[CANCER RESEARCH 49, 1671-1675, April 1, 1989]

Intracellular Hydroxyl Radical Production Induced by Recombinant Human Tumor

Necrosis Factor and Its Implication in the Killing of Tumor Cells in Vitro

Naofumi Yamauchi, Hiroshi Kuriyama, Naoki Watanabe, Hiroshi Neda, Masahiro Maeda, and Yoshiro NiitsuDepartment of Internal Medicine (Section 4), Sapporo Medical College, South-1, West-16, Chuo-ku, Sapporo, 060, Japan

ABSTRACT

This study investigated the effect of recombinant human tumor necrosisfactor (rh I NT') on hydroxyl radical production by established cell lines

in vitro, and its implication in the killing of tumor cells by rhTNF. Duringincubation of TNF sensitive mouse tumorigenic fibroblast L-M cells (2x 10"cells) in the presence of rhTNF (100 U), hydroxyl radical produc

tion as detected by the evolution of methane gas from dimethyl sulfoxideincreased gradually, at 18 h reaching 1.8 times that in the absence ofrhTNF. This increase was dependent on the concentration of rhTNF andwas effectively prevented by the simultaneous addition of anti-rhTNFmonoclonal antibody III 2F3, which inhibited both the binding of rhTNFto its receptor and the cytotoxic activity of rhTNF. The addition of ironchelator 2,2'-bipyridine, which inhibits iron-catalized Fenton reaction

and so inhibits hydroxyl radical generation, suppressed both the increaseof hydroxyl radical production and the cytotoxicity induced by rhTNF.A similar increase in hydroxyl radical production in the presence ofrhTNF was also detected with TNF-sensitive human myosarcoma-derivedKYM cells, but no such increase was detected with TNF insensitivehuman embryonic lung fibroblast HEL cells.

The results show that rhTNF induces increased hydroxyl radicalproduction in TNF-sensitive cells, and suggest that this plays an important role in the mechanism of tumor cell killing by rhTNF.

INTRODUCTION

TNF1 is a macrophage/monocyte-derived anticancer cytokine(1-3) with strong cytotoxicity to tumor cells in vitro (4-10).The mechanism of its lethal effect on tumor cells, however,remains largely unexplained, although some studies have suggested the involvement of lysosomal enzyme (11, 12) and hydroxyl radical (12-14), based on the observation of suppressedTNF cytotoxicity in the presence of various inhibitors.

Various studies have shown that TNF cytotoxicity is suppressed in the presence of the hydroxyl radical scavengersdimethyl sulfoxide (DMSO) (12-14) and promethazine (14),and also in the presence of an iron chelator (desferal) (13, 14),which is known to block hydroxyl radical production. Suchstudies are methodologically limited, however, as cytotoxicsuppression in itself does not provide a means of determiningthe kinetics of hydroxyl radical generation in TNF-sensitivecells, nor of detecting any changes in hydroxyl radical quantitiesin cells insensitive to TNF. It has thus not been possible todemonstrate a direct relationship between the cytotoxic effectof TNF and its stimulation of hydroxyl radical production.

We therefore attempted to measure the amount of hydroxylradical produced in several cell lines under various conditionsfollowing their exposure to rhTNF and investigated the relationbetween changes in this production and the cytotoxic effects ofrhTNF.

Received 7/12/88; revised 10/27/88; accepted 12/13/88.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.

1The abbreviations used are: TNF, tumor necrosis factor; DMSO, dimethylsulfoxide; rhTNF, recombinant human tumor necrosis factor; DETAPAC, di-ethylenetriaminopentaacetic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazineeth-anesulfonic acid; EMEM. Eagle's minimal essential medium; SOD, Superoxide

dismutase; FBS, fetal bovine serum.

