[CANCER RESEARCH 51. 794-798. February 1, 1991]

Production of Large Amounts of Hydrogen Peroxide by Human Tumor Cells1

Ted P. Szatrowski2 and Carl F. Nathan

Beatrice and Samuel A. Seaver Laboratory, Division of Hematology-Oncology, Department of Medicine, Cornell University Medical College, New York, New York10021

ABSTRACT

Few nonphagocytic cells are known to generate reactive oxygen intermediates. Based on horseradish peroxidase-dependent, catalase-inhibit-able oxidation of fluorescent scopoletin, seven human tumor cell linesconstitutive!)' elaborated H2O2 at rates (up to 0.5 nmol/104 cells/h) large

enough that cumulative amounts at 4 h nere comparable to the amountof IM)., produced by phorbol ester-triggered neutrophils. Superoxidedismutase-inhibitable ferricytochrome c reduction was detectable at muchlower rates. IM), production was inhibited by diphenyleneiodonium, aflavoprotein binder (concentration producing 50% inhibition, 0.3 Õ/M).and diethyldithiocarbamate, a divalent cation chelator (concentrationproducing 50% inhibition, 3 MM),but not by cyanide or azide, inhibitorsof electron transport, or by agents that inhibit xanthine oxidase, pol\ ani-ini1oxidase, or cytochrome P450. Cytochrome ¿559,present in human

phagocytes and lymphocytes, was undetectable in these tumor cells by asensitive spectrophotometric method. Mouse fibroblasts transfected withhuman tyrosinase complementary DNA made melanin, but not IM).,.Constitutive generation of large amounts of reactive oxygen intermediates, if it occurs in vivo, might contribute to the ability of some tumorsto mutate, inhibit antiproteases, injure local tissues, and therefore promote tumor heterogeneity, invasion, and metastasis.

INTRODUCTION

Appropriately stimulated PMNs3 (1-3) and monocytes (4-8)

produce large amounts of ROI (up to 1.5 nmol of hydrogenperoxide/10" cells/h) in a respiratory burst pattern. In phagocytes, a membrane-bound oxidase containing an unusual cytochrome b (cytochrome 6559)is responsible for the generation ofSuperoxide anión (9-11). The latter ROI dismutates to H2O2spontaneously or upon catalysis by Superoxide dismutase. Incontrast, few nonphagocytic cells can generate ROI. Rat glo-merular mesangial cells triggered by serum-treated zymosan(12), sea urchin ova triggered by fertilization (13), and porcinethyroid cells in the presence of NADPH (14) generate ROI.Human platelets (15), human endothelial cells stimulated byinterleukin 1 or 7-interferon (16), human fibroblasts stimulatedby interleukin 1 or tumor necrosis factor-«(17), and human B-cells (18) have also been shown to generate ROI. Recently,human B-lymphocytes were found to contain cytochrome A5S9(19). Constitutive generation of ROI by human tumor cells (20)and by fMLP-stimulated rat Walker carcinosarcoma cells (21,22) has been demonstrated but only at rates <0.05 nmol ofH2O2/104 cells/h, approximately 25-30 times slower than the

Received 8/16/90; accepted 11/20/90.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by National Cancer Institute Grant 5 PDICA33049-08.

2To whom requests for reprints should be addressed, at Division of Hematology-Oncology, Cornell University Medical College, 1300 York Avenue. Box 57,New York, NY 10021.

3The abbreviations used are: PMN, polymorphonuclear leukocyte; DDC,

diethyldithiocarbamate; cDNA, complementary DNA; DPI, diphenyleneiodonium; fMLP, formyl-methionyl-leucyl-phenylalanine; HPO, horseradish peroxi-dase; KRPG, Krebs-Ringer phosphate buffer with 5.5 mivi glucose; MSKCC,Memorial Sloan-Kettering Cancer Center; PMA, phorbol myristate acetate; ROI,reactive oxygen intermediates; rTNF-a, recombinant human tumor necrosis fac-tor-a; ICso, concentration producing 50% inhibition.

rate typically observed in neutrophils. Pretreatment with 7-interferon augmented Superoxide release from two human tumor cell lines from 0.04 nmol up to 0.20 nmol/104 cells/h (20).

