining for Nox4 in stellate, stroma, or inflammatory cells (Fig. 10C). Of note, mRNA for Nox4 was detected in the normal pancreas (8).

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Fig. 10. Nox4 and NADPH oxidase activity are present in pancreatic cancer tissues. Western blot analysis of Nox4 and FN (A), superoxide production (B), and immunofluorescence of Nox4 (C) were measured in the tissue samples of pancreatic adenocarcinoma of individual patients. Superoxide production was measured in the membrane fractions in the absence and presence of 100 µM NADPH by lucigenin-derived chemiluminescence. Values are means ± SE, n = 4. C: pancreatic cancer tissue sections were stained with primary antibody against Nox4 and FITC-conjugated secondary antibody (left and middle) or with secondary antibody only (right). In the middle, non-FITC fluorescence was subtracted from the image to the left. A and C: Western blot analysis and images are representative of samples obtained from 3 patients.
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DISCUSSION
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Our data show that the ECM proteins fibronectin and laminin and, to a lesser extent, collagen I stimulate ROS generation in pancreatic cancer cells. Preventing cell adhesion with polyHEMA caused a time-dependent decrease in ROS. The stimulatory effect of ECM proteins on ROS is mediated through integrin receptors.
Several lines of evidence indicate that the NADPH oxidase Nox4 mediates ECM-induced increases in ROS in pancreatic cancer cells: 1) fibronectin and laminin stimulate NADPH oxidase activity; 2) ECM-induced increases in cellular ROS are inhibited by DPI, an inhibitor of NADPH oxidase; 3) mitochondrial NADH dehydrogenase is not involved in ROS stimulation by ECM proteins; 4) Nox4 is present in pancreatic cancer cells; and 5) Nox4 AS oligonucleotides inhibited ROS production and NADPH oxidase activity in cells cultured on ECM proteins.
Our results show that 5-LO is another critical mediator of ECM-induced stimulation of ROS in pancreatic cancer cells. ECM-induced increases in cellular ROS are inhibited by the specific inhibitors of 5-LO and by transfection with 5-LO AS oligonucleotides. Inhibition of 5-LO completely prevented activation of NADPH oxidase by fibronectin, indicating that 5-LO mediates NADPH oxidase activation by this ECM protein. Thus one mechanism of 5-LO-induced ROS generation is through activation of NADPH oxidase. The pharmacological analysis indicated that, in addition to NADPH oxidase activation, 5-LO also mediates NADPH oxidase-independent pathways of ROS generation activated by fibronectin. The nature of NADPH oxidase-independent mechanisms of the ROS generation by 5-LO remains to be elucidated. LO, for example, could generate ROS as byproducts of arachidonic acid oxidation (22). The detailed signaling mechanisms linking 5-LO and NADPH oxidase are also not known, although LOs have been found to be involved in NADPH oxidase activation in several cell types (30, 34, 5153).
We recently showed that detachment from ECM stimulates pancreatic cancer cell death and that fibronectin and laminin markedly protect pancreatic cancer cells from death (47). We also demonstrated that the prosurvival effects of ECM proteins in pancreatic cancer cells are mediated through mechanisms different from those operating in normal cells. The present study demonstrates that this prosurvival effect of ECM proteins is mediated through ROS. Indeed, inhibitors of 5-LO and NADPH oxidase or the superoxide scavenger tiron all inhibited ROS and stimulated caspase activity and DNA fragmentation in cells cultured on fibronectin or laminin. Furthermore, as shown by AS oligonucleotide transfections, 5-LO and Nox4 are key mediators of the antiapoptotic effects of ECM proteins in pancreatic cancer cells.
5-LO has been shown to mediate proliferation and survival of pancreatic cancer cells (1113, 45). Our data suggest that the prosurvival effect of 5-LO is mediated, at least in part, through ROS stimulation by ECM proteins.
Our results in the present study and in Ref. 48 show that the two major environmental prosurvival factors, ECM proteins and growth factors, protect pancreatic cancer cells from death through a common pathway, namely, by stimulating cellular ROS. Activation of NADPH oxidase, in particular, Nox4, is a common mechanism through which both ECM proteins and growth factors stimulate ROS production and inhibit apoptosis in pancreatic cancer cells. The mechanisms used by ECM proteins and growth factors to stimulate NADPH oxidase, however, differ. Our results indicate that NADPH oxidase activation by fibronectin is fully mediated by 5-LO, whereas growth factors activate NADPH oxidase independently of 5-LO. The results of this study and those of Ref. 48 show that 5-LO is not involved in ROS stimulation by growth factors. Thus ECM proteins and growth factors generate ROS through overlapping but not identical mechanisms. A combination of both growth factors and ECM proteins produces a greater increase in ROS and inhibition of apoptosis than growth factors or ECM proteins alone. However, the effects of growth factors and fibronectin on ROS and apoptosis were not additive because the pathways they activated overlap.
We found that Nox4 is present in tissues from human pancreatic adenocarcinomas; furthermore, the membrane fractions of pancreatic adenocarcinomas display NADPH oxidase activity. Pancreatic adenocarcinomas are notoriously desmophilic (16, 29, 49, 54); thus the ECM proteins produced by the stromal cells surrounding the cancer cells may facilitate the survival of cancer cells through their effects on Nox4 in the cancer cells.
In conclusion, we have shown that fibronectin and laminin stimulate intracellular ROS in pancreatic cancer cells through activation of NADPH oxidase as well as through NADPH oxidase-independent pathways. 5-LO is an upstream mediator of both pathways. Although growth factors also stimulate ROS, they activate NADPH oxidase independently of 5-LO. Nox4 is the common mediator through which both ECM proteins and growth factors stimulate ROS. ECM-induced ROS stimulation is an important mechanism of the prosurvival, antiapoptotic effect of ECM proteins in pancreatic cancer cells. The results indicate that 5-LO and Nox4 are key molecules mediating pancreatic cancer cells' resistance to apoptosis and thus represent potential targets for therapeutic strategies to treat pancreatic adenocarcinoma.
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GRANTS
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This work was supported by the Department of Veterans Affairs Merit Review and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59926 (to A. S. Gukovskaya).
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ACKNOWLEDGMENTS
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The authors thank Dr. I. Gukovsky for advice and discussion and Mohammad Shahsahebi and Yoon Jung for help in the preparation of the manuscript.
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FOOTNOTES
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Address for reprint requests and other correspondence: A. Gukovskaya, VA Greater Los Angeles Healthcare System, West Los Angeles VA Healthcare Center, 11301 Wilshire Blvd., Bldg. 258, Rm. 340, Los Angeles, CA 90073 (e-mail: agukovsk{at}ucla.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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