MATERIALS AND METHODS

Materials. SOD, 2,2'-bipyridine, and DETAPAC were purchased

from Sigma (St. Louis, MO); catalase from Boeringer Mannheim (W.Germany); DMSO and HEPES from Wako Chem. Co., Ltd. (Osaka).

rhTNF and Monoclonal Antibody to rhTNF. rhTNF, produced inEscherichia coli and purified (99.9%) (15), and mouse anti-rhTNFmonoclonal IgG antibody III 2F3 which neutralized the rhTNF activity(16) were generously provided by Asahi Chemical Ind. Co., Ltd. (Tokyo). The rhTNF had a specific activity of 2.3 x 10" U/mg protein asdetermined by its cytotoxic activity against mouse L-M cells (16) and

a molecular mass of 51,000 as trimer.Radioiodination of rhTNF was performed by the method of Bolton

and Hunter (17) using Bolton and Hunter reagent (4000 Ci/mmol, 148TBq/mmol, NEN Research Products, Boston, MA) to obtain 125I-

labeled rhTNF with specific activity of 250 cpm/fmol and no significantloss in bioactivity (18).

Cell Culture. L-M (mouse tumorigenic fibroblast) cells and HEL(human embryonic fibroblast) cells were obtained from the AmericanType Culture Collection, Rockville, MD. These cells were cultured inEMEM (Flow Lab., Australia) with 5% or 10% heat-inactivated FBS(Flow Lab.), 100 units/ml of penicillin, 100 Mg/ml of streptomycin and10 HIMHEPES (pH 7.2) at 37'C in a 5% CO2 incubator. KYM cells

derived from human myosarcoma were kindly provided by Dr. M.Sekiguchi (Cancer Research Institute, Tokyo University) and werecultured in DM-160 medium (Kyokuto Pharmaceutical Ind., Tokyo)with 10% FBS, antibiotics, HEPES and other conditions as above.

In Vitro Assay for Cytotoxic Action. L-M cells, (1 x 10" cells/200

ii\) were added to well of 96-well microculture plate (Sumitomo Bakelite, Tokyo), and incubated at 37"C for 4 h in 5% CÛ2.Medium was

aspirated and both rhTNF (200 U/ml, 100 ¿/I)and one of the agentsindicated in Table 1 (100 ¿il)in EMEM were added, followed byincubation at 37°Cfor 24 h in 5% CO2. The cytotoxic activity was then

assessed by the dye uptake method using méthylèneblue (16).Binding Assay. Binding assays were performed in triplicate on mono-

layers of L-M cells (5 x IO5 cells/well) in 12-well culture clusters

(Costar, Cambridge, MA). The cells were incubated with 5 nM (586 U/ml)['"l]rhTNFfor 120 min at 4°Cin EMEM containing 0.5% gelatin

as binding medium. The cells were then washed three times with chilledbinding medium and lysed with 1 ml of 0.5 M NaOH, and 0.5 ml ofthe lysate was counted for radioactivity. Nonspecific binding was determined in the presence of a 1000-fold excess of unlabeled rhTNF protein.

Quantification of Hydroxyl Radical Formation. Hydroxyl radical production was measured by the formation of methane from DMSO, inaccordance with the method of Repine et al. (19). Each 2-ml reactionmixture was prepared in a siliconized 3.5-ml glass tube (Pierce, Rock-ford, IL) by the sequential addition of 1 ml of cell suspension (2 x IO7/

ml), 0.4 ml of DMSO solution (2 M) and 0.4 ml of solution containingbipyridine, III 2F3 or mouse IgG as applicable, each of these in theculture medium described above but containing 20 mM HEPES (pH7.2), followed by sealing of the tube with a silicon teflon septum andopen-top screw cap, and injection of 0.2 ml of the same 20 mM HEPESmedium containing rhTNF through the septum. The mixture wasvigorously mixed and then incubated up to 24 h at 37°Cin a shaking

water bath; the reaction was terminated by placing the tube on meltedice.