We have identified seven different human tumor cell lines offour histológica! types that can produce, without exogenousstimulation, amounts of H2O2 that after 4 h are as large asthose produced by stimulated PMN, and we have partiallycharacterized the nature and sources of hydrogen peroxideproduction in these cells.

MATERIALS AND METHODS

Cell Lines. The human melanoma cell lines SK-Mel 23, 28, 30, and131 (23-25) were provided by Dr. Alan Houghton (MSKCC, NewYork, NY). The human colon carcinoma cell lines HT 29 (26) andHCT 15 (27) and the human pancreatic carcinoma cell line AsPC 1(28, 29) were obtained from Dr. Sid Weite (MSKCC). Lan 1 humanneuroblastoma cells (30, 31) were provided by Dr. Nai-Kong Cheung(MSKCC). The human breast (CAMA-I) (32) and ovarian (SK-Ov 3)(26, 33) carcinoma cell lines were obtained from the late Dr. J. Fogh(Sloan-Kettering Institute, Rye, NY). Mouse fibroblasts (34) transfectedwith human tyrosinase cDNA were a gift from Dr. Houghton. All cellswere negative for Mycoplasma when stained using fluorescent bisben-zimide (Hoechst 33258; Aldrich Chemical Co., Milwaukee, WI).

All cells were adherent in culture and were maintained in 25-cm2

tissue culture flasks (Corning Glass Works, Corning, NY) in completemedium consisting of RPMI 1640 medium (Sigma Chemical Co., St.Louis, MO) with 7.5% heat-inactivated fetal bovine serum (HyCloneLaboratories, Logan, UT), 1% nonessential amino acids (Gibco Laboratories, Grand Island, NY), 1% L-glutamine (Hazleton Biologies,Lenexa, KS), and 1% penicillin-streptomycin (Hazleton). Cells weregrown in a humidified atmosphere of 5% CO2 in air at 37°C.Cells were

detached with trypsin (Flow Laboratories, McLean, VA) except Lan 1cells, which could be detached in phosphate-buffered saline (Gibco)alone.

PMN Isolation. Human blood drawn into heparinized Vacutainertubes using standard venipuncture techniques was layered onto Neutro-phil Isolation Medium (Los Alamos Diagnostics, Los Alamos, NM) andspun at 400 x g at 25°Cfor 30 min. The neutroph.il layer was removed

and washed twice in KRPG. This procedure (35) yielded >96% PMNs.ROI Assays. Tumor cells were seeded in complete medium (contain

ing phenol-free RPMI; Sigma) at 2xl05 cells/ml, 0.1 ml/well in 96-well microculture plates (Falcon Primaria; Becton-Dickson and Co.,Lincoln Park, NJ) and allowed to adhere overnight. The cells were thenwashed twice with warm KRPG and H2O2 measured using a scopoletinfluorescence assay, in which H2O2 oxidizes up to 35 ^M scopoletin(Sigma) to a nonfluorescent state in a reaction catalyzed by 1 purpu-rogallin unit/ml horseradish peroxidase (Sigma). The presence of 1 mivisodium azide (Fisher Scientific Co., Fair Lawn, NJ) in the reactionmixture prevents the competitive action of catalase and myeloperoxi-dase on H2O2. Fluorescence was measured using an automated platereader (MicroFluor MR6000; Dynatech Laboratories, Alexandria, VA)as previously described (36). Superoxide was measured using IO5cells

with or without preincubation with 0.1 mivi diethyldithiocarbamate(Sigma) by reduction of ferricytochrome c (Sigma) as previously described (37). The modified method of Ding and Nathan (38) was usedto measure cytochrome ¿>559content. Both of the latter two assays werecarried out using a Perkin-Elmer/Hitachi model 557 spectrophotome-ter with a 2-nm slit width.

794

Research. on February 24, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

PRODUCTION OF HYDROGEN PEROXIDE BY TUMOR CELLS

Reagents. Superoxide dismutase, metyrapone, a-naphthoflavone,chloramphenicol, ellipticene, PMA, and fMLP were obtained fromSigma. Sodium dithionite and potassium cyanide were from Fisher.rTNF-a was provided by Genentech Inc. (South San Francisco, CA).DPI, a nucleoside oxidase inhibitor and selective flavoprotein inhibitor(39), was a gift from Dr. Andrew R. Cross (University of Bristol,Bristol, United Kingdom).

