1 Departments of Internal Medicine and Cannon Research Center, Carolinas Medical Center, Charlotte, North Carolina 28232; 2 Division of Respiratory, Critical Care and Occupational (Pulmonary) Medicine, University of Utah, Salt Lake City, Utah 84132; 3 Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717; and 4 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Malignant melanoma cells spontaneously
generate reactive oxygen species (ROS) that promote constitutive
activation of the transcription factor nuclear factor-B (NF-
B).
Although antioxidants and inhibitors of NAD(P)H oxidases significantly
reduce constitutive NF-
B activation and suppress cell proliferation
(11), the nature of the enzyme responsible for ROS
production in melanoma cells has not been determined. To address this
issue, we now have characterized the source of ROS production in
melanoma cells. We report that ROS are generated by isolated,
cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H
oxidase inhibitors. The p22phox,
gp91phox, and p67phox
components of the human phagocyte NAD(P)H oxidase and the
gp91phox homolog NOX4 were demonstrated in
melanomas by RT-PCR and sequencing, and protein product for both
p22phox and gp91phox was
detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22phox and NOX4.
Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by
transfection of antisense but not sense oligonucleotides for
p22phox and NOX4. Also, the flavoprotein
inhibitor diphenylene iodonium inhibited constitutive DNA binding of
nuclear protein to the NF-
B and cAMP-response element consensus
oligonucleotides, without affecting DNA binding activity to activator
protein-1 or OCT-1. This suggests that ROS generated in autocrine
fashion by an NAD(P)H oxidase may play a role in signaling malignant
melanoma growth.
superoxide anion; diphenylene iodonium; p22phox; gp91phox; p67phox; NOX1; NOX4; nuclear factor-B; cAMP
response element; dicumarol
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INTRODUCTION |
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REACTIVE
OXYGEN SPECIES (ROS) generated by an NAD(P)H oxidase have been
recently recognized as important signaling molecules for proliferation
of normal cells. The role of a signaling NAD(P)H oxidase has been most
extensively explored in vascular smooth muscle cells, where both
p22phox and the unique
gp91phox homolog NOX1 have been shown to be
important for function of an NAD(P)H oxidase activity that mediates
angiotensin II-induced superoxide (O
Like normal cells, human tumor cells also produce substantial amounts
of ROS spontaneously (15, 37, 49), and evidence points to
a role for these ROS in signaling neoplastic proliferation. Mitogenic
signaling through both Ras (22) and Rac (26)
is mediated by O
We recently reported that endogenously produced ROS signal constitutive
activation of nuclear factor-B (NF-
B) and cellular proliferation
in M1619 malignant melanoma cells (11). On the basis of
inhibition of these events by the NAD(P)H:quinone oxidoreductase (NQO)
inhibitor dicumarol, we speculated that cytosolic NQO might provide the
enzymatic source of electrons for reduction of membrane ubiquinone to
ubiquinol, with subsequent generation of O
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MATERIALS AND METHODS |
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Materials.
Human malignant cell lines were obtained from American Type Culture
Collection (Rockville, MD). Human epidermal melanocytes, medium 154, and human melanocyte growth supplement (HMGS) were purchased from
Cascade Biologics (Portland, OR). RPMI medium 1640, HEPES,
antibiotic-antimycotic (10,000 U/ml penicillin, 10,000 µg/ml
streptomycin, and 25 µg/ml amphotericin B), and trypsin-EDTA solution
were purchased from the GIBCO-BRL division of Life Technologies (Grand
Island, NY). Fetal bovine serum (FBS) was purchased from Hyclone
Laboratories (Logan, UT). The intracellular oxidant sensitive probe
2',7'-dichlorofluorescin diacetate (DCFH-DA) was from Molecular Probes
(Eugene, OR). Electrophoretic mobility shift assay (EMSA) supplies were
purchased from Promega (Madison, WI). Supershift antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit
phospho-specific antibodies for inhibitor of NF-B (I
B
)
phosphorylated at serine 32 were purchased from New England Biolabs
(Beverly, MA). Protease inhibitors were from Sigma Chemical (St. Louis,
MO). All other materials were purchased from Sigma Chemical unless
otherwise specified.
Culture of malignant cell lines and cell culture treatments.