A 0.2-ml sample of the headspace gas from each tube was withdrawninto a gas-tight syringe (Hamilton, Reno, NV), after its vigorous mixingby depression and withdrawal of the syringe plunger at least 10 times.The methane concentration in the 0.2-ml gas sample was determinedon a Shimazu GC-9A gas Chromatograph (Shimazu, Tokyo) equippedwith a flame-ionization detector and a 3-mm x 2-m stainless steelcolumn packed with 60/80 mesh active carbon (Gasukuro Kogyo,

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HVDROXYL RADICAL PRODUCTION BY TNF

Tokyo). Nitrogen gas was used as carrier, at a flow rate of 50 ml/min;injector, column, and detector temperatures were 200, 150, and 200°C,

respectively. A calibration curve, linear over the range of 0.01-35 nmolmethane, was constructed with a 99.7% methane standard (GasukuroKogyo). The retention time for authentic methane under these conditions was 1 min. The methane content, expressed as pmol of methaneper 1.5 ml of headspace gas, was determined from the intersection onthe calibration curve of the peak area after subtraction of the areacorresponding to content of the laboratory air (14.2-18.2 pmol/0.2ml). Because of the known tendency for variations to occur in the resultsof biological assays, each experiment was accompanied by an assay, bythe method described above, for hydroxyl radical production by acontrol containing no rhTNF.

RESULTS

Suppression of rhTNF Cytotoxicity. The effects of varioushydroxyl radical scavengers, iron chelators and antioxidantenzymes on rhTNF cytotoxicity, as determined for L-M cells,are shown in Table 1. In the presence of DMSO (400 HIM)ashydroxyl radical scavenger and bipyridine (60 pM) as ironchelator and thus suppressor of hydroxyl radical formation(20), the cytotoxic effect of rhTNF was decreased to 24 and63%, respectively, ofthat observed with rhTNF alone. In contrast, no significant reduction in cytotoxic effect was observedin the presence of the SOD or catalase antioxidant enzyme orthe DETAPAC iron chelator, for all of which the cell membraneis impermeable (21-23). DETAPAC in fact apparently servedto enhance the rhTNF cytotoxicity for reasons which are as yetunknown.

Time Course of Cytotoxic Activity of rhTNF on L-M Cells.Following incubations at 37°Cfor 0-24 h with or without

rhTNF but in the absence of any hydroxyl radical scavenger, asshown in Fig. 1, cytotoxic assay by the dye uptake methodshowed no significant cell death at 0-15 h of incubation withrhTNF, but increasingly high levels of cell death at 18, 21, and24 h of incubation periods.

Influence of DMSO Concentration on Methane Formation.The amount of methane produced in 18 h of L-M cell incubation after addition of DMSO at concentrations of 0-600 mM isshown in Fig. 2. In the absence of rhTNF, methane productionreached a plateau at 100 mM DMSO, with no further increaseat higher concentrations. Additional methane production apparently induced by the presence of rhTNF (100 U/ml), asrepresented by the shaded area in Fig. 2, reached a similarplateau at 400 mM DMSO, which was therefore the DMSOconcentration utilized for all subsequent determinations ofmethane production.

Time Course of rhTNF-induced Methane Production. Thetime course of methane production in L-M cell incubation withand without rhTNF is shown in Fig. 3. In the absence of rhTNF,the cumulative methane production (O) increased during thefirst 21 h of incubation, and apparently reached a plateau atthat time. Methane production attributable to the presence ofrhTNF in similar incubation, represented by the shaded portionin Fig. 3 as the difference between the amounts found inincubation with (•)and without rhTNF, increased throughoutthe entire investigated incubation time of 24 h. A significantdifference of 15.5 ±5.3 pmol/2 x IO7cells was observed at 18

h, with further increases at 21 and 24 h.Influence of rhTNF Concentration on Methane Production.