RESULTS

Production of H2O2 by Tumor Cells. Seven adherent humantumor cell lines (two pigmented and two nonpigmented melanomas, one neuroblastoma, one colon carcinoma, and oneovarian carcinoma) elaborated hydrogen peroxide constitutivelyat rates ranging from 0.2 nmol to 0.5 nmol/10" cells/h (Fig. 1).

One colon, one breast, and one pancreatic carcinoma cell lineproduced <0.1 nmol of H2O2/104 cells/h. Human PMNs, when

stimulated by 10 ng/ml PMA, generated H2O2 at a rate ofabout 1.4 nmol/h (averaged over the first 2 h of the respiratoryburst) when tested simultaneously.

Fig. 2 shows that elaboration of hydrogen peroxide by SK-Mel 30 cells depended on the number of cells plated and wascontinuous at a constant rate over several hours until thescopoletin indicator became limiting. Such a continuous rate ofH2O2 production was observed in all experiments performedwith the tumor cell lines shown in Fig. 1 (data not shown).Dependency of H2O2 production on cell number was also observed for SK-Mel 28, SK-Mel 131, and Lan 1 cell lines (datanot shown). While the maximal rate of hydrogen peroxidegeneration by tumor cells was less than that of similar numbersof PMNs, cumulative amounts released by tumor cells surpassed those of triggered neutrophils after 4 h.

Neither PMA (dose range, 10-300 ng/ml), fMLP (0.5-10MM),nor rTNF-a (10-300 ng/ml), agents capable of triggeringa respiratory burst in PMNs in this system (35), could augmentthe observed H2O2 production rates in tumor cells as tested inSK-Mel 28, SK-Mel 30, SK-Mel 131, Lan 1, and HCT 15 celllines (data not shown).

The omission of 1 HIM azide from the assay medium, permitting endogenous catalase and peroxidase activity (8), resulted in a decrease in the H2O2 detected (Fig. 3). The addition

nmol H202/104 cells/h

0.5 1.0 1.5 2.0

(14)

Lan 1

HCT15HT

29AsPC

1SK-Ov

3CAMA-I223-

(6)iho)S^(2)HID

(2)

(2)

Fig. 1. Rates of H2O2 production by tumor cell lines. The rates of H2O2production/IO4 cells are shown for 10 different cell lines. Columns, means;

numbers in parentheses, numbers of experiments; error bars, SD calculated acrossexperiments. Tumor cell lines are shaded according to cell type (see "Methods").The rapid generation of H2O2 by PMNs in the first 2 h of a PMA-stimulatedrespiratory burst is displayed for comparison.

to4 x SK-M«I30 cell»

•4.e

NO

120 100

minutes240

Fig. 2. H2O2 Production as a function of time and tumor cell number. ,cumulative amounts of H2O2 produced as a function of time for five differenttumor cell densities: , representative PMA-stimulated PMN respiratoryburst for comparison. Insel, Ilo accumulation at 3 h is plotted versus tumor celldensity on a log scale. Results are the means of triplicates. SD at all points rangedbetween 0.01 and 0.09 nmol H2O2.

0.0

nmol H202/104 cells/h

0.2 0.4 0.6 0.8

Complete system

No azide

Cátalas«,no azide

No cells

NoHPO

Fig. 3. Evidence that tumor cell-dependent changes in fluorescence are due toH2O2. Plotted is the effect on measured H2O2production by SK-Mel 30 melanomacells of azide and catalase in the scopoletin-based assay. The change in fluorescence in the presence of tumor cells in the absence of horseradish peroxidase(HPO) is defined as 0 nmol H2O2/h/10* cells. Columns, means of triplicates;

error bars, SD.

of 3000 units/ml of exogenous catalase to the system resultedin the suppression of detected H2O2 to baseline, as defined bythe change in fluorescence in the absence of horseradish peroxidase.

Production of Superoxide Anión.Table 1 shows Superoxideanióngeneration as detected by a superoxide-inhibitable, ferri-cytochrome c reduction assay. The amounts of Superoxidegenerated constitutively appeared to be about one-tenth thoseof H2O2 on a per cell per hour basis. To test the hypothesis thatendogenous Superoxide dismutase might rapidly convert super-oxide to hydrogen peroxide, tumor cells were preincubated with100 MMDDC (37) for 2 h and then washed with KRPG. Thisaugmented modestly the amounts of Superoxide anión measured in the medium.

