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[Cancer Research 60, 3638-3644, July 1, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

Increased Tumor Necrosis Factor-{alpha} Sensitivity of MCF-7 Cells Transfected with NAD(P)H:Quinone Reductase1

Linda M. Siemankowski, Jeanne Morreale2, Brent D. Butts and Margaret M. Briehl3

Department of Pathology, University of Arizona, Tucson, Arizona 85724-5043


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence from a number of studies suggests that the mechanism by which tumor necrosis factor (TNF) kills transformed cells involves oxidative stress. NAD(P)H:(quinone acceptor) oxidoreductase (NQO1) is an antioxidant enzyme with particular relevance to cancer. The MCF-7 breast cancer cell line was stably transfected with rat NQO1 cDNA to determine whether increased NQO1 activity alters sensitivity to TNF-induced apoptosis. Five clones, with a range of NQO1 enzyme activities from 5- to 50-fold greater than the MCF-7 line, and two control transfectants were examined. Northern blot hybridization analyses and reverse transcription-PCR demonstrated that the increase in NQO1 activity in the transfectants was attributable to expression from the transfected rat sequence. Based on sulforhodamine B assays for the number of viable cells, the NQO1 clones showed increased sensitivity to EO9, an indoloquinone that undergoes bioactive reduction by NQO1. Viability studies also demonstrated that the NQO1 transfectants were significantly more sensitive to TNF than the control transfectants or MCF-7 parent. This increased sensitivity could not be explained by changes in superoxide dismutase or catalase activity or to increased sensitivity to oxidative stress in general, as assessed by response to hydrogen peroxide and paraquat treatment. Using dichlorodihydrofluorescein diacetate as a probe, we found that the NQO1 transfectants had no difference in baseline level of oxidative stress compared to the control cells but did exhibit greater intracellular oxidative stress after TNF treatment. We conclude that NQO1 can affect the TNF-mediated pathway to apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress has been implicated in the mechanism by which TNF4 kills transformed cells. Increased levels of reactive oxygen species (1, 2, 3) and oxidative damage (4 , 5) are observed after treatment of different cell types with TNF. A common response to TNF treatment is the induction of an antioxidant defense enzyme, MnSOD (6) . MnSOD is localized to mitochondria and catalyzes the reduction of superoxide anion radical to hydrogen peroxide, which can be converted to water by glutathione peroxidases. Increased MnSOD expression is apparently a protective response because transfection with MnSOD increases resistance to TNF-mediated cytotoxicity (7, 8, 9) .

The effect of MnSOD on TNF-induced cytotoxicity raises the question of whether the status of other antioxidant defense enzymes influences susceptibility to TNF. NQO1 (EC 1.6.99.2) is an antioxidant enzyme with particular relevance to cancer. Originally named DT-diaphorase, it is a flavoprotein that uses NADH or NADPH to catalyze the two-electron reduction of quinones. The unique, obligatory, two-electron reduction mechanism of NQO1 avoids the formation of unstable quinones that can spontaneously autooxidize, producing superoxide (10 , 11) . A characteristic of quinones is their propensity to undergo reduction-oxidation cycles to form mutagens (12) . Quinones are relatively abundant in foods and are produced upon combustion of organic compounds (e.g., tobacco and engine fuel). Given the level of human exposure to these compounds, their conversion to relatively stable compounds is significant to cancer prevention (13 , 14) . Furthermore, the chemopreventive effects of oltipraz and the antioxidant food additive 2(3) -tert-butyl-4-hydroxyanisole have been attributed at least in part to the induction of NQO1 gene expression and activity (15 , 16) .

NQO1-mediated metabolism of some compounds generates unstable and potentially cytotoxic products. This has led to the discovery of novel, potential chemotherapeutic agents that undergo bioreductive activation by NQO1. Specificity with this chemotherapeutic approach may be possible in light of reported differences in NQO1 levels between normal and tumor tissues (12 , 17 , 18) . Marin et al. (17) measured NQO1 activity in tumor and macroscopically normal tissue from 20 breast cancer patients. Significantly increased levels of NQO1 were found in 70% of the tumor samples, with an average of 12-fold higher activity in the tumor versus normal tissue. In a sampling of 17 breast tumors and paired normal tissue, Schlager and Powis (18) found significantly greater NQO1 activity in the cancer tissue: 165 ± 43 versus 50 ± 11 nmol/min/mg of protein, respectively.

Given the potential role of NQO1 in the prevention and treatment of cancer, we sought to determine whether increased NQO1 activity alters the sensitivity of the MCF-7 breast cancer cell line to apoptosis. We determined previously that these cells undergo apoptosis in response to TNF (19) . We demonstrate here that MCF-7 clones isolated after stable transfection with rat NQO1 cDNA are more sensitive to TNF than the parental cells. Our results provide further support for the importance of oxidative stress to the mechanism of TNF-induced apoptosis and may be significant to the role of NQO1 in cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The human breast adenocarcinoma-derived MCF-7 cell line was obtained from the American Type Culture Collection (Manassas, VA) and maintained at 37°C under 5% CO2 in DMEM supplemented with 5% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. All cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). Tests were performed periodically to ensure that the cultures remained Mycoplasma-free.

