Nitric Oxide Enhances the Manganese Superoxide ... - CiteSeerX

Vol. 10, 655– 664, September 1999

Cell Growth & Differentiation

Nitric Oxide Enhances the Manganese Superoxide Dismutasedependent Suppression of Proliferation in HT-1080 Fibrosarcoma Cells1 J. Andres Melendez,2 Raichal P. Melathe, Ana M. Rodriguez, Joseph E. Mazurkiewicz, and Kelvin J. A. Davies Departments of Biochemistry and Molecular Biology [J. A. M., A. M. R.] and Microbiology, Immunology, and Molecular Genetics [J. E. M.], Albany Medical College, Albany, New York 12208; Montfiore Medical Center, Department of Radiation Oncology, Bronx, New York [R. P. M.]; and Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, California 90089-0191 [K. J. A. D.]

morpholinosydnonomine, and (Z)-1-[2-aminethyl)-N-(2ammonioethyl)amino]diazen-1-ium-1,2-diolate in a dose-dependent manner. In addition, the NOz toxicity is blocked by oxyhemoglobin, a NOz scavenger. Our findings suggest that NOz may play a role in the reversal of tumorigenicity associated with MnSOD overexpression.

Introduction Abstract The overexpression of manganese superoxide dismutase (MnSOD), an enzyme that catalyzes the removal of superoxide (O2. ) from the mitochondria, has been shown to be closely associated with tumor regression in vivo and loss of the malignant phenotype in vitro. To investigate the mechanism by which MnSOD overexpression mediates this reversal, we have established 29 independent, clonal MnSODoverexpressing HT-1080 fibrosarcoma cells. MnSOD activity is inversely correlated with cell proliferation in our cell lines. Incubating cells in 3% oxygen can prevent the inhibition of cellular proliferation mediated by MnSOD, suggesting that oxygen is a prerequisite component of the MnSOD-dependent proliferative inhibition. Confocal laser microscopy was used in combination with the oxidant-sensitive fluorescent dyes dihydrorhodamine-123, dihydroethidium, and 2*,7*-dichlorodihydrofluorescein diacetate to determine the oxidizing capacity of the MnSOD-overexpressing cells. When compared with parental or control cell lines, there was a significant decrease in the rate of oxidation of the fluorophores in the MnSODoverexpressing cell lines. Thus, an increase in the oxidizing capacity of the cells does not appear to mediate the inhibition of proliferation associated with MnSOD overexpression. Superoxide dismutase has also been shown to enhance the cytotoxic activity of NOz toward tumor cells. In this study, we have shown that MnSOD overexpression enhances the cytostatic action of the NOz donors, sodium nitroprusside, 3-

Received 3/30/99; revised 7/27/99; accepted 7/27/99. 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 NIH National Institute of Environmental Health Science Grant ES03598 (to K. J. A. D.) and a National Cancer Institute minority career development award Grant CA77063 (to J. A. M.). 2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, MC10, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208.

MnSOD3 is strategically localized in the mitochondrial matrix to catalytically remove O2. produced as a result of electron leakage during normal electron transport at complexes I and III. Superoxide or its reduced byproducts, H2O2 and the HOz, can directly damage lipids, proteins, and nucleic acids, leading to cell damage or even death. A significant body of evidence suggests that MnSOD may serve as a tumor suppressor. The involvement of MnSOD in tumor suppression may be due to its ability to dismutate O2. to H2O2 and oxygen. Compounds that generate O2. and other reactive oxygen species have been shown to promote skin tumors in mice, whereas treatment with antioxidants that serve to terminate the chain reactions initiated by reactive oxygen species antagonize this process (1). MnSOD levels have also been shown to be abnormally low in malignant tissue as compared with the corresponding normal tissue (2– 6). Thorough analyses of numerous SV40-transformed fibroblast cell lines show a strong incidence of transformants containing a deletion of the long arm of chromosome 6, where the MnSOD gene is mapped (7). Similarly, an increase in MnSOD levels in many transformed cells is associated with a decrease in the tumorigenicity of the affected cell. Thus, it appears that elevated MnSOD may be detrimental to the transformed cells. This concept is supported by studies demonstrating that overexpression of MnSOD in numerous transformed cell lines leads to reversion of tumorigenicity in vivo (8 –12) or of the malignant phenotype in vitro (8 –14). The mechanisms underlying the tumor-suppressing ability of MnSOD have not been defined. The currently prevailing theory is that an imbalance in the redox state of the cell leads to an inhibition of cell proliferation. In theory, scavenging of O2. by MnSOD leads to an increase in H2O2, and without a concomitant increase in the peroxide-scavenging enzymes, this oxygen metabolite may become toxic. In the mitochondrial microenvironment, excess H2O2 would be available to

3 The abbreviations used are: MnSOD, manganese superoxide dismutase; GPX, glutathione peroxidase; CuZnSOD, CuZn superoxide dismutase; TNF, tumor necrosis factor; CMV, cytomegalovirus; DCF, dichlorofluorescein; DR123, dihydrorhodamine-123; FBS, fetal bovine serum; DETANONOate, (Z)-1-[2-aminethyl)-N-(2-ammonioethyl)amino]diazen-1ium-1,2-diolate.