In contrast, when cells were preincubated with varying con-

795

Research. on February 24, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

PRODUCTION OF HYDROGEN PEROXIDE BY TUMOR CELLS

centrations of DDC prior to assay with scopoletin, measuredhydrogen peroxide production was markedly inhibited, with anICso of about 100 pM (Fig. 4). When DDC was present in thescopoletin assay medium, the degree of suppression of detectable H2O2 was even larger (IC5o, about l /UM)and considerablylarger than one would expect from Superoxide dismutase inhibition (since Superoxide dismutates rapidly to H2O2 even without catalysis) or from weak interference by DDC in the scopoletin assay demonstrated in Fig. 4, inset. Thus, DDC may haveinhibited Superoxide dismutase-mediated conversion of super-oxide to hydrogen peroxide, but there may be an additionalmechanism by which DDC can affect H2O2 production.

Mechanism of ROI Production. The best known enzymaticsource of large amounts of ROI in mammalian cells is theNADPH:O2 oxidoreductase that contains cytochrome ¿»559(9-11). The upper-most spectrophotometric tracing in Fig. 5 showsa peak at 559 nm when the technique was applied to 3 x IO7

PMNs, after double reduction by dithionite to eliminate theabsorbance contributed by hemoglobin and by mitochondria!cytochromes (38). The lower tracings reflect the same techniquewhen applied to IO7neuroblastoma cells (an equivalent amount

of cellular protein). No peak corresponding to cytochrome ¿>559was detectable; the lower limit of sensitivity of the techniquewas estimated to be about 1 pmol of cytochrome A559(38).Cytochrome ¿»559was likewise not detectable in SK-Mel 28 orin SK-Mel 30 cells (tracings not shown).

Table I Production of Superoxide aniónThe amount of Superoxide anióngenerated was measured by ferricytochrome

c reduction with and without Superoxide dismutase. Shown is the effect ofpreincubation with 100 »JMDDC for 2 h prior to assay, for seven different tumorcell lines.

Superoxide (nmol/105 cells/h)

SK-Mel30SK-Mel28SK-Mel131SK-Mel23HT29HCT15Un1No

cellsControl0.24

±0.07'0.1

5±0.020.17±0.140.28

±0.090.13±0.120.01±0.070.07

±0.040.02±0.03DDCpreincubation0.30

±0.030.23±0.010.24±0.13Not

done0.14±0.110.13±0.120.13±0.120.02

±0.031Mean ±SD.

20 -

15

• •DDC in osioy lysttmO Opr«-incubation with DOC

CMO

CM

0

C

0.0

DDC Concentration

Fig. 4. Effect of DDC on tumor cell H2O2 production. The effect of preincubation of Lan I neuroblastoma cells with varying concentrations of DDC (O)compared to like concentrations of DDC in the assay mixture (•).Inset, measuredH2O2 at 4 h when DDC and 4 nmol H2O2 are placed in the assay mixture, withoutcells. Points, means of triplicates; error bars, SD.

0.002AU

500 600Wavelength (nm)

Fig. 5. Lack of detectable cytochrome ft!59in tumor cells. The absorbance peakat 559 nm demonstrated for 3 x IO7 PMNs is not detectable for IO7 Lan 1

neuroblastoma cells, representing an equivalent amount of cell protein.

o o Lan 1

• »PMN+PMA

0.0

DPI Concentration

Fig. 6. Effect of DPI on tumor cell H¡O¡production. HjO¡generated by Lan1 neuroblastoma cells (O) or by PMA-stimulated PMN (•)is shown as a functionof DPI concentration in the assay mixture. Points, means of triplicates; error bars,SD.

In the scopoletin-based assay system, DPI inhibited tumorcell H2O2 production with an IC50 of about 0.3 pM (Fig. 6),suggesting a role for a flavoprotein in the generation of H2O2by tumor cells. DPI also inhibited the neutrophil respiratoryburst (IC5o, about 0.1 MM),as has been previously reported (39).Incubation with 10 MMDPI was not toxic to cells, as judged bytheir ability to exclude trypan blue.