Isolation of Stable Transfectants.
The NQO1 expression vector, pA-RDT10, was obtained from Dr. Gerald Forrest (City of Hope, Duarte, CA); it had been constructed by insertion of the rat NQO1 protein coding sequence into the HindIII site of pHßApr-1-neo. The latter plasmid allows for expression under the control of the ß-actin promoter and selection for neomycin resistance (neor; Ref. 20 ). pHßApr-1-neo was used for control transfections. MCF-7 cells (5 x 105) were plated into 100 mm tissue culture dishes and incubated overnight. Transfections were carried out using the calcium phosphate-based Mammalian Transfection Kit from Stratagene (La Jolla, CA), following the manufacturer’s instructions. After three days, selection was begun in 700 µg/ml Geneticin (G418; Life Technologies, Inc.). After 16 days of selection, isolated colonies were picked onto small squares of sterile Whatman 3 M paper (Fisher Scientific, Pittsburgh, PA) that had been saturated with trypsin solution and were then transferred to fresh G418-supplemented complete medium. For maintenance of the selected clones, 300 µg/ml of G418 was used. Transfectants were grown in G418-free medium for at least 8 days before being analyzed

Enzyme Assays.
Cultures were harvested for NQO1 assays when approximately 50% confluent. Cell monolayers were washed three times with PBS and lysed directly on the plate in a solution of 0.25 M sucrose, 10 mM Tris-HCl pH 7.5, 1.0 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF and 1.0% Triton X-100. After incubation at 4°C for 30 min, the lysates were centrifuged at 6,000 x g for 15 min. The supernatant fractions were collected and stored at –80°C until assayed. NQO1 activity was measured following the method described by Ernster (21) , except that the reactions were initiated by addition of NADPH rather than cell lysates. Assay mixtures contained 40 mM Tris-HCl, pH 7.8, 250 µM NADPH, 40 µM 2,6-dichlorophenol-indophenol and 0.07% BSA, with or without 50 µM dicumarol. Dicumarol inhibits NQO1 and was used to correct for other NAD(P)H reductases in the cell lysates. Catalase and SOD activities were measured as described previously (19) .

Analysis of NQO1 Gene Expression.
Northern blot hybridization analyses were carried out using standard procedures (22) . For preparing probes, pA-RDT10 was digested with HindIII to release the 1.2 kb rat NQO1 cDNA insert and phagemid HHCMC32 (American Type Culture Collection, Rockville, MD) was digested with EcoRI to release the 1.2 kb human GAPDH cDNA fragment. The restriction digests were subjected to electrophoresis through low-melting temperature agarose and the appropriate fragments purified using a GeneClean kit (Bio101, Vista, CA). Probes were made using a Random Primers DNA Labeling System, following the manufacturer’s instructions (Life Technologies, Inc.). After hybridization, measurements of transcript levels were carried out using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The values obtained with the GAPDH probe were used to normalize differences caused by gel loading or transfer.

For analysis of NQO1 expression by RT-PCR, cDNA was synthesized from 2.5 µg of total RNA using Superscript reverse transcriptase (Life Technologies, Inc.), as directed. After heat inactivating the enzyme at 70°C for 15 min, 1 µl of the 20 µl reaction was used as template for the PCR. Three oligos were used together in the PCR reaction: an upstream primer that amplifies from both the human and rat NQO1 sequence (5'-GGCTGGTTTGAGAGAGTG-3'); and downstream primers specific for either the human sequence (5'-GCACGAATACGGTCGATTC-3') or rat sequence (5'-GTCGGCTGGAATGGACTTG). The primer combinations amplify a fragment from position 393 to position 1017 (625 bp) in human NQO1 cDNA (GenBank accession number J03934) and a fragment from position 391 to 849 (459 bp) in rat NQO1 cDNA (GenBank accession number J02640). PCR mixes contained 60 mM Tris-HCl (pH 8.5), 15 mM (NH4)SO4, 1.5 mM MgCl2, 250 µM each dNTP, 0.5 pmol each primer and 1 U Taq polymerase (Life Technologies, Inc.) in a 50-µl reaction (final volume). The mixtures were heated to 80°C before addition of the cDNA template. PCR was carried out in a Perkin-Elmer (Norwalk, CT) 480 thermal cycler starting with a 2-min incubation at 94°C, followed by 35 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min, plus a 3 s extension/cycle and a final incubation at 72°C for 7 min. PCR products were electrophoresed through 2% agarose and visualized by ethidium bromide staining.

Sensitivity to EO9.
Cells (9000 per well) were seeded into 96-well microtiter dishes. After an overnight incubation, they were treated with vehicle alone (DMSO) or EO9 at concentrations ranging from 25 to 1000 nM. In each experiment, triplicate wells were used for each drug concentration. EO9 was a generous gift of Dr. Hans R. Hendriks at the European Organization for Research and Treatment of Cancer New Drug Development Office (Amsterdam, the Netherlands). The medium was removed after 3–4 h, and the cells were rinsed once with PBS and refed with fresh complete medium. Three days later, the number of viable cells was measured using a SRB assay as described (23) , with the modification that the cells were first washed twice with PBS, fixed in cold 10% trichloroacetic acid for 30 min at 4°C, washed four times with tap water, and air dried before staining with SRB for 15–20 min.