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react with Fe21, contained in multiple Fe-S electron transport enzymes (15), leading either to the production of HOz via the metal-catalyzed Haber-Weiss reaction or of ferryl or perferryl species (16). Li et al. (14) have been able to enhance the proliferation of MnSOD-overexpressing MCF-7 cells through the addition of pyruvate (9), which acts as an antioxidant and protects from H2O2 toxicity in cultured cells (17). However, other studies from the same laboratory have demonstrated that excess pyruvate is not required for the proliferation of MnSOD-overexpressing squamous carcinoma cells (11) and that pyruvate decreases the proliferation rate of MnSODoverexpressing, SV40-transformed human fibroblasts (13). One interpretation of the decrease in proliferation is that pyruvate may increase the cellular H2O2 catabolizing capacity. Oberley and coworkers have shown that cells can adapt to MnSOD overexpression by compensatory increases in the levels of the H2O2 scavenging enzymes, catalase (10) and GPX (11, 14). However, this compensatory adaptation does not reverse the antiproliferative effect of MnSOD overexpression. Superoxide is also an oxidant, and as such, one molecule of O2. when reduced produces one molecule of H2O2. Liochev and Fridovich (15) have proposed that when O2. is dismuted, two O2. yield one H2O2. As a result, by increasing SOD levels, H2O2 production from O2. should be halved. This hypothesis has been supported recently by the finding that CuZnSOD overexpression leads to a decrease in steadystate hydrogen peroxide levels in V79 Chinese hamster cells (19). Thus, it is not possible to conclude whether H2O2 production contributes to the antiproliferative effects of MnSOD overexpression. This study is directed at further defining the mechanism(s) by which MnSOD inhibits the proliferation of transformed cell lines in vitro. We have characterized numerous stable transfectants with varying levels of MnSOD overexpression and used these clonal transfectants to determine how MnSOD reverses the in vitro malignant phenotype. Our findings indicate that the MnSOD-associated proliferative inhibition is oxygen dependent and is not due to an increase in the oxidizing capacity of the cell. Furthermore, we have demonstrated that MnSOD overexpression dose dependently enhances the sensitivity of fibrosarcoma cells to NOz.

Results Isolation and Initial Proliferation Characterization of MnSOD Transfectants. HT-1080 fibrosarcoma cells were transfected with either a MnSOD expression vector or a control vector lacking the insert. Stable transfectants were isolated, and MnSOD levels were characterized using nondenaturing PAGE (19). The level of MnSOD was determined by performing scanning densitometry, and the ratio of MnSOD to CuZnSOD was used as an index of MnSOD activity, because CuZnSOD levels were not affected by MnSOD overexpression (Fig. 1B). The colony-forming ability of the independent clones was then determined in vitro using a clonogenic assay (13). Relative MnSOD activity versus colony formation is plotted in Fig. 1A. Regression analysis shows an association between increased MnSOD activity and a decreased ability to form colonies. These initial findings are in complete agreement with published studies on

Fig. 1. Characterization of MnSOD activity and clonogenic activity. SOD activities of 29 independent stable MnSOD transfectants were determined using nondenaturing PAGE. A, MnSOD and CuZnSOD band intensities of this gel were determined using JANDEL image analysis software. Band intensities were plotted in arbitrary units. MnSOD activity is shown as a ratio of MnSOD:CuZnSOD activity of each cell and is plotted versus the colony-forming ability of each cloned cell line in a 7-day fix and stain assay. Activities for both MnSOD and CuZnSOD were used only if the values were within the linear detection range of SOD activity as determined using the image analysis software. B, representative cross-sections of MnSOD overexpressors were further characterized. Upper gel, nondenaturing SOD PAGE of HT-1080 (parental) and CMVA, CMV2 (control transfectants) before and after a 4-h TNF-a (10 ng/ml) treatment. Lower gel, nondenaturing SOD PAGE of MnSOD-overexpressing transfectants. Values above each lane represent fold-increase in MnSOD activity relative to parental and control cells as determined spectrophotometrically. Twenty-five mg of total cell protein were loaded per lane. C: top left, MnSOD immunoreactive protein in overexpressing cell line; top center, MitoTracker Red staining in an overexpressing cell; top right, top left and center panels are superimposed, showing very close association between the immunoreactive MnSOD and MitoTracker Red. Bottom left, MnSOD immunoreactive protein in control cell, and bottom right, overexpressing cell line at identical confocal parameters. White bar: 10 mm in top panels and 25 mm in bottom panels.


Table 1

Antioxidant enzyme activities of MnSOD-overexpressing cellsa

Cells

Catalase (units/mg protein)

Glutathione peroxidase (units/mg protein)

HT1080 CMV HT2 HT3 HT8 HT9 HT14 HT15

16.2 6 1.8 14.8 6 1.2 14.1 6 2.0 14.4 6 2.1 10.5b 6 1.1 14.5 6 1.2 14.5 6 0.9 15.4 6 0.7

27.7 6 1.1 34.4 6 4.5 38.2 6 3.8 43.1b 6 2.1 38.5 6 4.6 34.0 6 7.8 45.6b 6 3.6 48.0b 6 5.5

Values represent mean 6 SE; n 5 4 –7; antioxidant enzyme activities were measured as described in “Materials and Methods.” P , 0.05 when compared with HT-1080 cells. ANOVA with a 5 0.05 was used for processing the data. Paired t tests were used as post-tests. a