Inhibitors of electron transport (potassium cyanide, sodiumazide), xanthine oxidase (allopurinol), monoamine oxidase(pargyline, clorgyline, nialamide, phenylzine), polyamine oxidase (diphenhydramine, quinacrine), or cytochrome P450 (me-tyrapone, a-naphthoflavone, chloramphenicol, ellipticene) atconcentrations up to 10 mM were all unable to block thedetection of tumor cell-generated H2O2 in the scopoletin assay(data not shown).

Mouse fibroblasts transfected with human tyrosinase cDNAand producing melanin (34) did not produce H2O2 in thescopoletin assay (data not shown).

DISCUSSION

It was surprising that several tumor cell lines, representing avariety of tissue types, constitutively produced large amountsof H2O2. Hydrogen peroxide production continued for at least4 h at a constant rate, its detection eventually limited by the

796

Research. on February 24, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

PRODUCTION OF HYDROGEN PEROXIDE BY TUMOR CELLS

assay indicator; the cumulative amount of H2O2 produced after4 h was comparable to the magnitude of the respiratory burstof similar numbers of PMNs. Superoxide anión was detectedat production rates an order of magnitude smaller than thosefor H2O2, even when DDC was used to block Superoxide dis-mutase-mediated conversion of Superoxide to H2O2. While it ispossible that stoichiometric quantities of Superoxide are beingmade by these cells but are not detected by the assay used, thesefindings are also consistent with a mechanism by which H2O2is produced directly. Several enzymes, such as glucose oxidasein fungi and uric acid or amino acid oxidase in mammaliancells, produce H2O2 directly. Likewise, the respiratory' burst of

fertilization in sea urchin ova forms H2O2 but not Superoxide(40).

The source of H2O2 generation is unclear. The degree ofinhibition of H2O2 generation by DDC was striking, whetherDDC was present only in the scopoletin assay mixture or whencells were pretreated with DDC and washed prior to the scopoletin assay. This suggests that DDC may be affecting H2O2production by a mechanism distinct from inhibition of super-oxide disimilase. Since DDC is a chelator of divalent cations,the H2O2 production pathway may be dependent upon a divalent metal ion.

The ability of DPI, an agent thought to bind to a specificcomponent of the PMN membrane oxidase (39), to inhibitH2O2 production suggests a role for a flavoprotein and/or anucleoside oxidase in the mechanism of H2O2 generation. Sincethese tumor cells appear to contain <1 pmol/107 cells of the

cytochrome ¿>559responsible for ROI production in humanneutrophils (9-11), tumor cells may possess a different type offlavoprotein oxidase.

That transfection with cDNA for tyrosinase permits mousefibroblasts to make melanin (34), but was not sufficient to allowthem to produce H2O2, suggests that the source of H2O2 isindependent of the melanin synthesis pathway.

While H2O2 is generated in several human biosynthetic pathways, including those for certain monoamines and polyamines,and in metabolic pathways such as that for xanthine, agentsknown to be able to inhibit enzymes in these pathways wereunable to slow the H2O2 production by tumor cells noted inthis system. Similarly, agents that can block the cytochromeP450 system had no effect on the ability of these cells to produceH202.

The generation of large amounts of hydrogen peroxide, if itwere to occur in vivo, could be important pathophysiologicallyfrom several aspects. The finding that tumor cells themselvescan produce reactive oxygen intermediates may help explainwhy some tumor cells are resistant to oxidative cytolysis (32).The same ovarian cancer cell lines shown in the present workto produce H2O2, for example, have been demonstrated to beresistant to lysis by H2O2 in concentrations up to 10 HIM(32).That tumor cells possess specific antioxidant defenses (41)suggests at least one mechanism by which these tumor cells donot undergo irreversible damage themselves in the course ofconstitutive ROI production.

Since ROI can induce DNA strand breaks (42), damage toguanine or thymine bases (43, 44), and sister chromatid exchanges (45), endogenous production of ROI might be a mechanism whereby tumors enhance their own genetic instability.Genetic instability, in turn, has been correlated with augmentedmalignant potential (46, 47).

Certain protease inhibitors, including «i-proteinase inhibitor,a2-macroglobulin, plasminogen activator inhibitor, and «2-plas-

min inhibitor are susceptible to oxidation at methionine residues of the active site (48, 49). Inactivation of «i-proteinaseinhibitor, plasminogen activator inhibitor, and/or «2-macro-globulin by oxidation favors unrestrained action of variousproteases, such as elastase, plasminogen activator, and plasmin,which are capable of considerable tissue destruction. Plasminogen activator is produced by many tumors and is stronglyimplicated in tumorigenicity and metastasis (50-52).