Assessment of Sensitivity to TNF, Paraquat, and Hydrogen Peroxide.
For determining response to TNF, cells were grown to a confluency of 60–70%, released by trypsinization and plated in 96-well microtiter plates. Initial experiments demonstrated that the extent of cell killing in response to TNF was affected by the density at which the cells were plated. Cells were plated, therefore, at densities of 3, 6, and 9 x 103 cells/well. Each cell dilution was seeded into 14 wells. Two h after plating, TNF (Roche Molecular Biochemicals, Indianapolis, IN) was added to seven of the wells for a final concentration of 20 ng/ml; the remaining seven wells were left untreated. In each microtiter dish, medium alone was delivered to a row of 12 wells; the results from these were subtracted as background in the assay. The plates were incubated for 3 days. Protein content in each well was then measured using the SRB assay as described above. The absorbance readings from each set of 7 samples (minus the background value) was averaged. At each plating density, sensitivity to TNF was calculated as the average value from +TNF samples, over the average value from the –TNF samples. Finally, an average TNF sensitivity was determined for each cell type, using the values from the three plating densities. The same method was followed for determining sensitivity to paraquat and hydrogen peroxide except that cells were plated at densities of 9, 18, and 24 x 103 cells/well and incubated overnight before treatment. The treatment time was reduced to 24 h. Paraquat was used at a final concentration of 700 µM, and hydrogen peroxide was used at concentrations of 300, 600, and 900 µM.

Measurement of Intracellular Oxidative Stress.
Cells were grown to a confluency of 60–70%, released by trypsinization, and plated in 24-well microtiter plates at a density of 1 x 106 cells/well. Fourteen wells were plated per cell type. Additional wells contained medium without cells; results from these were used to correct for background fluorescence in the assay. Two h after plating, TNF was added to seven wells/cell type (final concentration, 20 ng/ml). After 20 h of further incubation, the medium was gently removed by blotting on paper towels, and the wells were rinsed with 500 µl of DMEM containing 0.5% serum and no phenol red or L-glutamine. The cells were then incubated with the same medium containing 20 µM DCFH-DA (Molecular Probes, Eugene, OR) for 30 min at 37°C. The medium was removed, and 500 µl of PBS were added to each well. Fluorescence was read with excitation and emission wavelengths of 485 and 530 nm, respectively, using a model 7620 microplate fluorometer (Packard Instrument Co., Inc., Meriden, CT). To normalize for cell number, after the fluorescence readings, the plates were centrifuged for 10 min at 700 x g to bring down any floating cells. Saturated trichloroacetic acid was added to a final concentration of 12%. The plates were incubated for 30 min at 4°C. Precipitates were collected by centrifuging for 10 min at 2000 x g and 4°C, as above. The samples were washed four times with deionized H2O and allowed to dry. Protein content was determined using SRB assays as described above, except that the samples were diluted 1:10 before reading. Fluorescence readings were corrected for background fluorescence and normalized for protein content.

Statistical Analyses.
Data were analyzed using either the two-sample Student’s t test, assuming unequal variances between two unpaired samples, or one-way ANOVA, using Bonferroni multiple comparison to determine which groups were significantly different from each other. Statistical analysis was performed using StataQuant 4.0 (Stata Corp., College Station, TX).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of NQO1 Expression in Stable Clones.
Increased NQO1 activity in MCF-7 cells was achieved by stable transfection of an expression vector containing the rat NQO1 cDNA under the control of the ß-actin promoter and encoding for neor. Five clones, with a range of NQO1 enzyme activities from 5- to 50-fold greater than the parental line (Table 1)Citation , were chosen for further study. Two neor control transfectants were also analyzed. Northern blot hybridization analyses showed faint to nearly undetectable signals for NQO1 message in the neor control transfectants and MCF-7 parent (Fig. 1)Citation . Two transcript sizes were seen, corresponding to the 2.7- and 1.7-kb mRNAs seen by Jaiswal et al. (24) in their studies of NQO1 expression in a human Hep-G2 hepatoblastoma-derived cell line. The two transcripts are likely attributable to use of different polyadenylation signals (24) . Message levels in the NQO1 transfectants ranged from approximately 20-fold to more than 200-fold greater than the MCF-7 cells (Fig. 1)Citation . The Northern blot also showed that, in samples from the NQO1 transfectants compared to the neo transfectants, the smaller NQO1 transcript migrated more slowly through the gel. This apparent increase in transcript size may be attributable to additional sequence having been transcribed from the expression vector, either upstream or downstream of the protein coding region. The relative levels of expression seen in DT1, DT6, and DT15 correlate well with the measured NQO1 enzyme activities (Table 1)Citation . The transcript levels seen for DT9 and DT12 do not correlate as well; the reason for this is not clear. RT-PCR was next used to confirm expression from the transfected gene. An upstream primer was chosen so that it would amplify both the rat and human NQO1 cDNA and downstream primers were chosen to be species specific. With these primers, 624- and 465-bp fragments are predicted to be amplified from the human and rat sequences, respectively. RNA extracted from the neor control transfectants or MCF-7 cells, gave a 624-bp PCR fragment only (Fig. 2)Citation . A 465-bp fragment was seen after the RT-PCR with mRNA from the NQO1 transfectants. The 624-bp fragment corresponding to the human transcript was also present in the samples from the NQO1 transfectants, but at a reduced level compared to the rat-derived fragment. For the DT1 and DT15 samples, the human band was barely detectable. This is likely attributable to higher levels of rat transcripts outcompeting human transcripts for the shared PCR primer. Taken together, these results suggest that the increased NQO1 activity in the transfectants is most likely attributable to expression from the transfected rat sequence. Although it is possible that transfection of the rat gene may have increased NQO1 activity through an effect on the endogenous promoter, this possibility is not consistent with the relative levels of the rat and human transcripts seen by the PCR analyses.