b

the effects of MnSOD overexpression on tumor proliferation in vitro (8, 9, 13). Our key finding is that as MnSOD levels increase significantly, the ability of the cell to grow in vitro is diminished. This was demonstrated using 29 independent stable transfectants. To further characterize the levels of MnSOD in the transfected cells, we chose to focus on 10 –12 cell lines with varying levels of MnSOD. Both spectrophotometric and nondenaturing-PAGE SOD activity assays were used to more accurately determine the levels of MnSOD. The spectrophotometric activity assay agreed well with the native PAGE results, and the fold increase in MnSOD activity over control cell lines as determined spectrophotometrically is indicated above each sample in Fig. 1B. TNF-a is a potent inducer of MnSOD activity in many cell types (20) and was able to induce MnSOD activity in parental cells or cells transfected with vector alone (Fig. 1B). A 2-fold increase was observed in MnSOD activity after a 4-h treatment with TNF-a. The level of induction of MnSOD activity by TNF-a varies among cell lines and has been reported to be as high as 30-fold after 24-h treatment (20 –22). We have isolated a number of MnSOD overexpressors, the levels of which are increased anywhere from 2- to 15-fold and are reflective of the levels observed after physiological stimuli, such as TNF-a. For the remainder of this study, the cell lines will be referred to by their foldincrease in MnSOD activity over control cell lines, e.g., HT14.3 indicates that this stable transfectant has a 14.3 (61.64 units/mg protein) fold increase in MnSOD activity levels relative to the parental cell line (4.3 units/mg protein). A number of investigators have reported that mammalian cells can modulate antioxidant enzyme levels either in response to oxidative stress (23, 24) or overexpression of antioxidant enzymes (10, 11, 14). To address whether the levels of either catalase or glutathione peroxidase were altered in response to MnSOD overexpression, the activities of these enzymes were characterized in a number of the transfected cell lines (Table 1). The levels of catalase were decreased in the HT8 cells, whereas the levels of GPX were increased in the HT3, HT14, and HT15 cells when compared with the HT-1080 parental cell line. No statistically significant differences were observed in the levels of either catalase or GPX when the MnSOD-overexpressing cell lines were compared with the transfection controls (CMV).

Overexpressed MnSOD Is Localized to Mitochondria. The localization of MnSOD in the mitochondria is critical to its role in removing O2. at the site of its production. Therefore, we set out to localize the MnSOD by cellular fractionation. In parental, control, and MnSOD-overexpressing cells, we found the majority of MnSOD activity in the mitochondrial fraction (data not shown). The presence of low levels of MnSOD in the cytosol, nuclear, and peroxisomal fractions could reflect incomplete processing of the overexpressed enzyme or leakage of the enzyme from organelles during fractionation. We subsequently demonstrated that the overexpressed MnSOD immunoreactive protein colocalizes with a mitochondria-specific stain, MitoTracker Red (Fig. 1C) and that there is a significant increase in MnSOD immunoreactive protein in the MnSOD-overexpressing cells (Fig. 1C, bottom right) relative to the parental cell lines (Fig. 1C, bottom left). Reversal of the MnSOD-mediated Inhibition of Proliferation by Low Oxygen. In studies involving overexpression of MnSOD, it is often assumed that any effects, whether beneficial or detrimental, are attributable to decreased or increased O2. , respectively. If the proliferation inhibitory effects of MnSOD are dependent on O2. , decreased O2. production should reverse any observed effects. The production of O2. is dependent on cellular oxygen consumption (25, 26) and can be diminished by decreasing available oxygen. In Fig. 2A, cells were seeded at a low density and allowed to grow in either 3 or 21% oxygen for 5 days. The growthinhibitory effects of high MnSOD overexpression could be partially overcome when cells were incubated in low (3%) oxygen. In all cases, there was a significant increase in the ability of the high MnSOD overexpressors to grow in 3% O2 as compared with 21% O2. This is the first demonstration that the effects of MnSOD overexpression on tumor cell proliferation are directly dependent on oxygen availability. The inhibition of proliferation associated with MnSOD overexpression may be due to several parameters, such as a decrease in doubling time, viability, or plating efficiency of the cells. To determine population doubling time, single cells of either control or MnSOD-overexpressing cells were seeded into Terasaki culture plates using conditioned media, and cells were counted for 6 days. Both the parental and control transfectants showed similar population doubling numbers over 5 days of 4.68 6 0.2 and 4.54 6 0.1, respectively, whereas the HT14.3 and HT8.6 cell lines had doubling numbers of 1.81 6 0.4 and 2.54 6 0.3. This decrease in doubling number of the MnSOD-overexpressing cells may account for at least 50% of the inhibition that is observed in both the cloning efficiency and cell growth assays. Trypan blue exclusion was performed to assess cellular viability. No differences in the percentage of viability were observed between control and any of the MnSOD overexpressors during routine cell culture. In addition, there were no observed differences in plating efficiency observed between the parental or control cell lines and the MnSOD overexpressors during routine cell culture in contrast to the dramatic differences that were observed in the clonogenic assays. The observed differences in cellular clonogenic activity and proliferation between control and MnSOD overexpressors may be, in part, attributed to an increase in the doubling time of these

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Fig. 2. Effects of 3% O2 and conditioned medium on cellular proliferation. A, control and MnSOD-overexpressing cells were seeded (103/well) in 96-well plates and incubated in 3 or 21% oxygen and stained with crystal violet to assess viability at the indicated days. Cell lines were grouped into control, .2-fold, or .8-fold MnSOD overexpression. Each data point represents the mean of 16 or more individual determinations; bars, SD. pp, P , 0.001 when compared with the indicated 21% O2 MnSOD overexpressor. B, clonogenic activity is plotted versus fold-increase in MnSOD activity of cells incubated in: l, 21% O2 in MEM 1 10% FBS; f, 3%O2 in MEM 1 10% FBS; Œ, 21% O2 in 50% conditioned media; and F, 3% O2 in 50% conditioned media. Results from one experiment are shown where n 5 4 for each cell line; bars, SE. p, P , 0.05; pp, P , 0.005; ppp, P , 0.001 when compared with the indicated 21% O2 and complete medium MnSOD overexpressor.