Thus, constitutive generation of hydrogen peroxide by tumorcells could potentially enhance neoplastic behavior by augmenting both genetic instability of a tumor and its capacity toinjure and penetrate host tissues.

ACKNOWLEDGMENTS

We thank Drs. Alan Houghton, Sid Weite, and Nai-Kong Cheungfor cell lines. Dr. Andrew R. Cross for diphenyleneiodonium, andGenentech, Inc. for supplying rTNF-a.

REFERENCES

1. Babior, B. M. Oxygen-dependent microbial killing by phagocytes. N. Engl.J. Med.. 298: 659-725, 1978.

2. Klebanoff, S. J. Antimicrobial mechanisms in neutrophilic polymorphonu-clear leukocytes. Semin. Hematol., 12: 117-142, 1975.

3. Babior, B. M., Kipnes, R. S., and Curnutte, J. T. Biological defense mechanisms: the production by leukocytes of Superoxide, a potential bactericidalagent. J. Clin. Invest., 52: 741-744, 1973.

4. Johnston, R. B., Jr., Lehmeyer, J. E., and Guthrie, L. A. Generation ofSuperoxide anión and chemiluminescence by human monocytes duringphagocytosis and on contact with surface-bound immunoglobulin G. J. Exp.Med.. 143: 1551-1556, 1976.

5. Reiss, M., and Roos, D. Differences in oxygen metabolism of phagocytosingmonocytes and neutrophils. J. Clin. Invest., 61: 480-488, 1978.

6. Weiss, S. J., King, G. W., and LoBuglio, A. F. Superoxide generation byhuman monocytes and macrophages. Am. J. Hematol., 4: 1-8, 1978.

7. Kitagawa, S., Takaku, F., and Sakamoto, S. A comparison of the superoxide-releasing response in human polymorphonuclear leukocytes and monocytes.J. Immunol.. 125: 359-364, 1980.

8. Nakagawara, A., Nathan, C. F., and Cohn, Z. A. Hydrogen peroxide metabolism in human monocytes during differentiation in vitro. J. Clin. Invest.,68: 1243-1252. 1981.

9. Segal, A. W. and Jones, O. T. G. Novel cytochrome b system in phagocyticvacuoles of human granulocytes. Nature (Lond.). 276: 515-517, 1978.

10. Cross, A. R.. Jones. O. T. G., Harper, A. M.. and Segal, A. W. Oxidation-reduction properties of the cytochrome b found in the plasma-membranefraction of human neutrophils. Biochem. J., 194: 599-606, 1981.

11. Royer-Pokora, B.. Kunkel. L. M., Monaco, A. P.. et al. Cloning the gene foran inherited human disorder—chronic granulomatous disease—on the basisof its chromosomal location. Nature (Lond.), 322: 32-38, 1986.

12. Baud, L., Hagege, J.. Sraer, J.. Rondeau, E., Perez, J., and Ardaillou, R.Reactive oxygen production by cultured rat glomerular mesangial cells duringphagocytosis is associated with stimulation of lipoxygenase activity. J. Exp.Med., 158:1836-1852, 1983.

13. Foerder, C. A., Klebanoff, S. J., and Shapiro, B. M. Hydrogen peroxideproduction, chemiluminescence, and the respiratory burst of fertilization:interrelated events in early sea urchin development. Proc. Nati. Acad. Sci.USA, 75:3183-3187. 1978.

14. Nakamura, Y., Ogihara, S., and Ohtaki. S. Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasmamembranes. J. Biochem., 102: 1121-1132, 1987.

15. Marcus, A. Pathways of oxygen utilization by stimulated platelets andleukocytes. Semin. Hematol., 16: 188-195, 1979.

16. Matsubara, T., and Ziff. M. Increased Superoxide aniónrelease from humanendothelial cells in response to cytokines. J. Immunol., 137: 3295-3298,1986.

17. Meier, B., Radeke. H. H., Selle, S., et al. Human fibroblasts release reactiveoxygen species in response to interleukin-1 or tumour necrosis factor-a.Biochem. J., 263: 539-545. 1989.