Malignant melanoma cell lines were cultured and passaged as previously
described (11). Human epidermal melanocytes were cultured
in medium 154 supplemented with HMGS according to the supplier's
instructions and passed with 0.05% trypsin and 0.53 mmol/l EDTA.
Growth rates of melanocytes and M1619 melanoma cells were compared by
measuring proliferation (as described in Measurement of
proliferation in cell cultures) every 24 h for 72 h. Intracellular generation of ROS by melanocytes or M1619
cells was measured by oxidation of DCFH-DA to 2',7'-dichlorofluorescein
(DCF) by H2O2. The effect of NAD(P)H oxidase
inhibitors and blockade of the flavoprotein-dependent enzymes xanthine
oxidase and nitric oxide synthetase on proliferation of malignant cell
lines was studied in cultures stimulated with 10% FBS and grown for
48 h before proliferation was measured. The effect of NAD(P)H
oxidase activity on transcriptional activation was studied by
incubation of 70% confluent cultures with 0-50 µmol/l of the
flavoprotein inhibitor diphenylene iodonium (DPI) for 24 h before
measurement of DNA binding by EMSA, detection of constitutive NF-B
nuclear activation by immunohistochemical staining for the p65
component, or immunoassay of levels of active transcription
factor component in nuclear protein.
Measurement of proliferation in cell cultures. Proliferation of cultured cells seeded into 24-well uncoated plastic plates (Costar) at 50,000 cells/well (except where indicated) was quantitated as previously described (11) by using a colorimetric method based on metabolic reduction of the soluble yellow tetrazolium dye 3-(4,5,-dimethylthiazol)-2-yl-2,5-diphenyltetrazolium bromide (MTT) to its insoluble purple formazan by the action of mitochondrial succinyl dehydrogenase. For studies with a final cell density of less than about 40,000 cells/well, direct cell counts were performed on 10 random fields/well of Wright's-modified Geimsa-stained monolayers viewed at a ×100 magnification with a 0.01-cm2 ocular grid.
Measurement of ROS generation by intact cells. Intracellular production of ROS by M1619 cells or epidermal melanocytes was measured by oxidation of DCFH-DA to DCF (42). DCFH-DA is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the nonfluorescent polar derivative DCFH and thereby trapped within the cells. In the presence of H2O2, DCFH is oxidized to the highly fluorescent DCF. Approximately 1 × 106 M1619 cells or human epidermal melanocytes were incubated in the dark for 10 min at 37°C with 50 µmol/l DCFH-DA, harvested, and resuspended in plain medium. Fluorescence was analyzed by using a FACScan (Becton Dickinson, Sunnyvale, CA) flow cytometer with excitation at 488 nm and emission at 530 nm.
Measurement of ROS generation by cell membranes.
The method of Pagano et al. (38), with centrifugation
speeds modified according to the work of Mohazzab-H and Wolin
(36), was used to prepare membranes for measurement of
ROS. M1619 cells from six near-confluent T-75 flasks were harvested
with cell dissociation solution (Sigma), washed once with ice-cold
Dulbecco's phosphate-buffered saline (DPBS), and centrifuged for 5 min
at 675 g. The pellet was resuspended in 500 µl of ice-cold
Tris-sucrose buffer [pH 7.1; composed of (in mmol/l) 10 Trizma base,
340 sucrose, 1 phenylmethylsulfonyl fluoride, 1 EDTA, and 10 µg/ml
protease inhibitor cocktail (Sigma)] and sonicated by four 15-s
bursts. The cell sonicate was centrifuged at 1,475 g and
4°C for 15 min in an Eppendorf microfuge to remove nuclei and
unbroken cells. The supernatant was then centrifuged at 29,000 g and 4°C for 15 min in a Beckman Optima TL
ultracentrifuge. The pellet was discarded, and the supernatant was
further centrifuged at 100,000 g and 4°C for 75 min. The
pellet was resuspended in 100 µl of Tris-sucrose buffer and stored at
80°C. Supernatant from the last centrifugation was also saved as a
representative of lactate dehydrogenase-containing soluble elements of
cytoplasm (36). Generation of ROS was measured by
superoxide dismutase (SOD)-inhibitable lucigenin chemiluminescence, as
recently reported (38, 56), in 500 µl of 50 mmol/l
phosphate buffer (pH 7.0) containing 1 mmol/l EGTA, 150 mmol/l sucrose,
5 µmol/l lucigenin, 15 µg of cell membrane protein, 50 µg of
cytosolic protein, and 100 µmol/l NADH or NADPH as substrate.