cells. We have also observed that the high MnSOD-overexpressing cells are sensitive to seeding at low density. When MnSOD-overexpressing cells are seeded at a very low density, their rate of proliferation is significantly decreased relative to when they are seeded at a higher density. However, in either case, the proliferative capacity of the high MnSODoverexpressing cells is decreased relative to the control cells. The clonogenic activity of all of the MnSOD-overexpressing cell lines was also assessed in conditioned media in 3 or 21% oxygen (Fig. 2B). Conditioned medium is commonly used to enhance growth of cells when seeded at low density. When MnSOD-overexpressing cells were maintained in conditioned medium, there was a significant increase in clonogenic activity relative to cells grown in normal medium. When the MnSOD-overexpressing cells were grown in conditioned medium and 3% oxygen, the inhibition of clonogenic activity was completely overcome. The growth-promoting effects of low oxygen and conditioned medium was additive in the ability to promote clonogenic survival of the MnSOD-overexpressing cells. Both high seeding density and conditioned medium have similar effects and are able to reverse the inhibition of clonogenic activity and/or proliferation associated with MnSOD overexpression. This is not surprising because seeding cells at high density would serve to rapidly condition the medium with any necessary

growth factors that would be supplied by the addition of conditioned medium. The ability of low oxygen to further compensate for the MnSOD-associated proliferative defect indicates that oxygen or a byproduct is a critical component of the proliferative inhibition. MnSOD Overexpression Decreases the Oxidizing Capacity of the HT-1080 Fibrosarcoma Cells. Of the numerous cloned cell lines that we have analyzed, only cells with an 8-fold or greater increase in MnSOD activity show a significantly decreased rate of proliferation. In addition, the finding that this alteration in cell proliferation is partially overcome by incubation in low oxygen suggests that some toxic metabolite may accumulate in response to MnSOD overexpression and affect the proliferative characteristics of the fibrosarcoma cells. Studies have demonstrated that the elevated levels of SOD may become toxic as a result of the generation of the product of the dismutation reaction, H2O2 (27, 28). Thus, we used three well-characterized, oxidant-sensitive fluorescent compounds to assess the redox status of the cell. Dihydroethidium, when oxidized, is converted to ethidium, and the dehydrated ethidium fluoresces blue in the cytosol and red in the nucleus, where it binds DNA (29, 30). Studies have also demonstrated that dihydroethidium is more sensitive to oxidation by O2. than by H2O2 (31, 32). Confocal laser microscopy was used to determine the rate of oxidation of this fluorophore in both control and MnSODoverexpressing cells. In all cases, there was a decrease in the rate of oxidation of dihydroethidium by the MnSOD-overexpressing cell lines relative to the control or parental cell line without the addition of any stimuli. The 6-fold decrease in the rate of oxidation of the HT15 cell line was representative of the change that was observed in all of the high MnSOD overexpressors (Fig. 3, A and B). Dihydroethidium does not appear to be taken up preferentially in the control cell line because a detectable fluorescent signature is not observed until 3 min after addition of the fluorophore in both cell lines. DR123 is oxidized to membrane-impermeable, fluorescent rhodamine-123 in the presence of H2O2 (33, 34). As with the dihydroethidium, the MnSOD-overexpressing cells showed a decrease in the rate of oxidation of dihydrorhodamine-123 relative to either the parental or MnSOD-overexpressing cell line (Fig. 3, C and D). The most dramatic difference in the oxidizing capacity of the MnSOD overexpressors was observed with the oxidative indicator dihydroethidium. Dihydroethidium is preferentially sensitive to O2. and may be more sensitive to changes in O2. levels mediated by increases in MnSOD. It has been shown that dihydroethidium does not detect SOD-generated H2O2, as in the present system, but does detect high nonphysiological concentrations of H2O2 (35). The difference in the rate of oxidation of DR123 between control and MnSOD-overexpressing cells was less (3-fold) pronounced than that observed for the dihydroethidium and may reflect the decrease in sensitivity of this dye for O2. . The difference in the rate of oxidation of the fluorophore DR123 also indicates that MnSOD decreases the levels of intracellular peroxides. The cell permeant carboxy-29-79-dichlorodihydrofluorescein diacetate is oxidatively converted to its fluorescent derivative in the presence of H2O2 and cellular peroxidases (36 –38). The oxidation of carboxy-29-79-dichlo-


Fig. 3. MnSOD overexpression decreases the ability of HT-1080 fibrosarcoma cells to oxidize the redox-sensitive fluorescent probes dihydroethidine, DR123, or 29,79-dichlorodihydrofluorescein diacetate. A, 250 confocal fluorescent images were taken from t 5 0 min to t 5 9 min. The ethidium fluorescent intensity at every time point for each individual cell in the confocal field was determined using InterVision 2-D analysis software. The mean fluorescence intensity of all of the cells in a field at each time point was determined and plotted starting at 200 s. B, total rhodamine123 fluorescent intensity was determined for the entire stack in each individual cell at 90-s intervals. The mean fluorescence intensity for all of the cells in a field at each time point was determined and plotted. A representative experiment of which three to five were performed for each cell line is shown for each of the probes. C, oxidized DCF fluorescence was determined as described in “Materials and Methods.” Values represent mean relative fluorescence intensity/mg protein from three independent experiments; bars, SE.