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  		Table 1 NQO1 activity in MCF-7 stable transfectants

 


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  		Fig. 1. Expression of NQO1 in the MCF-7 parent, control, and NQO1 transfectants. Northern blot hybridization analyses of total RNA extracted from the indicated cell type at approximately 50% confluency. The blots were probed with 32P-labeled rat NQO1 and then stripped and reprobed with labeled human GAPDH cDNA. The numbers above and below the results from the NQO1 probing indicate the intensities of the upper and lower transcript bands, respectively, relative to the same NQO1 band in the lane containing RNA from MCF-7 cells. The numbers have been corrected for differences in gel loading and transfer as determined by the relative intensities seen with the GAPDH probe.

 


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  		Fig. 2. Increased NQO1 message from the stably transfected rat gene sequence. RT-PCR analysis with total RNA extracted from the MCF-7 parent, control, and NQO1 transfectants. The reaction mixes contained primers that allow for amplification of 624- and 465-bp fragments from the human and rat NQO1 cDNA sequences, respectively (see "Materials and Methods"). MW, 100-bp ladder molecular weight marker; the bright band in the middle of the ladder corresponds to 600 bp.

 
NQO1 Transfectants Are More Sensitive to EO9.
EO9 is an indoloquinone that was synthesized as an analogue of the anticancer drug mitomycin C (25) . Subsequently, it was found to be reduced by NQO1 to an unstable intermediate that in the presence of air forms oxygen- and drug-based radicals (26) . A significant correlation between NQO1 activity and sensitivity to EO9 is observed across the National Cancer Institute tumor cell line panel (27) and other cancer cell lines (28 , 29) . Thus, increased NQO1 expression in the MCF-7 transfectants would be expected to lead to increased cell killing upon treatment with EO9. This was tested by calculating the IC50 values after a 3-day treatment of the cells with 0, 25, 50, 100, 250, 500, and 1000 nM EO9. Compared to the MCF-7 line or neor control transfectant, the NQO1 transfectants showed a significantly increased sensitivity to EO9 (Table 2)Citation . Despite expressing varying levels of NQO1 activity (Table 1)Citation , the three transfectants that were tested showed a similar response to EO9.


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  		Table 2 Sensitivity to the indoloquinone EO9

 
Elevated NQO1 Expression Is Associated with Increased Sensitivity to TNF.
In previous experiments, we used morphological assessments to verify that TNF treatment of the MCF-7 cell line leads to death by apoptosis (19) . The percent of cells with apoptotic morphology at any one sampling time is relatively small, however, so that a large number of cells must be scored to test for significant differences in TNF sensitivity. As an alternative approach to counting the number of cells seen with apoptotic morphology, we used a SRB assay (23) to assess cell viability after treatment of control and NQO1 transfectants with 20 ng/ml TNF. Each cell type was plated into microtiter plates at three plating densities. TNF was added to half of the wells; the remainder of the wells were used as the –TNF control and received no additional treatment. After 3 days, the SRB assay was performed. TNF sensitivity was calculated as the ratio of absorbance measured in the +TNF samples to that measured in the –TNF samples. As shown in Fig. 3Citation , the NQO1 transfectants were all found to be significantly more sensitive to TNF than the neor control transfectants. The control transfected clones were not significantly different from the MCF-7 parent in their TNF sensitivity (data not shown).



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  		Fig. 3. Increased sensitivity to TNF in the NQO1 transfectants. Results from SRB assays for relative cell growth after incubation of the different cell types in the absence ({square}) or presence ({blacksquare}) of 20 ng/ml TNF for 3 days (see "Materials and Methods"). The height of the columns indicates the average absorbance readings calculated from seven microtiter plate wells; bars, SE. Data for the Neor graph were combined from the neo2 and neo7 transfectants. Average +TNF/–TNF values indicate the average ratio for the three plating densities. Asterisks, significant differences in the +TNF/–TNF value compared to the neo control (*, P < 0.01; **, P < 0.05). The data shown are from one of three experiments that gave the same results.

 
No correlation was seen between fold increase in enzyme activity and TNF sensitivity of the NQO1 transfectants. The greatest decrease in viable cell number with TNF treatment was seen for DT6, which exhibited an intermediate fold increase in NQO1 activity. DT15, with the highest NQO1 activity, showed the smallest change in viable cell number after treatment with TNF. There was no statistical difference in TNF sensitivity between the NQO1 transfectants.

While culturing the DT15 transfectant, we observed that it took longer to reach confluency than the other cell types. Using SRB assays to monitor cell growth, we calculated the doubling time for the parental MCF-7 cells and the transfectants. The results confirmed that the DT15 transfectants had a significantly (P < 0.01) longer mean doubling time (59 h) than the other cell types (28 h). The latter doubling time is consistent with previous measurements of the growth of MCF-7 cells (30) . By measuring TNF response as the difference in the number of viable cells between untreated and treated cultures, we controlled for differences in growth rate. Thus, the longer doubling time of DT15 does not explain its sensitivity to TNF.

The selection for stable transfectants may have led to changes in the cells that influence TNF sensitivity. Stable transfection of one antioxidant defense gene has been reported to lead to changes in other antioxidant defense enzyme activities (31 , 32) . If increased NQO1 activity caused a consistent change in another antioxidant defense enzyme, this could be the cause of the observed, altered TNF sensitivity. In particular, sensitivity to TNF has been correlated with SOD expression in several cell types, including the MCF-7 line (6 , 7 , 33, 34, 35) . SOD and catalase activities in the control and NQO1 transfectants were measured to determine whether there had been any changes in these. Significantly lower levels of SOD were found in DT12 and DT15 compared to the other cell types, and DT15 also had significantly lower catalase activity (Table 3)Citation . Catalase and SOD activity were not significantly altered in DT9 cells compared to MCF-7 or neo controls. Because increased NQO1 activity is the single consistent change in the antioxidant defenses of the different transfectants, we believe that it underlies their increased sensitivity to TNF rather than changes in other antioxidant defenses, as seen in DT12 and DT15.