Chemiluminescence (in arbitrary light units) was measured by using a
Turner model 20/20D luminometer (Turner Designs, Sunnyvale, CA) at 30-s
intervals for 5 min with and without addition of 300 units of SOD to
determine dependence of light generation on O
RT-PCR detection of NAD(P)H oxidase components. To probe for the presence of p22phox, gp91phox, p47phox, and p67phox components of the putative analog of neutrophil NAD(P)H oxidase and the newly described gp91phox homologs NOX1 (2, 4, 48) and NOX4 (12, 17, 30, 45), we performed semiquantitative RT-PCR, as recently described (10), on triplicate near-confluent cultures of proliferating cells grown in 25-mm plastic dishes. Cell monolayers were washed twice with DPBS and lysed with 4 mol/l guanidine thiocyanate, 25 mmol/l sodium citrate, and 0.5% N-lauroylsarcosine. After scraping, lysates were sheared with four passes through a pipette. RNA was extracted by the phenol-chloroform method (13) and quantitated spectrophotometrically at 260 and 280 nm. RNA (2 µg) was reverse transcribed by using 200 units of M-MLV revere transcriptase (Promega) in a reaction mixture containing 1 mmol/l dATP, dCTP, dGTP, and dTTP, 40 units of RNase inhibitor, 25 µmol/l random hexamers, 5 mmol/l MgCl2, 500 mmol/l KCl, and 100 mmol/l Tris · HCl (pH 8.3) in a total volume of 50 µl. The resultant cDNA was PCR amplified for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), p22phox, gp91phox, p47phox, p67phox, NOX1, and NOX4 by using human gene-specific sense and antisense primers based on sequences published in GenBank: GAPDH-5', ACCACCATGGAGAAGGCTGG; GAPDH-3', CTCAGTGTAGCCCAGGATGC; p22phox-5', ATGGAGCGCTGGGGACAGAAGCACATG; p22phox-3', GATGGTGCCTCCGATCTGCGGCCG; gp91phox-5', TGGTACACACATCATCTCTTTGTG; gp91phox-3', AAAGGGCCCATCAAGCGCTATCTTAGGTAG; NOX1-5', CTGGGTGGTTAACCACTGGTTT; NOX1-3', ACCAATGCCGTGAATCCCTAAG; NOX4-5', TAACCAAGGGCCAGAGTATCACT; NOX4-3', CCGGGAGGGTGGGTATCTAA; p47phox-5', ACCCAGCCAGCACTATGTGT; p47phox-3', AGTAGCCTGTGACGTCGTCT; p67phox-5', CGAGGGAACCAGCTGATAGA; and p67phox-3', CATGGGAACACTGAGCTTCA. PCR was carried out on a Perkin-Elmer DNA thermal cycler 480. Except where indicated, amplification was carried out for 30 cycles for GADPH, 32 cycles for p22phox, 34 cycles for NOX4, and 36 cycles for all other primers at 95°C for 1 min, 58°C for 1 min, and 72°C for 2 min, followed by an extension step at 72°C for 10 min. PCR-amplified DNA was separated on 1.2% agarose gel, stained with ethidium bromide, and visualized and photographed under ultraviolet light. PCR products from defined bands were purified with QiaQuick gel extraction kits (Qiagen, Chatsworth, CA) and sequenced automatically with an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA) by using the same respective primers for sequencing as for PCR.
Immunoassay for p22phox, gp91phox,
IB
, phosphorylated I
B
, and p65 component of NF-
B.
To measure protein expression of p22phox and
gp91phox in the cytosol and in the 100,000 g plasma membrane fraction of M1619 cells, we performed
immunoassays as detailed earlier (10, 11) using previously
described rabbit polyclonal antibodies prepared against whole human
p22phox (R3179) (24) and
gp91phox COOH-terminal peptide (R2089)
(40) at dilutions of 1:1,000. To assess nuclear
translocation of the cytosolic transcription factor NF-
B, we
similarly immunoassayed the p65 NF-
B component in nuclear protein
that was isolated as outlined below. For immunoassay of the NF-
B
inhibitor I
B
or for I
B
phosphorylated at serine 32, we
followed the same procedure previously reported (11), with
cells lysed in boiling buffer to which 50 mM dithiothreitol had been
added as a reducing agent.