rodihydrofluorescein diacetate to DCF was not affected by MnSOD overexpression (Fig. 3E). The failure of DCF to detect differences in the levels of intracellular peroxides may be due to its decreased sensitivity for intracellular peroxides. A sub-

tle decrease in the levels of cellular peroxides was observed in several of the MnSOD overexpressors, as determined by DCF fluorescence; however, these differences were not statistically significant. The oxidatively sensitive fluorophores indicate that the MnSOD overexpression decreases the oxidizing capacity of HT-1080 fibrosarcoma cells. Thus, the proliferative inhibition is not likely due to an increase in the prooxidant status of the MnSOD-overexpressing cells. MnSOD Overexpression Sensitizes HT-1080 Fibrosarcoma Cells to the Cytostatic Effects of NOz Donors. NOz has been shown to be responsible for the cytotoxic activity of macrophages toward human cancer cell lines (39 – 41). Superoxide dismutase has also been shown to enhance the cytotoxic activity of NOz toward human ovarian cancer (42) and hepatoma (43) cell lines. For these reasons, we chose to test the sensitivity of the MnSOD overexpressors to NOz donors. For our studies, the NOz donors SIN-1, SNP, and DETANONOate were used. SIN-1 generates NOz, O2. , and peroxynitrite, whereas SNP produces NOz and the nitrisonium ion (44). Our initial studies indicated that NOz donors were equally cytotoxic to both control and MnSOD-overexpressing cell lines. Further analyses demonstrated that addition of the NO donors (0.3–10 mM) to parental or control cells in a 96-well plate caused complete cytotoxicity of only treated cells. However, when MnSOD-overexpressing cells were treated in the same fashion, cells in wells distal to the treatment wells were also affected. The wells in close proximity to the NOz donor had no adherent cells remaining after crystal violet staining, whereas cells distal to the NOz donor showed a visible but reduced level of staining relative to their control well (Fig. 4, top plate). A representative experiment is shown in Fig. 4. The degree of growth inhibition was also dependent on the dose of the NOz donor added to the treatment wells. These initial experiments suggest that the NOz donors generate a diffusible molecule (gas), which travels up and out of the treatment well into adjacent wells and preferentially affects the MnSOD-overexpressing cells. The ability of SNP and SIN-1 to generate a diffusible toxic molecule is not surprising because it has been shown that the biotransformation, in biological medium, of gylceryl trinitrate and SNP leads to the release of NOz into the gaseous headspace (45). Thus, NOz may be released in to the gaseous headspace of the 96-well microtiter plates (Fig. 4). Interestingly, neither the wild-type (HT-1080) nor control transfectants (CMV) were as sensitive to the NOz-mediated proliferative inhibition. HTIL1a, with a 100-fold increase in interleukin 1a, was also included as a control to rule out the possibility that protein overexpression may account for the observed affects of the NOz donors. All of the MnSODexpressing cells were sensitive to SNP, whereas SIN-1 had less of an effect on the low MnSOD overexpressors. The difference in the toxicity of the NOz donors on the MnSODcontaining cells may be due to the profile of reactants that are generated by SIN-1 and SNP. To demonstrate that the diffusible reactant was indeed NOz, a specific NOz scavenger, oxyghemoglobin (5mM), was added to all untreated wells prior to addition of the NOz donors. As seen in the lower panels of Fig. 4, oxyhemoglobin protected the cells from the diffusible molecule generated from the wells treated with either of the

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Fig. 4. MnSOD overexpression sensitizes HT-1080 fibrosarcoma cells to the inhibition of cellular proliferation associated with the NOz donors, SNP and SIN-1. Cells were seeded as described in Fig. 2A and stained with crystal violet on day 5 after seeding. Cell lines were seeded in replicate wells horizontally on a 96well plates. The respective NOz donor (3 mM) was added to the first row of cells indicated by the small arrows adjacent to the plates marked with the (1). The top two plates remained untreated and serve as control for cell growth (2). Oxyhemoglobin (5 mM) was added to the untreated wells of the two bottom 96-well plates. The HTIL-1a was engineered to overexpress interleukin 1a and serves as an additional control. Large arrows, diffusional gradient of the gas, NOz, which travels up and out of the treated wells and exerts its effect on cells in distal wells.

nitric donors. The reductive metabolism of SNP can lead to the generation of another diffusible and potentially toxic molecule, cyanide (46, 47). Hemoglobin also binds CN2 and could explain the protection afforded to the SNP-treated cells. However, oxyhemoglobin protects MnSOD-overexpressing cells from SIN-1, which does not generate CN2, indicating that the diffusible molecule is likely NOz. These data indicate that MnSOD overexpression sensitizes fibrosarcoma cells to the cytostatic effects of NOz. To determine the degree of sensitization, the incorporation of [3H] thymidine was used as more quantitative measurement of proliferation after treatment with SNP (Fig. 5A). MnSOD overexpression enhanced the sensitivity of the fibrosarcoma cells to SNP by as much as 10-fold (HT9). The degree of sensitivity to SNP was also dependent on the level of MnSOD overexpression, because the HT2.6 and HT3.3 were less sensitive to the effects of SNP. A similar MnSOD dosedependent effect on cellular proliferation was observed when the cell lines were treated with the strict NOz donor, DETANONOate (Fig. 5B).