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  		Table 3 Antioxidant defense enzyme activities in MCF-7 stable transfectants

 
Increased Oxidative Stress in NQO1 Transfectants after TNF Treatment.
In light of evidence that the mechanism of TNF-induced apoptosis involves oxidative stress (reviewed in Ref. 36 ), increased NQO1 activity may affect TNF sensitivity by raising the level of oxidative stress in the cell. Specifically, metabolism of a cellular substrate by NQO1 could generate oxidizing species. The NQO1 substrate may be present constitutively or generated in response to TNF. This model raises the following questions: (i) whether the transfectants are more sensitive to oxidative stress in general; and (ii) whether higher levels of oxidative stress are seen in the transfectants in either the absence or presence of TNF. To address the first question, we compared the response of the different cell types to hydrogen peroxide and paraquat. Intracellular metabolism of paraquat generates superoxide anion (37) . We saw no differences in sensitivity to hydrogen peroxide between MCF-7, neo6, neo7, DT9, DT12, and DT15 cells, and when cells were treated with paraquat, only DT6 was found to be significantly more sensitive (P < 0.02, data not shown). These results suggest that increased sensitivity to oxidative stress in general does not explain the altered TNF response of the NQO1 transfectants.

We next addressed the question of whether there are differences in the intracellular levels of oxidative stress between the NQO1 and control transfectants. DCFH-DA is a colorless, nonfluorescent cell-permeable dye. It is deacetylated by esterases within the cell, forming DCFH, which is no longer permeable to the cell membrane and becomes fluorescent upon reaction with oxidants in the cell (38) . Using DCFH-DA as a probe, we observed that in the absence of TNF, the level of oxidants in the NQO1 transfectants was slightly, but not significantly, higher than in the control transfectant (Fig. 4)Citation . After TNF treatment, significantly greater fluorescence was seen in the NQO1 transfected cells compared to the MCF-7 or control transfectants (Fig. 4)Citation . As a control for differences in esterase activity between the cell types, the fluorescence in the samples was rechecked after 2 h. No increase in fluorescence was seen (data not shown). This indicates that in all of the cell types, there was sufficient esterase to cause complete hydrolysis of DCFH-DA at the concentration used. The greater fluorescence seen in the NQO1 transfected cells, therefore, is attributable to an increased rate of oxidation of the DCFH. The highest levels of fluorescence were observed in TNF-treated DT6 and DT15 cells; a smaller, but still significant, increase was seen after treatment of DT9 cells. The observed variation in intracellular oxidants for DT9 cells versus the other NQO1 transfectants, after TNF treatment, may be explained by other changes that occurred during the selection of stable clones. For example, a small but significant decrease in catalase activity was seen for DT15 (Table 3)Citation , whereas DT12 and DT15 were found to have significantly lower total SOD activity. Overall, the results suggest that increased NQO1 activity does not raise baseline levels of oxidative stress in the cells but is consistently associated with a greater state of oxidative stress after treatment with TNF.



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  		Fig. 4. Increased level of oxidative stress in NQO1 transfectants after TNF treatment. The indicated cell types were incubated in the absence ({square}) or presence ({blacksquare}) of 20 ng/ml TNF for 20 h. Levels of intracellular oxidants were measured based on fluorescence with DCFH. The fluorescence data were normalized to protein content in the samples. Values shown are the mean of seven replicates; error bars, SE. Asterisks, significant difference in the amount of fluorescence relative to the control.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have found that overexpression of NQO1 in MCF-7 cells increases TNF-mediated cell killing. TNF treatment caused greater oxidative stress in the NQO1 versus control transfectants. In the absence of TNF, levels of oxidants in the transfectants were no higher than in the vector only-transfected controls. This suggests that raising the level of NQO1 in the cell did not increase the baseline amount of oxidative stress resulting from aerobic metabolism. Because most of the NQO1 transfectants were no more sensitive to hydrogen peroxide or paraquat than the control transfectants or MCF-7 parent, it does not appear that their altered TNF response is explained by increased susceptibility to oxidative stress in general. We interpret the increased TNF sensitivity of the NQO1-transfected MCF-7 cells as evidence that the mechanism of apoptosis is sensitive to perturbations in the cellular redox state, which can be modulated by NQO1 levels.

NQO1 can function as either an antioxidant or a prooxidant, depending upon the chemical nature and reactivity of the hydroquinone produced in the reactions it catalyzes (39) . Recent reports indicate that NQO1 maintains {alpha}-tocopherolquinone (40) and ubiquinones (41, 42, 43) in their reduced states. These molecules are found in all cell membranes and act as hydrophobic phase antioxidants to prevent lipid peroxidation. The initial phase in TNF signaling for apoptosis involves the assembly of a protein complex on the cytoplasmic face of the plasma membrane (44) . Studies of protein-protein interactions suggest that binding of TNF to 55-kDa plasma membrane receptors (designated p55 or TNF-R1) leads to trimerization of the receptors followed by the sequential recruitment of TNFR-associated death domain, Fas-associated death domain, and caspase-8 proteins. Assembly of this death-inducing signaling complex could be sensitive to the oxidation state of the membrane. If NQO1 influences apoptosis at this point in the signaling pathway, we would predict that recruitment of caspase-8 is facilitated when plasma membrane {alpha}-tocopherolquinone or ubiquinone is present in its reduced state. This possibility does not preclude oxidative stress playing a role in the mechanism of TNF-induced apoptosis further downstream of the initial signaling events.