Transfection protocol for p22phox and NOX4 sense and antisense treatment of M1619 cells. To transfect antisense oligonucleotides for p22phox, M1619 cells were culture in six-well plates at a density of 20,000 cells/well and grown in RPMI 1640 containing 10% FBS. After 24 h, wells were washed once with DPBS, and 800 µl of RPMI 1640 (serum and antibiotic free) were added to each well. Previously reported (33) p22phox sense (5'-GGTCCTCACCATGGGGCAGATC-3') or antisense (5'-GATCTGCCCCATGGTGAGGACC-3') oligonucleotides (2 µg) were mixed with 5 µl of Lipofectace reagent (Life Technologies) and 200 µl of serum- and antibiotic-free RPMI 1640 at room temperature for 15 min. This mixture was then added to each well, and cells were incubated at 37°C. After 6 h the transfection mixture was gently removed and replaced with 2.5 ml of RPMI 1640 containing 10% FBS. Cell were incubated an additional 48 h before staining with hematoxylin and eosin for photography or quantitation of growth with the MTT assay. M1619 cells were transfected with NOX4 sense (5'-TCGAGGAGGTCCTGTGTCGG-3') or antisense (5'-AGCTCCTCCAGGACACAGCC-3') oligonucleotides based on gene-specific unique sequences published in GenBank (accession no. NM 016931). The transfection protocol was identical to that used for p22phox oligonucleotides, except that 10 µl of Lipofectin reagent (Life Technologies) were used instead, and cells were photographed with a phase-contrast microscope and a green filter.
EMSAs.
To assess DNA binding of NF-B or the cAMP response element (CRE)
family of binding proteins, nuclear protein was isolated and EMSAs were
performed as previously reported (11). The consensus binding oligonucleotides 5'-AGTTGAGGGGACTTTCCCAGGC-3' and
3'-TCAACTCCCCTGAAAGGGTCCG-5' for the p50 component of NF-
B,
5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and 3'-TCTCTAACGGACTGCAGTCTCTCGATC-5'
for CRE, 5'-CGCTTGATGAGTCAGCCGGAA-3' and 3'- GCGAACTACTCAGTCGGCCTT-5'
for AP-1, and 5'-TGTCGAATGCAAATCACTAGAA-3' and
3'-ACAGCTTACGTTTAGTGATCTT-5' for OCT-1 were used in binding reactions after end-labeling by phosphorylation with
[
-32P]ATP and T4 polynucleotide kinase. Competition
experiments were performed with 10× respective unlabeled wild-type
oligonucleotide sequences, and supershift experiments were carried out
by incubating the binding reaction with 1 µg of supershift antibody.
Immunohistochemical localization of NF-B.
Constitutive activation of NF-
B was also studied by qualitatively
assessing nuclear localization of the p65 component by immunohistochemical staining, as described previously
(11).
Transduction protocols for IB
gene transfer.
To repress activation of NF-
B, cells were transduced with adenoviral
(Ad serotype 5) vectors that were E1a/E1b-deleted and expressed a
superrepressor of NF-
B (AdI
B
SR; 2 × 1011
plaque forming units/ml) under the regulation of the cytomegalovirus (CMV) immediate-early promoter region (6) or expressed the CMV immediate-early promoter region alone (AdCMV-3; 2.05 × 1011 plaque forming units/ml, control vector). These
adenoviral vectors were constructed in the Vector Core Laboratory
[Gene Therapy Center, University of North Carolina (UNC), Chapel Hill,
NC] and were generous gifts, respectively, from Dr. Albert S. Baldwin
of the UNC Lineberger Comprehensive Cancer Center and Dr. Andrew Ghio of the U.S. Environmental Protection Agency (UNC Human Health Effects
Center). Transduction was performed by using previously published
protocols (6). M1619 cells were seeded onto 24-well plates
at a density of 25,000 cells/well and grown for 6 h in RPMI 1640 with 10% FBS. Medium was removed and replaced with 200 µl of
complete medium containing ~1.25 × 106-2.0 × 107 colony forming units
of AdI
B
SR or AdCMV-3. After overnight incubation, the
vector-containing medium was removed, and cells were washed once with
warm DPBS and reincubated with fresh complete medium. After an
additional 24 h, proliferation was assessed with the MTT assay.