18. Volkman, D. J.. Buescher, E. S., Gallin, J. I., and Fauci, A. S. B cell lines asmodels for inherited phagocytic diseases: abnormal Superoxide generation inchronic granulomatous disease and giant granules in Chediak-Higashi syndrome. J. Immunol.. 133: 3006-3009, 1984.

19. Kobayashi, S., Imajoh-Ohmi, S.. Nakamura, M., and Kanegasaki, S. Occurrence of cytochrome bKt in B-cell lineage of human lymphocytes. Blood, 75:458-461, 1990.

20. Aune. T. M., and Pogue, S. L. Inhibition of tumor cell growth by interferon-gamma is mediated by two distinct mechanisms dependent upon oxygentension: induction of tryptophan degradation and depletion of intracellular

797

Research. on February 24, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

PRODUCTION OF HYDROGEN PEROXIDE BY TUMOR CELLS

nicotinamide adenine dinucleotide. J. Clin. Invest., 84: 863-875. 1989.21. Leroyer, V., Werner, L., Shaughnessy. S., Goddard. G. J.. and Orr, F. W.

Chemiluminescence and oxygen radical generation by Walker carcinosar-coma cells following chemotactic stimulation. Cancer Res., 47: 4771-4775,1987.

22. Shaughnessy, S., Buchanan, M. R., Turple. S., Richardson, M., and Orr,F. W. Walker carcinosarcoma cells damage endothelial cells by the generationof reactive oxygen species. Am. J. Pathol., 134: 787-796, 1989.

23. Houghton, A. N., Albino, A. P., Cordon-Cardo, C., Davis, L. J., and Eisinger,M. Cell surface antigens of human melanocytes and melanoma: expressionof adenosine deaminase binding protein is extinguished with melanocytetransformation. J. Exp. Med., ¡67:197-212, 1988.

24. Carey, T. E., Takahashi, T., Resnick, L. A., Oettgen. H. F., and Old, L. J.Cell surface antigens of human malignant melanoma. I. Mixed hemadsorp-tion assays for humoral immunity to cultured autologous melanoma cells.Proc. Nati. Acad. Sci. USA, 73: 3278-3282, 1967.

25. Eisinger, M. and Marko, O. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc. Nati.Acad. Sci. USA, 79: 2018-2022, 1982.

26. Fogh, J., and Trempe, G. New human tumor cell lines. In: J. Fogh (ed.),Human Tumor Cells in Vitro, pp. 115-159. New York: Plenum Press, 1975.

27. Dexter, D. L., Barbosa, J. A., and Calabresi, P. AyV-Dimethylformamide-induced alteration of cell culture characteristics and loss of tumorigenicity incultured human colon carcinoma cells. Cancer Res., 39: 1020-1025, 1979.

28. Chen, W. H., Horoszewicz. S. J., Leong, S. S., et al. Human pancreaticadenocarcinoma: in vitro and in rim morphology of a new tumor lineestablished from ascites. In Vitro. IS: 24-28, 1982.

29. Tan, M. H., Shimano, T., and Chu, T. M. Differential localization of humanpancreas-cancer-associated antigen and carcinoembryonic antigen in homologous pancreatic tumoral xenograft. J. Nail. Cancer Inst., 67: 563-566,1981.

30. Cheung, N. K. V., Saarinie, U. M., Neely, J. E., et al. Monoclonal antibodiesto glycolipid antigen on human neuroblastoma cells. Cancer Res.. 45: 2642-2649, 1985.

31. Munn, D. H. and Cheung, N. K. V. Phagocytosis of tumor cells by humanmonocytes cultured in recombinant macrophage colony-stimulating factor.J. Exp. Med., 772: 231-237, 1990.

32. O'Donnell-Tormey, J., DeBoer. C. J., and Nathan, C. F. Resistance of humantumor cells in vitro to oxidative cytolysis. J. Clin. Invest., 76: 80-86, 1985.

33. Fogh, J., Wright, W. C., and Loveless, J. D. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J. Nati. Cancer Inst.,58: 209-214, 1977.

34. Bouchard, M.,Fuller, B. I).. Vijayasaradhi. S., and Houghton, A. N. Inductionof pigmentation in mouse fibroblasts by expression of human tyrosinasecDNA. J. Exp. Med., 169: 2029-2042, 1989.