Discussion It has been clearly demonstrated by numerous investigators that MnSOD plays a role in preventing tumor cell proliferation both in vitro and in vivo (8 –13). However, the mechanisms by which this occurs have not been elucidated. Oberley and coworkers (8, 10, 11, 13, 48) have demonstrated that inhibition of tumor cell proliferation is more pronounced in cells overexpressing higher levels of MnSOD. Our studies also indicate that a severe inhibition of cell proliferation occurs in

cells with an 8 –15-fold increase in MnSOD activity. Furthermore, the ability of 3% O2 to reverse the antiproliferative effects of MnSOD implicates O2. as a key component of this process. Increases in MnSOD activity also decrease the ability of the HT-1080 cells to oxidize redox-sensitive fluorophores. A decrease in the production of H2O2 may be attributed to an elevation in either of the major peroxidative enzymes, catalase or GPX. Several of the MnSOD-overexpressing cells show a significant increase in GPX activity over that of the HT-1080 parental cells. These same cells show a decrease in DR123 (HT14; Fig. 3, C and D) and DCF fluorescence (HT3, HT14, and HT15; Fig. 3E). Interestingly, all of the transfected cell lines, including the vector control, show a basal elevation in GPX levels as compared with the parental cells and a marked but statistically insignificant decrease in oxidized DCF levels (Fig. 3). Li et al. (14) have shown that levels of intracellular peroxides detected by DCF are increased, even in the presence of elevated GPX activity. If elevated GPX does not interfere with the detection of hydrogen peroxide by redox-sensitive fluorophores, then hydrogen peroxide levels may be decreased as a result of MnSOD overexpression in this model system. It has been suggested that elevated levels of SOD would serve to decrease H2O2 concentrations by preventing free radical chain oxidations initiated by O2. (15). The targets of O2. include reduced glutathione, ascorbate, and the [4Fe-4S] clusters of dehydratases. When O2. acts as an oxidant, each O2. is reduced to H2O2. In contrast, when O2. is dismuted by SOD, it takes two O2. to yield one H2O2. This hypothesis has been substantiated recently by studies demonstrating that the overexpres-


Fig. 5. MnSOD overexpression enhances the inhibition of cellular proliferation mediated by the NOz donors SNP and DETANONOate. A, cells were treated as described in “Materials and Methods” with the indicated concentrations of SNP. Thymidine incorporation is expressed as a percentage of untreated control. n 5 3 for each data point; bars, SE. Inset: p, P , 0.01; pp, P , 0.005; p, P , 0.001 for the indicated MnSOD overexpressor when compared with HT-1080 parental cell line at the same [SNP]. B, cells were treated as above with the indicated concentrations of DETANONOate. Cells were stained with a 0.2% crystal violet, 10% ethanol solution, followed by washing in distilled H20. The stain was solubilized in 33% acetic acid. The OD595 was measured, and cellular proliferation was expressed as a percentage of untreated control for each cell line.

sion of CuZn-SOD is associated with a reduction in H2O2 levels in Chinese hamster cells (18). Because of the elevation in GPX activity in some MnSOD-overexpressing cells, we cannot confidently conclude that hydrogen peroxide levels are decreased as a result of MnSOD overexpression. Nonetheless, elevated GPX does not protect from the proliferative inhibition associated with MnSOD overexpression and indicates that hydrogen peroxide is not the principle mediator of this effect. The argument could be raised that the increase in GPX is not sufficient to detoxify the hydrogen peroxide that is generated as a result of the dismutation reaction. This is unlikely, because any excess hydrogen peroxide would be detected by the redox-sensitive fluorophores in our system. The rate constant for the enzymatic dismutation reaction is nearly diffusion limited and is commonly exemplified in biochemistry texts as one of the most efficient catalytic enzymes known. It is unclear why only dramatically elevated levels of MnSOD alter the proliferative characteristics of the fibrosarcoma cells. The possibility exists that mitochondrial O2. may protect tumor cells from molecules that inhibit cell

growth. Our data suggest that a likely inhibitory molecule is NOz. The rate constant for the reaction of NOz with O2. approaches 2 3 1010 M21s21 (49), whereas that of the enzymatic dismutation reaction is on the order of 2 3 109 M21s21. Only when MnSOD is in great excess, as observed with the high MnSOD overexpressors, would it effectively compete for superoxide in the presence of NOz. Under the above conditions, we have observed that MnSOD dramatically enhances the cytostatic effect of NOz donors on fibrosarcoma cells. The ability of activated macrophages to inhibit tumor cell proliferation in vitro has long been know to be a consequence of NOz production (39, 40). NOz has been shown to target and inactivate a number of mitochondrial proteins, including components of the electron transport system (40, 50, 51) and aconitase (40). Superoxide generated in the mitochondria may play a role in protecting these sites from reactions with NOz. It is unclear how MnSOD enhances the reactivity of NOz, but one might speculate that MnSOD may effectively remove O2. and decrease the amount of peroxynitrite generated, which in the mitochondrial environment may be less toxic to cells. It is also possible that MnSOD itself may be toxic to the cell in the presence of NOz-generating compounds by its ability to catalyze peroxynitrite-dependent nitration reactions (52). However, we do not believe this to be the case because DR123 is also a sensitive indicator of peroxynitrite in cells (53, 54), and the levels of its oxidized form were diminished in the MnSOD-overexpressing cells (Fig. 3). Superoxide dismutase has also been shown to enhance the toxicity of NOz donors toward ovarian carcinoma (42) and human hepatoma cell lines (43). The enhanced toxicity associated with NOz and H2O2 has been attributed to the generation of potent oxidants such as the HOz in the presence of trace metals (42). This occurs by a multistep mechanism whereby NOz reduces Fe(III) to Fe(II), followed by the subsequent reduction of H2O2 to generate the HOz. In the present study, the increase in sensitivity to NOz does not appear to be attributed to elevated H2O2 as a result of MnSOD overexpression because we were unable to detect increased levels of oxidants using either dihydroethidium, DR123, or DCF. However, it has been demonstrated that NOz itself may elevate H2O2 levels through the inhibition of mitochondrial respiration or via the inhibition of the peroxidedetoxifying enzyme, catalase (42). It is quite intriguing that the cell line (HT8) that showed a significant decrease in basal catalase activity was also equally sensitive to the strict NOz donor, DETANONOate, as the highest (HT15) MnSOD overexpressor (Fig. 5B). Studies are presently under way to define whether the enhanced sensitivity to NOz is attributable to an elevation in H2O2 in response to NOz. It is clear that MnSOD overexpression sensitizes fibrosarcoma cells to NOz. The data from Figs. 4 and 5 suggest that NO donors can either kill MnSOD-overexpressing cells preferentially when used at relatively high concentrations (1–10 mM) or inhibit cell growth at low physiological concentrations (1–10 mM). In the diffusion assay (Fig. 4), the cytotoxic nature of the NOz donor is observed in wells relatively close to the NOz donor, whereas in wells distal to the NOz donor, cells become cytostatic. In contrast, in Fig. 5, low-dose treat-