Statistical analysis. Data are expressed as means ± SE for a minimum number of four observations, unless indicated. Differences between two groups were compared using the unpaired Student's t-test. Two-tailed tests of significance were employed. Differences between multiple groups were compared using one-way analysis of variance. The post hoc test used was the Newman-Keuls multiple comparison test. Significance was assumed at P < 0.05.
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RESULTS |
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Malignant melanoma cells produce intracellular ROS.
We have previously reported that proliferation of M1619 malignant
melanoma cells is strongly inhibited by a number of antioxidants, including the H2O2 scavenger catalase, the
sulfhydryl donor N-acetylcysteine, and the glutathione
peroxidase mimetic ebselen, suggesting the importance of oxidants in
regulating growth of this cell line (11). We have also
previously demonstrated that cultured melanoma cells release
OB activation, interleukin-8 and GRO-
secretion,
O
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Malignant melanoma cell membranes produce ROS.
To further study the source of O-nitro-L-arginine, 100 µmol/l) (data not shown). Chemiluminescence generation was likewise
reduced by direct addition of dicumarol to the membrane preparation in
the absence of cytosol. This raises the possibility dicumarol inhibits
plasma membrane NAD(P)H oxidase activity, perhaps by disrupting
substrate binding in a fashion similar to the mechanism by which it
inhibits cytosolic NQO1 (34).
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Malignant melanoma cells and melanocytes express NAD(P)H oxidase
components that are necessary for proliferation.
The best-described membrane NAD(P)H oxidase is that of the neutrophil.
To determine whether components of this NAD(P)H oxidase or its reported
homologs are also expressed in melanomas and nonmalignant melanocytes,
we performed RT-PCR on RNA extracted from proliferating cells
stimulated by 10% FBS (melanomas) or HMGS (melanocytes). M1619
melanoma cells (Fig. 3 A,
M1619 melanoma, lane 2) and other malignant melanomas (Table
1) strongly expressed the -subunit of
cytochrome b558, p22phox,
initially detected at 32 cycles. The 252-base pair PCR product obtained
was sequenced and is identical to bases 221-372 of the reported
human mRNA sequence (accession no. XM 008040). The cytochrome- and
flavin-bearing
-subunit of the cytochrome,
gp91phox, was also present in malignant
melanomas (Fig. 3A, M1619 melanoma, lane 3, and
Table 1), but only after 36 cycles. This 527-base pair product was
sequenced and corresponds to bases 630-1158 of the reported human
mRNA sequence (accession no. NM 000397). By immunoassay, both
p22phox and gp91phox were
easily detectable in the 100,000 g plasma membrane fraction of M1619 melanoma cells (Fig. 3B). No evidence was found for
the NOX1 homolog of gp91phox (Fig.
3A, M1619 melanoma, lane 6). However, NOX4 was
easily detectable at 34 cycles in M1619 and other malignant melanoma
cells (Fig. 3A, M1619 melanoma, lane 7, and
3C, lanes 1-5) as a 564-base pair product,
with sequences corresponding to bases 197-741 of the reported
human mRNA sequence (accession no. NM 016931). Melanocytes also
expressed p22phox (Fig. 3A,
melanocytes, lane 2) and NOX4 (Fig. 3A,
melanocytes, lane 7) but did not contain mRNA for
gp91phox (Fig. 3A, melanocytes,
lane 3). The p67phox cytosolic
component was observed in M1619 cells (Fig. 3A, M1619 melanoma, lane 5) as a 747-base pair product with sequences
identical from bases 556-1283 of the reported human mRNA sequence
(accession no. BC 001606) and was also observed in two other malignant
melanoma cell lines (Table 1). Faint PCR product was also
detected in M1619 cells for the p47phox
cytosolic component of the leukocyte NADPH oxidase (Fig.