An alternative possibility is that NQO1 increases TNF sensitivity by acting as a prooxidant. Engagement of TNF-R1 by TNF activates a neutral sphingomyelinase that can hydrolyze plasma membrane sphingomyelin and thereby release ceramide (reviewed in Ref. 45 ). Incubation of rat hepatocytes with a permeable analogue, C2-ceramide, increases hydrogen peroxide levels in the cells and the level of ceramide in mitochondria (46) . When C2-ceramide is incubated with isolated mitochondria, direct production of hydrogen peroxide is observed. Results with inhibitors of the mitochondrial electron transport chain have suggested that the hydrogen peroxide is generated by blockage of electron flow at the ubiquinone pool (46) . Higher levels of NQO1 in the cell may result in more electrons being passed from NADH to ubiquinone. With electron flow from ubiquinone to the cytochrome b-c1 complex inhibited by ceramide, this may increase the rate of superoxide production because electrons will be passed to oxygen instead.

Although MCF-7 stable transfectants with variable levels of NQO1 were isolated, enzyme activities were not found to correlate with sensitivity to TNF or EO9. Gustafson et al. (47) stably transfected CHO cells with human NQO1. EO9 sensitivity among the clones was found to be proportional to the level of enzyme activity. The clones that we have isolated exhibit NQO1 activity similar to the clones of Gustafson et al. (47) but are much more sensitive to EO9. The IC50 for the MCF-7 transfectants was {approx}25 nM, compared to {approx}220 nM EO9 for the CHO transfectants. The NQO1 activity in the parental CHO cells was undetectable by the same assay that we used. Rat NQO1 has been shown to metabolize EO9 at a significantly higher rate than human NQO1 (48) . Small amounts of rat NQO1 in the MCF-7 transfectants may bioactivate all of the EO9 made available so that expression of more NQO1 does not result in any greater sensitivity to EO9. Likewise, the substrate for NQO1 during TNF-induced apoptosis may also become limiting once a minimal amount of the more active rat enzyme is expressed.

The ability of NQO1 to affect apoptosis sensitivity is potentially significant in the prevention and treatment of cancer. NQO1 could be particularly important if the point at which it exerts an effect is common to apoptosis induced by other agents. By increasing NQO1 levels, chemopreventive agents may lower the threshold at which damaged, precancerous cells undergo apoptosis. Tumors with elevated NQO1 levels could be identified through enzyme profiling (14) ; these may respond more favorably to apoptosis-inducing chemotherapeutic agents.


   ACKNOWLEDGMENTS
 
We thank Drs. Denise Roe and Haiyan Cui in the Biometry Shared Resource at the Arizona Cancer Center for invaluable assistance with the statistical analyses, Dr. Hans R. Hendriks at the European Organization for Research and Treatment of Cancer New Drug Development Office for providing EO9, and Dr. Margaret Tome for her critical review of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Phoenix Friends of the Arizona Cancer Center, by United States Army Breast Cancer Research Program Award DAMD17-94-J-4296, and by NIH Postdoctoral Training Grants T32 CA09213 and T32 ES07091 (to L. M. S.). Back

2 Present address: Department of Radiation Oncology, Stanford University, Stanford, CA 94305. Back

3 To whom requests for reprints should be addressed, at Department of Pathology, University of Arizona, P. O. Box 24-5043, Tucson, AZ 85724-5043. Phone: (520) 626-6827; Fax: (520) 626-1027; E-mail: mmbriehl{at}u.arizona.edu Back

4 The abbreviations used are: TNF, tumor necrosis factor {alpha}; CHO, Chinese hamster ovary; DA, diacetate; DCFH, dichlorodihydrofluorescein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MnSOD, manganese superoxide dismutase; NQO1, NAD(P)H:(quinone acceptor) oxidoreductase; RT, reverse transcription; SRB, sulforhodamine B. Back