35. Nathan, C. F. Neutrophil activation on biological surfaces: massive secretionof hydrogen peroxide in response to products of macrophages and lympho

cytes. J. Clin. Invest., 80: 1550-1560, 1987.36. De la Harpe, J., and Nathan. C. F. A semi-automated micro-assay for H2O:

release by human blood monocytes and mouse peritoneal macrophages. J.Immunol. Methods, 78: 323-336, 1985.

37. Tsunawaki, S., and Nathan, C. F. Enzymatic basis of macrophage activation.J. Biol. Chem., 259: 4305-4312, 1984.

38. Ding, A. H., and Nathan, C. F. The measurement of cytochrome ¿559inpolymorphonuclear leukocytes and macrophages in the presence of hemoglobin or mitochondrial cytochromes. Anal. Biochem., 175: 22-29, 1988.

39. Cross, A. R., and Jones, O. T. G. The effect of the inhibitor diphenyleneio-donium on the superox¡de-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase. Biochem. J., 237: 111-116,1986.

40. Heinecke. J. W., and Shapiro, B. M. Respiratory burst oxidase of fertilization.Proc. Nati. Acad. Sci. USA, 86: 1259-1263, 1989.

41. Nathan, C. F., Arrick, B. A.. Murray, H. W., DeSantis. N. M., and Conn. Z.A. Tumor cell anti-oxidant defenses. Inhibition of the glutathione redox cycleenhances macrophage-mediated cytolysis. J. Exp. Med., 153:766-782,1980.

42. Birnboim, H. C. DNA strand breakage in human leukocytes exposed to atumor promoter, phorbol myristate acetate. Science (Washington DC), 215:1247-1249, 1982.

43. Jackson. J. H., Gajewski, E.. Schraufstatter, I., et al. Damage to the bases inDNA induced by stimulated human neutrophils. J. Clin. Invest., 84: 1644-1649, 1989.

44. Lewis, L. G., and Adams. D. O. Induction of 5,6-ring-saturated thyminebases in NIH-3T3 cells by phorbol ester-stimulated macrophages: role ofreactive oxygen intermediates. Cancer Res., 45: 1270-1275. 1985.

45. Weitberg, A. B., Weitzman, S. A., Destrempes, M., Latt, S. A., and Stessei,T. P. Stimulated human phagocytes produce cytogenetic changes in culturedmammalian cells. N. Engl. J. Med., 308: 26-30. 1983.

46. Cifone, M. A., and Fidler, I. J. Increasing metastatic potential is associatedwith increasing genetic instability of clones isolated from murine neoplasms.Proc. Nati. Acad. Sci. USA, 78: 6949-6952, 1981.

47. Megidish, T., and Mazurek, N. A mutant protein kinase C that can transformfibroblasts. Nature (Lond.), 342: 807-811, 1989.

48. Swaim, M. W., and Pizzo, S. V. Methionine sulfoxide and the oxidativeregulation of plasma proteinase inhibitors. J. Leukocyte Biol., 43: 365-379,1988.

49. Johnson. D., and Travis, J. The oxidative inactivation of human alpha-1-proteinase inhibitor. Further evidence for methionine at the reactive center.J. Biol. Chem.. 254: 4022-4026, 1979.

50. Mullins, D. E., and Rohrlich, S. T. The role of proteinases in cellularinvasiveness. Biochim. Biophys. Acta, 695: 177-214, 1983.

51. Daño,K., Andreasen, P. A., Grondahl-Hansen, J., Kristensen, P., Nielsen,L. S., and Skriver, L. Plasminogen activators, tissue degradation, and cancer.Adv. Cancer Res., 44: 139-266, 1985.

52. Ossowski, L.. and Reich, E. Antibodies to plasminogen activator inhibithuman tumor metastasis. Cell. 35: 611-619, 1983.

798

Research. on February 24, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

1991;51:794-798. Cancer Res   Ted P. Szatrowski and Carl F. Nathan  Tumor CellsProduction of Large Amounts of Hydrogen Peroxide by Human

  Updated version

  http://cancerres.aacrjournals.org/content/51/3/794

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/51/3/794To request permission to re-use all or part of this article, use this link

Research. on February 24, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from