3A, M1619 melanoma, lane 4), but this product was
not sufficiently well expressed to be sequenced. Neither
p67phox nor p47phox was
found in epidermal melanocytes. Thus three known membrane components
(p22phox and two possible partners,
gp91phox and NOX4) and the
p67phox cytosolic component of the NAD(P)H
oxidase are present in proliferating M1619 and other melanoma cells,
and p47phox may be expressed at low levels. In
contrast, when proliferating, normal melanocytes expressed only
p22phox and NOX4.
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NF-B is constitutively expressed in melanoma cells and may be
regulated by the NAD(P)H oxidase.
We have previously reported that NF-
B is constitutively activated in
M1619 melanoma cells (11). NF-
B is also constitutively activated in proliferating human epidermal melanocytes
(35). The flavoprotein-dependent NAD(P)H oxidase inhibitor
DPI reduced constitutive activation of NF-
B in melanoma cells as
studied by NF-
B DNA binding activity (Fig.
5, A and B),
immunohistochemically detectable p65 in nuclei (Fig. 5, C
vs. D), and immunoassay of the p65 NF-
B component in
nuclear protein (Fig. 5E). DPI treatment of melanoma cells
also decreased phosphorylation of the NF-
B inhibitor I
B
(Fig.
5F). Taken together, these findings and those reported
previously (11) suggest that a flavoprotein-containing NAD(P)H oxidase may play a role in stimulating constitutive NF-
B transcriptional activity in these cells through generation of ROS.
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Inhibition of NF-B does not impair melanoma proliferation.
Inhibition of NF-
B by antisense strategies reduces tumorigenicity of
fibrosarcomas (20), and overexpression of the NF-
B inhibitor I
B
blocks tumor cell growth of Hodgkin's disease
(5), squamous cell lung cancer (6), squamous
cell head and neck cancer (16), and breast cancer
(47) cells. We therefore infected M1619 cells with an
adenoviral vector encoding a superrepressor version of the NF-
B
inhibitor I
B
(AdI
B
SR) to determine whether selectively
inhibiting NF-
B could reduce M1619 melanoma cell proliferation.
Infection with AdI
B
SR resulted in dose-related increases
I
B
SR expression (Fig.
6A). However, infection with even the highest dose (1 × 107 colony forming units)
of AdI
B
SR did not substantially reduce M1619 melanoma
proliferation (Fig. 6B), especially compared with the
profound growth inhibitory effect shown by antioxidants and NAD(P)H
oxidase inhibitors in Fig. 1. Therefore, oxidant generation by a growth
regulatory NAD(P)H oxidase must regulate proliferation through other
signal transduction pathways.
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Inhibition of NAD(P)H oxidase in melanoma cells reduces DNA binding
activity for CRE.
Another family of redox responsive transcription factors important for
melanoma proliferation are activating transcription factor/CRE-binding
(ATF/CREB) proteins that bind CRE. The transcription factor
CREB and its associated family member ATF-1 promote tumor growth,
metastasis, and survival through CRE-dependent gene expression (58), and expression of the dominant negative KCREB
construct in melanoma cells decreases their tumorigenicity and
metastatic potential in nude mice (23). We therefore
explored whether inhibition of NAD(P)H oxidase in melanoma cells might
reduce DNA binding of transcription factors to CRE. As reported
previously for the MeWo melanoma cell line (23, 58),
proliferating M1619 cells displayed prominent DNA binding activity for
CRE, composed of ATF-1, ATF-2, and CREB-1 transcription factors (Fig.
7A). Treatment of
proliferating M1619 cells overnight with DPI inhibits CRE-binding activity in a dose-dependent manner (Fig. 7, B and
C) but does not change DNA binding activity of nuclear
protein for the transcription factors AP-1 (Fig. 7D) or
OCT-1 (Fig. 7E). Thus the growth regulatory NAD(P)H oxidase
may signal melanoma proliferation in part through activation of
CRE-mediated transcriptionally related events.