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ments led to cellular stasis. Thus, the ultimate effect on the cell is dependent on the NOz donor dose. It is not known if NOz is involved in the inhibition of proliferation associated with MnSOD overexpression. In our studies, a combination of low oxygen and preconditioned medium completely abolished the MnSOD-associated proliferative inhibition. Balin et al. (55) have demonstrated previously that human fibroblast cultures grow optimally at an O2 concentration of 3–18%. Increased sensitivity to O2 was also shown to be dependent on seeding density and inhibited upon the addition of conditioned medium. Whorton et al. (56) has shown that NOz production is decreased in vascular endothelial cells incubated in low oxygen. A decrease in NOz levels under low oxygen may be one of the factors that allow the MnSOD overexpressors to overcome their proliferative inhibition (Fig. 2). In addition, treatment of cells with preconditioned medium decreases the severity of the NOz-mediated proliferative inhibition (data not shown). It has been demonstrated that a variety of factors found in serum or produced by cells can alter the reactivity of NOz. Early studies by Braughler et al. (57) demonstrated that at levels of NOz that inactivate purified guanylate cyclase, crude preparations of the enzyme remain active. The protection from inactivation of the crude preparation of guanylate cyclase by NOz was attributed to the presence of factors that serve as scavengers or sinks for NOz (57). Colon carcinoma cells have also been shown to release a soluble factor that inhibits NOz production from endothelial cells (58). It is possible that incubation of MnSOD-overexpressing cells in low oxygen may decrease endogenous NOz levels, and addition of conditioned medium may further decrease NOz via a scavenging mechanism. However, this hypothesis has yet to be confirmed. A mitochondrial form of NOz synthase has been discovered recently that positions NOz in close proximity to many known molecular targets (59, 60). Giulivi (61) has demonstrated the inactivation of cytochrome oxidase by nanomolar concentrations of mitochondrial derived NOz. It has been demonstrated that atmospheric levels of NOz (120 –1000 ppb) are effective in activating soluble guanylate cyclase only in the presence of SOD (62). Thus, it is feasible that levels of NOz that would normally not affect cellular proliferation would become inhibitory in the absence of O2. . MnSOD may play a critical role in the maintenance of mitochondrial respiration by enhancing the reactivity of NOz toward molecules that would normally be precluded from this chemistry in the presence of O2. . In conclusion, the common decrease in MnSOD levels that is observed in numerous tumor and transformed cells lines suggests that superoxide may provide a selective proliferative advantage to these cells. It is believed that the antitumoral immune response is in part mediated by the release of NOz from immune cells. In the tumor microenvironment, a decrease in MnSOD activity would serve to elevate intramitochondrial O2. and in the presence of NOz would generate peroxynitrite. Nitration events catalyzed by peroxynitrite may be less toxic to tumor cells than nitric oxide or its byproducts. It is exciting to speculate that protection from NOz may be the rationale for the decrease in MnSOD activity in many tumor

cells, and efforts are being directed toward addressing the role of NOz in this model system.