Methane production amounts as measured after 18 h of L-Mcell incubation were clearly dependent on the rhTNF concentration throughout the range investigated, as shown in Fig. 4.The values with rhTNF at 1, 10, 100, and 1000 U/ml were,respectively, 2.0, 2.4,2.8, and 3.2 times that found after similarincubation with no rhTNF.

Effect of Bipyridine on Methane Production. As shown in Fig.5, the presence of bipyridine during L-M cell incubation clearlyresulted in a decrease in the difference between the methaneproduction levels with and without rhTNF (shaded bars). In theabsence of bipyridine, methane production with rhTNF washigher than that without by 37.0 ±9.1 pmol/2 x IO7 cells. In

its presence (60 /¿M),this difference was effectively reduced (P< 0.05) to 17.5 ±2.9 pmol/2 x IO7 cells. This effect, by a

known inhibitor of hydroxyl radical production, clearly indicates that the increased level of methane production observedin the presence of rhTNF throughout this study is actuallyattributable to an increased level of hydroxyl radical production.

Effect of III 2F3 on Hydroxyl Radical Production. The inhibiting effect of the monoclonal IgG antibody HI 2F3 on rhTNFreceptor binding and cytotoxicity to L-M cells is shown inTable 2. Its effect on hydroxyl radical production by L-M cellsin the presence of rhTNF is indicated in Fig. 6. Without III2F3, the addition of rhTNF at 100 U/ml resulted in an apparentdoubling of methane production during L-M cell incubation,from 22.0 ±4.4 (bar a) to 48.0 ±3.8 (bar b) pmol/2 x IO7

cells. With HI 2F3 (10 Mg/ml), this increase was completelysuppressed (bar c). In contrast, as shown by bar d, mouse IgG(10 iig/ml) apparently had no effect on the heightening ofmethane production by the presence of rhTNF.

It was further found that methane production in incubationof the cell-free medium and DMSO with rhTNF (100 U/ml)

was no higher than that without rhTNF, as shown by bars fand

Table I Effect of reactive oxygen scavengers on L-M cell susceptibility to rhTNFL-M cells (1 x IO4cells/200 jil) were added to wells of 96-well microculture plate, and incubated at 37°Cfor 4 h. Medium was aspirated and both rhTNF (200 U/

ml, 100 i¡\)and one reactive oxygen scavenger (100 ^1) in EMEM were added, followed by incubation at 37*C for 24 h in 5% CO2. Cytotoxicity was assessed by dye

uptake method as described previously.

AgentNone

SODCatalaseSOD +catalaseDMSO

BipyridineDETAPACFunctionSuperoxide

aniónscavengingHydrogen peroxide scavengingInhibition of hydroxyl radical

generationHydroxyl radical scavengingIron chelatingIron chelatingConcentration1.25

ng/ml750 units/ml

0.63 Mg/ml +325 units/ml

400 mM60MM60

MMrhTNF

cytotoxicity''(%)70.4

±1.87C

66.3 ±0.7068.4 ±0.9366.0 ±1.3616.6±

1.24''44.0 ±0.19'86.5 ±1.48'Relative

sensitivity*

to rhTNF1.00

0.940.970.940.24

0.631.23

' Calculated as cytotoxicity = 100 x I 1 - OP of rhTNF treated cells\OD of rhTNF free cells /'

rhTNF cytotoxicity with agentCalculated as relative sensitivity = .rhTNF cytotoxicity without agent

' Mean ±SE of three separate experiments.* ' Values significantly (ä,P < 0.001; ',P< 0.005) different from value with no agent added.

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HYDROXYL RADICAL PRODUCTION BY TNF

È 60O

X

e 40O

Ü 20

O 12 1l 24

INCUBATION TIME (hours)

Fig. 1. Time course of cytotoxic activity of rhTNF on L-M cells. L-M cells (1x IO4 cells/200 i/1) were added to wells of 96 well microculture plate, andincubated at 37*C for 4 h. Medium was aspirated and 200 «IEMEM containingrhTNF (100 U/ml) was added, followed by incubation at 37'C for the indicated

periods in 5% CO... Cytotoxicity was assessed by dye uptake method as describedpreviously. Percentage cytotoxicity was calculated by the formula shown in Table1. Values are means ±SD of triplicate wells. *, **, values significantly (*, P <0.005, **, P < 0.001) different from value without rhTNF.