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DISCUSSION |
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In this report we demonstrate evidence for a growth regulatory oxidase activity in human malignant melanoma cells. By using RT-PCR, we found clear evidence in M1619 melanoma cells for mRNA expression of the NAD(P)H oxidase components p22phox, gp91phox, the gp91phox homolog NOX4, p67phox, and possibly p47phox (Fig. 3, A and C). Expression of oxidase components is not necessarily a transforming event in melanomas, because p22phox and NOX4 were also expressed in normal melanocytes. However, several oxidase components appear critically important for malignant growth, because melanoma proliferation is reduced by transfection of antisense but not sense oligonucleotides p22phox (Fig. 4A) and NOX4 (Fig. 4B). Thus the NAD(P)H oxidase is a normal component of signaling machinery that may be parasitized to serve malignant proliferation.
The putative melanoma NAD(P)H oxidase shares some of the functional
properties of the NAD(P)H oxidase of phagocytes but with important
differences. Like the phagocyte oxidase, the initial enzyme product
appears to be O
In phagocytes, the NAD(P)H oxidase consists of two membrane proteins,
gp91phox and p22phox,
that bind a flavin adenine nucleotide (FAD) and form a cytochrome with
a redox midpoint potential of 245 mV and a reduced-minus-oxidized difference spectrum of 558 (3). At least two and possibly
three cytosolic proteins (p47phox,
p67phox, and p40) are also essential, and
several other cytosolic components participate, including the small
GTPases Rac1 or Rac2. The oxidase is thought to contain all the factors
necessary for transporting electrons from the donor substrate NADPH via
FAD to generate O
A potentially large number of signal transduction and gene expression
systems might be influenced by a growth regulatory NAD(P)H oxidase
(2), among which is the redox-regulated transcription factor NF-B. We have previously shown that antioxidants reduce I
B
phosphorylation and constitutive NF-
B activation in
malignant melanoma cells (11). We now demonstrate that the
flavoprotein inhibitor DPI reduces I
B
phosphorylation (Fig.
5F) and constitutive NF-
B activation (Fig. 5,
A-E), suggesting that ROS from an NAD(P)H oxidase
contribute to constitutive NF-
B activation. NF-
B has been
recently demonstrated to be important for malignant proliferation of a
variety of cancers (5, 6, 9, 47, 55). Repression of
NF-
B interferes with normal and transformed cell proliferation (21, 27), and inhibition of NF-
B by antisense
strategies (20) or overexpression of the NF-
B inhibitor
I
B
(5, 6, 16, 47) blocks tumor growth. In malignant
melanomas NF-
B is activated as a result of enhanced constitutive
I
B kinase activity (44) and is thought to play a
significant role in autocrine generation by melanomas of the chemokines
MGSA-
/GRO-
and interleukin-8 (56). However, in
contrast to results with other tumor types, we were unable to suppress
growth of M1619 melanoma cells by expression of a superrepressor form
of the NF-
B inhibitor I
B
(Fig. 6). Thus NF-
B activation in
this melanoma cell line may play a greater role in conferring
resistance of the tumor to apoptosis, chemotherapy, and
radiation through upregulating expression of antiapoptotic Bcl-2
family proteins (7, 52, 53). As shown by the significant inhibition by DPI of DNA binding to CRE (Fig. 7, B and
C), an alternative group of transcription factors that could
be redox regulated by the NAD(P)H oxidase is the ATF/CREB family
(1, 29). Molecular disruption of ATF/CREB-mediated
transcription has been previously shown to reduce proliferation,
metastatic potential, and radiation resistance of malignant melanomas
(14, 23, 41, 57, 58).
Others have previously shown the importance of OB activation, and proliferation in
melanomas (11). In this report we show that dicumarol
inhibits lucigenin chemiluminescence by melanoma plasma membranes in an
in vitro system lacking cytosolic components (Fig. 2), suggesting
inhibition of membrane NAD(P)H oxidase activity. Other coumarins have
previously been reported to block O
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ACKNOWLEDGEMENTS |
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This work was funded by grants from the Charlotte-Mecklenberg Health Care Foundation and in part by National Institutes of Health Grants HL-40665 (to J. R. Hoidal), HL-61377 (to A. R. Whorton), and AR-42426 and HL-66767 (to M. T. Quinn).
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. P. Kennedy, Carolinas Medical Center, 410 Cannon Research Center, PO Box 32861, Charlotte, NC 28232 (E-mail: tkennedy{at}carolinas.org).
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.
First published January 2, 2002;10.1152/ajpcell.00496.2001
Received 16 October 2001; accepted in final form 17 December 2001.
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