Received 11/12/99. Accepted 5/ 3/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Meier B., Radeke H. H., Selle S., Younes M., Sies H., Resch K., Habermehl G. G. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-{alpha}. Biochem. J., 263: 539-545, 1989.[Medline]
  2. Yamauchi N., Kuriyama H., Watanabe N., Neda H., Maeda M., Niitsu Y. Intracellular hydroxyl radical production induced by recombinant human tumor necrosis factor and its implication in the killing of tumor cells in vitro. Cancer Res., 49: 1671-1675, 1989.[Abstract/Free Full Text]
  3. Hennet T., Richter C., Peterhans E. Tumour necrosis factor-{alpha} induces superoxide anion generation in mitochondria of L929 cells. Biochem. J., 289: 587-592, 1993.
  4. Matthews N., Neale M. L., Jackson S. K., Stark J. M. Tumour cell killing by tumour necrosis factor: inhibition by anaerobic conditions, free-radical scavengers and inhibitors of arachidonate metabolism. Immunology, 62: 153-155, 1987.[Medline]
  5. Zimmerman R. J., Chan A., Leadon S. A. Oxidative damage in murine tumor cells treated in vitro by recombinant human tumor necrosis factor. Cancer Res., 49: 1644-1648, 1989.[Abstract/Free Full Text]
  6. Wong G. H. W., Goeddel D. V. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science (Washington DC), 242: 941-944, 1988.[Abstract/Free Full Text]
  7. Wong G. H. W., Elwell J. H., Oberley L. W., Goeddel D. V. Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell, 58: 923-931, 1989.[Medline]
  8. Hirose K., Longo D. L., Oppenheim J. J., Matsushima K. Overexpression of mitochondrial manganese superoxide dismutase promotes the survival of tumor cells exposed to interleukin-1, tumor necrosis factor, selected anticancer drugs, and ionizing radiation. FASEB J., 7: 361-368, 1993.[Abstract]
  9. Manna S. K., Zhang H. J., Yan T., Oberley L. W., Aggarwal B. B. Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-{kappa}B and activated protein-1. J. Biol. Chem., 273: 13245-13254, 1998.[Abstract/Free Full Text]
  10. Lind C., Hochstein P., Ernster L. DT-diaphorase as a quinone reductase: a cellular control device against semiquinone and superoxide radical formation. Arch. Biochem. Biophys., 216: 178-185, 1982.[Medline]
  11. Prochaska H. J., Talalay P., Sies H. Direct protective effect of NAD(P)H: quinone reductase against menadione-induced chemiluminescence of postmitochondrial fractions of mouse liver. J. Biol. Chem., 262: 1931-1934, 1987.[Abstract/Free Full Text]
  12. Belinsky M., Jaiswal A. K. NAD(P)H. quinone oxidoreductase1 (DT-diaphorase) expression in normal and tumor tissues. Cancer Metastasis Rev., 12: 103-117, 1993.[Medline]
  13. Chesis P. L., Levin D. E., Smith M. T., Ernster L., Ames B. N. Mutagenicity of quinones: pathways of metabolic activation and detoxification. Proc. Natl. Acad. Sci. USA, 81: 1696-1700, 1984.[Abstract/Free Full Text]
  14. Riley R. J., Workman P. DT-diaphorase and cancer chemotherapy. Biochem. Pharmacol., 43: 1657-1669, 1992.[Medline]
  15. Clapper M. L. Chemopreventive activity of oltipraz. Pharmacol. Ther., 78: 17-27, 1998.[Medline]
  16. Benson A. M., Hunkeler M. J., Talaly P. Increase of NAD(P)H: quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc. Natl. Acad. Sci. USA, 77: 5216-5220, 1980.[Abstract/Free Full Text]
  17. Marin A., Lopez de Cerain A., Hamilton E., Lewis A. D., Martinez-Penuala J. M., Idoate M. A., Bello J. DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours. Br. J. Cancer, 76: 923-929, 1997.[Medline]
  18. Schlager J. J., Powis G. Cytosolic NAD(P)H. (quinone-acceptor)oxidoreductase in human normal and tumor tissue: effects of cigarette smoking and alcohol. Int. J. Cancer, 45: 403-409, 1990.[Medline]
  19. Siemankowski L. M., Morreale J., Briehl M. M. Antioxidant defenses in TNF-treated MCF-7 cells: selective increase in MnSOD. Free Radical Biol. Med., 26: 919-924, 1999.[Medline]
  20. Gunning P., Leavitt J., Muscat G., Ng S-Y., Kedes L. A human b-actin expression vector system directs high-level accumulation of antisense transcripts. Proc. Natl. Acad. Sci. USA, 84: 4831-4835, 1987.[Abstract/Free Full Text]
  21. Ernster L. DT diaphorase. Methods Enzymol., 10: 309-317, 1967.
  22. Kingston, R. E. CsCl purification of RNA from cultured cells. In: F. M. Ausubel, R. Brent, R. E. Kingston, Moore, D. D., Seidman, J. G., Smith, J. A., and Struhb, K. (eds.) Current Protocols in Molecular Biology, pp. 4.2.3–4.2.5 New York: John Wiley & Son, Inc., 1994.
  23. Skehan P., Storeng R., Scudiero D., Monks A., McMahon J., Vistica D., Warren J. T., Bokesch H., Kenney S., Boyd M. R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst., 82: 1107-1112, 1990.[Abstract/Free Full Text]
  24. Jaiswal A. K., McBride O. W., Adesnik M., Nebert D. W. Human dioxin-inducible cytosolic NAD(P)H: menadione oxidoreductase. cDNA sequence and localization of gene to chromosome 16. J. Biol. Chem., 263: 13572-13578, 1988.[Abstract/Free Full Text]
  25. Oostveen E. A., Speckamp W. N. Mitomycin analogs I: indoloquinones as (potential) bisalkylating agents. Tetrahedron, 43: 255-262, 1987.