100 200 300 400 500 600DMSO CONCENTRATION (mM)

Fig. 2. Effect of DMSO on the production of methane by L-M cells incubatedwith rhTNF. Each 3.5-ml tube contained 2 X IO7 L-M cells and the indicated

concentration of DMSO with (•)or without (O) 100 U/ml of rhTNF and wasincubated at37'Cforl8h. The concentration of methane in 200 nI of headspace

gas was measured by gas chromatography and total produced methane wascalculated as described in "Materials and Methods.11 •¿�produced methane when

medium containing only DMSO was incubated. Shaded area, produced methaneinduced by the presence of rhTNF. Values are mean ±SE of six separatepreparations. *, **, ***, values significantly (*, P < 0.05; **, P < 0.005; ***, P <0.001) different from value without rhTNF.

e. respectively, thus tending to preclude the possibility thatrhTNF itself functions enzymatically or catalytically in theproduction of hydroxyl radical.

Hydroxyl Radical Production with KYM and HEL Cells. Theeffect of rhTNF addition on two other lines of different TNFsensitivity are shown in Table 3. The results with rhTNF-sensitive KYM cells were similar to those described above forL-M cells. Methane production amounts in the presence andabsence of rhTNF were, respectively, 39.3 ±5.6 and 15.7 ±4.7pmol/2 x IO7 KYM cells, a difference of approximately 2.5times. With rhTNF-insensitive HEL cells, on the other hand,no significant difference in methane production was observedbetween incubations with and without rhTNF, which yieldedvalues of 13.3 ± 1.7 and 13.2 ±3.5 pmol/2 x IO7 cells,

respectively.

DISCUSSION

The hydroxyl radical is a highly potent reactive oxygen knownto function as a direct mediator in the cytolytic effects ofanticancer quinone (24), NK cells (25), neutrophils (26), andradiological exposure (27).

0

QOCE0.

IHUl

10-

INCUBATION TIME (hours)

Fig. 3. Time course of the production methane by L-M cells incubated withrhTNF. L-M cells (2 x IO7 cells/2 ml) were incubated at 37'C in medium

containing 400 mM DMSO with (•)or without (O) the addition of rhTNF (100U/ml). At indicated times, produced methane was assessed by gas chromatography as described in Fig. 1. No methane was detected when medium containingonly 400 mM DMSO was incubated (•).Shaded area, produced methane inducedby the presence of rhTNF. Values are means ±SE of eight separate preparations.*, **, values significantly (*,/»<0.05; **,/"< 0.001 ) different from value without

rhTNF.

60

5°

O

O

QOo:o.

UZ

III

40

30

01 10 100 1000

rhTNF CONCENTRATION (U/ml)

Fig. 4. Production of methane by L-M cells as a function of rhTNF concentration. L-M cells (2 x IO7cells/2 ml) were indicated in medium containing 400mM DMSO with indicated concentration of rhTNF at 37"C for 18 h. Produced

methane was assessed by gas chromatography, as described in Fig. 1. Values aremean ±SE of eight separate preparations.

TNF, on the other hand, reportedly stimulates the expulsionof Superoxide aniónby neutrophils (28), and hydrogen peroxideby macrophages (29). Its cytotoxic effect also reportedly issuppressed by hydroxyl radical scavengers (12-14), and by ironchelator, an inhibitor of hydroxyl radical production (13, 14),thus suggesting that hydroxyl radical production is involved inthe mechanism of TNF cytotoxicity.