Materials and Methods Cell Culture. Human fibrosarcoma HT-1080 cells were cultured in MEM supplemented with 10% FBS. Cells were treated with recombinant human TNF-a (R & D Systems), SIN-1, or SNP and DETANONOate (Alexis Biochemicals). Construction of recombinant plasmids and transfection was described previously in detail by Melendez and Davies (63). Enzyme Assays. Cells were grown to confluence in 25-cm2 culture flasks and incubated with or without TNF-a for 4 h. Cells were then washed twice and harvested from each flask with 2 ml of PBS (pH 7.2), plus 1 mM EDTA. After a brief centrifugation, the cell pellet was resuspended in 200 ml of potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA and sonicated for 15 s. The lysate was centrifuged at 10,000 3 g for 10 min, and the supernatant was collected. This centrifugation was repeated twice more, and the supernatants were saved. The protein concentration of the final supernatant was determined using the BCA protein reagent (Pierce Chemicals Co.). SOD activity was assayed according to the method of Beauchamp and Fridovich (19). Lysate supernatants were analyzed by electrophoresis in a discontinuous polyacrylamide gel, consisting of a 5% stacking gel (pH 6.8) and a 10% running gel. To visualize SOD activity, the gels were incubated for 15 min in the dark with 2.5 mM nitroblue tetrazolium, 30 mM TEMED, 0.028 mM riboflavin, and 50 mM phosphate buffer (pH 7.8), washed twice in deionized water, and then exposed to fluorescent light until clear zones of SOD activity were distinctly evident. Catalase activity was measured as described by Claiborne (64). GPX activity was determined as described by Tappel (65). Total superoxide dismutase activity was assayed by the method of McCord and Fridovich (66) using xanthine/xanthine oxidase as the source for superoxide radicals. One enzyme unit of superoxide dismutase is defined as the amount that inhibits cytochrome c reduction by 50%, at room temperature at pH 7.8. CuZnSOD activity was measured as the cyanide inhibitable fraction of total SOD activity. Immunofluorescence. Cells were grown to subconfluency in MEM containing 10% FBS on glass coverslips. Cells were then incubated for 30 min with 10 nM of MitoTracker Red and washed twice with PBS. Coverslips were incubated in 3.7% formaldehyde at 37°C for 10 min. Adherent cells were then washed two times with PBS and incubated for 5 min in 0.5% Triton X-100 at room temperature. Cells were washed three times as above and incubated for 10 min in PBS containing glycine (10 mg/ml). Cells were then washed three times and incubated for 1 h with a 1:1500 dilution of an anti-human kidney MnSOD polyclonal antibody (gift of Dr. Larry W. Oberley, University of Iowa). Cells were washed two times and incubated with a 1:1000 dilution of FITC-conjugated secondary goat anti-rabbit antibody (Amersham). The cells were washed three times and examined with an NORAN-Oz confocal laser scanning system. Detection of Intracellular Oxidants. Cells were grown to subconfluency in MEM containing 10% FBS on glass coverslips. For DR123 (10mM) or DCF (5mM), cells were pretreated for 30 min in MEM containing 10% FBS. Cells were then washed two times with PBS containing 0.1 mg/ml of both calcium chloride and magnesium chloride and immediately analyzed. For dihydroethidium, cells were treated with 20 mg/ml in PBS containing 0.1 mg/ml of both calcium chloride and magnesium chloride from a freshly thawed aliquot of 10 mg/ml for each individual time course and immediately analyzed. The fluorescence intensity in the cells was monitored with respect to time and quantified by fluorescence confocal microscopy for dihydroethidium and DR123. DCF fluorescence was measured as described by Brigham et al. (67). Confocal Microscopy. Images of cells, preloaded with the fluorescent dyes, DR123 or dihydroethidium, were collected on a confocal scanning microscope (Noran OZ; Noran Instruments, Middleton, WI) equipped with a krypton/argon laser (running under InterVision software; Noran Instruments) coupled to a Nikon Diaphot 200 inverted microscope. Samples were imaged using either a 320 0.95NA air objective lens (dihydroethidium: lex, 488 nm and lem, .590 nm) or a 360 1.4NA oil objective lens (DR123: lex, 488 nm and lem, .500 nm). For dihydroethidium, the cells were preloaded with the dye, placed on the confocal microscope in a temperature-controlled incubation chamber (Medical Systems Corp., Greenville, NY), and maintained at 30°C. A single optical section was imaged throughout the time series; 250 images were collected over an


8-min, 53-s interval. Each image represents the jump average of eight images (512 3 480 pixels each) collected at the rate of 3.75 images/s. Samples were similarly prepared and analyzed for dihydrorhodamine, except that a z-series (20 images, 0.25 mm steps) was taken every 90 s, for a total of six. Data were analyzed with the InterVision 2D analysis software. Colony Formation in Vitro. To determine the ability of MnSOD transfectants and control cell lines to form colonies, a 7-day fix and stain assay was used. Cells were grown to confluence in 25-cm2 culture flasks and were harvested in 2.0 ml of PBS (pH 7.2), plus 1 mM EDTA. Approximately 36.5 cells/cm2 were suspended in either MEM supplemented with 10% FBS or in conditioned medium (collected from parental cell line) and seeded onto 24-well culture plates. The cells were grown for 7 days, and the plates were stained using 0.2% crystal violet in 10% ethanol for 10 min, and the excess stain was washed off with water. Colonies of 0.5 mm or greater diameter were counted. In Vitro Proliferation Assay. Cells were grown to confluence in 25cm2 culture flasks and harvested in 2.0 ml of PBS (pH 7.2) containing 1 mM EDTA. Approximately 1000 cells were seeded onto 96-well culture plates and incubated in a FORMA variable oxygen incubator in an environment of 5% CO2 and either 21% O2 or 3% O2 and compensated with N2 accordingly. Plates were stained on days 0, 2, 4, or 5 using 0.2% crystal violet in 10% ethanol for 10 min, and the excess stain was washed off with water. The stained cells were solubilized with 33% acetic acid, and optical absorbance was measured at 595 nm. For experiments with the NOz donors, cells were seeded as indicated above and then treated 24 h after seeding and stained on day 5. Determination of [3H]Thymidine Incorporation. Cells were plated at 3125 cells/cm2 on 24-well culture plates and incubated for 24 h in a 0.5-ml volume at 37°C in a humidified 5% CO2 incubator. Then, cells were treated with the indicated concentrations of SNP for 48 h. Cultures were incubated for an additional 18 h with 0.5mCi/well of [methyl-[3H]thymidine (NEN Boston, MA). Cells were washed with 2 ml of cold PBS, incubated for 1 min in 1 ml of cold 5% trichloroacetic acid, followed by removal and subsequent addition of 1 ml of cold methanol for 1 min. Methanol was removed, and wells were air dried for 10 min. Cells were solubilized in 0.5 ml of 0.1 N NaOH, and 100 ml were counted in 3 ml of Ecoscint scintillation mixture (National Diagnostics). Statistical Analysis. ANOVA with a 5 0.05 was used for processing the data. Paired t tests were used as post-tests for Fig. 2B, two-sample t test was used as post-tests in Figs. 2A and 5A.

Acknowledgments We thank Tom Jeitner and Pauline M. Carrico for informative discussions and expert editorial assistance.

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