  26. Bailey S. M., Lewis A. D., Knox R. J., Patterson L. H., Fisher G. R., Workman P. Reduction of the indoloquinone anticancer drug EO9 by purified DT-diaphorase: a detailed kinetic study and analysis of metabolites. Biochem. Pharmacol., 56: 613-621, 1998.[Medline]
  27. Fitzsimmons S. A., Workman P., Grever M., Paull K., Camalier R., Lewis A. D. Reductase enzyme expression across the National Cancer Institute tumor cell line panel: correlation with sensitivity to mitomycin C and EO9. J. Natl. Cancer Inst., 88: 259-269, 1996.[Abstract/Free Full Text]
  28. Robertson N., Haigh A., Adams G. E., Stratford I. J. Factors affecting sensitivity to E09 in rodent and human tumour cells in vitro: DT-diaphorase activity and hypoxia. Eur. J. Cancer, 30A: 1013-1019, 1994.
  29. Smitskamp-Wilms E., Peters G. J., Pinedo H. M., Van Ark-Otte J., Giaccone G. Chemosensitivity to the indoloquinone E09 is correlated with DT-diaphorase activity and its gene expression. Biochem. Pharmacol., 47: 1325-1332, 1994.[Medline]
  30. Sgambato A., Zhang Y.-J., Ciaparrone M., Soh J. W., Cittadini A., Santella R. M., Weinstein I. B. Overexpression of p27Kip1 inhibits the growth of both normal and transformed human mammary epithelial cells. Cancer Res., 58: 3448-3454, 1998.[Abstract/Free Full Text]
  31. Li N., Oberley T. D., Oberley L. W., Zhong W. Inhibition of cell growth in NIH/3T3 fibroblasts by overexpression of manganese superoxide dismutase: mechanistic studies. J. Cell. Physiol., 175: 359-369, 1998.[Medline]
  32. Li N., Oberley T. D., Oberley L. W., Zhong W. Overexpression of manganese superoxide dismutase in DU145 human prostate carcinoma cells has multiple effects on cell phenotype. Prostate, 35: 221-233, 1998.[Medline]
  33. Zyad A., Benard J., Tursz T., Clarke R., Chouaib S. Resistance to TNF-{alpha}, and adriamycin in the human breast cancer MCF-7 cell line: relationship to MDR1, MnSOD, and TNF gene expression. Cancer Res., 54: 825-831, 1994.[Abstract/Free Full Text]
  34. Li J.-J., Oberley L. W. Overexpression of manganese-containing superoxide dismutase confers resistance to the cytotoxicity of tumor necrosis factor {alpha} and/or hyperthermia. Cancer Res., 57: 1991-1998, 1997.[Abstract/Free Full Text]
  35. Sakashita A., Epstein C. J., Carlson E., Koeffler H. P. Hematopoietic progenitor cells of transgenic mice with increased copper/zinc-superoxide dismutase activity are resistant to tumor necrosis factor. J. Cell. Physiol., 160: 233-238, 1994.[Medline]
  36. Briehl M. M., Baker A. F. Modulation of the antioxidant defence as a factor in apoptosis. Cell Death Differ., 3: 63-70, 1996.
  37. Krall J., Bagley A. C., Mullenbach G. T., Hallewall R. A., Lynch R. E. Superoxide mediates the toxicity of paraquat for cultured mammalian cells. J. Biol. Chem., 263: 1910-1914, 1988.[Abstract/Free Full Text]
  38. Hempel S. L., Buettner G. R., O’Malley Y. Q., Wessels D. A., Flaherty D. M. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate and dihydrorhodamine 123. Free Radical Biol. Med., 27: 146-159, 1999.[Medline]
  39. Cadenas E. Antioxidant and prooxidant functions of DT-diaphorase in quinone metabolism. Biochem. Pharmacol., 49: 127-140, 1995.[Medline]
  40. Siegel D., Bolton E. M., Burr J. A., Liebler D. C., Ross D. The reduction of {alpha}-tocopherolquinone by human NAD(P)H: quinone oxidoreductase: the role of {alpha}-tocopherolhydroquinone as a cellular antioxidant. Mol. Pharmacol., 52: 300-305, 1997.[Abstract/Free Full Text]
  41. Beyer R. E., Segura-Aguilar J., Di Bernardo S., Cavazzoni M., Fato R., Fiorentini D., Galli M. C., Setti M., Landi L., Lenaz G. The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems. Proc. Natl. Acad. Sci. USA, 93: 2528-2532, 1996.[Abstract/Free Full Text]
  42. Landi L., Fiorentini D., Galli M. C., Segura-Aguilar J., Beyer R. E. DT-diaphorase maintains the reduced state of ubiquinones in lipid vesicles thereby promoting their antioxidant function. Free Radical Biol. Med., 22: 329-335, 1997.[Medline]
  43. Beyer R. E. The relative essentiality of the antioxidative function of coenzyme Q-the interactive role of DT-diaphorase. Mol. Aspects Med., 15(Suppl.): s117-s129, 1994.
  44. Ashkenazi A., Dixit V. M. Death receptors: signaling and modulation. Science (Washington DC), 281: 1305-1308, 1998.[Abstract/Free Full Text]
  45. Kolesnick R., Golde D. W. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell, 77: 325-328, 1994.[Medline]
  46. Garcia-Ruiz C., Colell A., Mari M., Morales A., Fernandez-Checa J. C. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. J. Biol. Chem., 272: 11369-11377, 1997.[Abstract/Free Full Text]
  47. Gustafson D. L., Beall H. D., Bolton E. M., Ross D., Waldren C. A. Expression of human NAD(P)H: quinone oxidoreductase (DT-diaphorase) in Chinese hamster ovary cells: effect on the toxicity of antitumor quinones. Mol. Pharmacol., 50: 728-735, 1996.[Abstract]
  48. Beall H. D., Murphy A. M., Siegel D., Hargreaves R. H. J., Butler J., Ross D. Nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase (DT-diaphorase) as a target for bioreductive antitumor quinones: quinone cytotoxicity and selectivity in human lung and breast cancer cell lines. Mol. Pharmacol., 48: 499-504, 1995.[Abstract]



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