The present study provides the first quantitative evidence ofa correspondence between hydroxyl radical production andTNF concentration, cell sensitivity, and cytotoxic effect, by themeasurement of methane production from DMSO during incubation of three cell lines under various degrees of hydroxylradical suppression and rhTNF concentration.

We first applied this method to investigation of the kineticsof hydroxyl radical production in incubation of TNF sensitiveL-M cells. The results show that the increse in hydroxyl radical

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HYDROXYL RADICAL PRODUCTION BY TNF

8̂0£2g£

70ì„"

60C-si^

50^Q1

4°Og

30gLUK

10LU0'

T--T-•.--TT•--_Table 3 rhTNF-induced methane production by KYM and HEL cells

Cytotoxicity was determined by the dye uptake method previously described.Methane production per 2x10' cells in 18 h was assessed by gas chromatography

as described in Fig. 1.

ab cdFig. 5. Effect of iron chelator bipyridine on the production of methane by I .-

M cells incubated with rhTNF. L-M cells (2 x 10' cells/2 ml) were incubated for18 h at 37'C in medium containing 400 mM DMSO with (b,d) or without (a,c)

rhTNF (100 U/ml), in the presence (c,d) or absence (a,b) of bipyridine (60 JIM).Produced methane was assessed by gas chromatography as described in Fig. 1.Values are mean ±SE of six separate preparations.

Table 2 Effect ofanti-rhTNF monoclonal antibody III 2F3 on cytotoxicity andcell surface binding of rhTNF

Cytotoxicity was determined by the dye uptake method previously described.Total and nonspecific binding of [125I]rhTNF were assessed as follows. L-M cells(5 x 10s cells/500 n\ binding medium) were incubated for 2 h at 4'C with [12!I]

TNF (5 nin, 586 U/ml) in the presence or absence of unlabeled rhTNF (5 JIM).The cells were then washed, followed by elution with 0.5 M NaOH and determination of the surface bound radioactivity.

TreatmentNone

III 2F3*MouseIgG*"

Calcula«* 10 ng/lC

or l'"I]TNF.60

n150gì408S

ÕÎ30u

11^20IUS

,00Total

bindingCytotoxicity"

rhTNF Nonspecific binding(mean ±SE, (mean ±SE, of [125I]rhTNF

%) cpm) (mean ±SE,cpm)78.2

±2.5 28981.2 ±4.3 255

76.5 ±3.1 2686±

103.2 266 ±1.7±5.3 252 ±4.2±87.6 261 ±3.5:d

from the formula shown in Table 1.«U rhTNF of IgG solution was added simultaneously withrhTNF-TTTT-

Fig. 6. Effect of anti-rhTNF monoclonal antibody III 2F3 on the productionof methane by L-M cells incubated with rhTNF. L-M cells (2 x IO7 cells/2 ml)were incubated at 37*C for 18 h in medium containing 400 mM DMSO alone (a),

with rhTNF at 100 U/ml (¿>),with rhTNF at 100 U/ml and III 2F3 at 10 ^g/ml(c), or with rhTNF at 100 U/ml and mouse IgG at 10 jig/ml (d). L-M cell-freemedium (2 ml) containing 400 mM DMSO and no rhTNF (e) or rhTNF (/) at100 U/ml was incubated at 37°Cfor 18 h. Produced methane was assessed by

gas chromatography as described in Fig. 1.

CellL-MKYMHELrhTNF(U/ml)010001000100Cytotoxicity"(%)070.4076.000Numberof

experimentsg8688gProduced

methane(pmol/2 x IO7

cells/18h)22.0±4.4*-,P<

0.00148.0±3.8—15.7

±4.8—,P<0.00139.3

±5.6—13.2

±3.5^NSC13.3±

1.7—" Calculated from the formula shown in Table 1.* Mean ±SE of indicated number of experiments.c NS, not significant.

production effected by the presence of rhTNF, in terms of thedifference between its production with and without rhTNF, isdependent on both incubation time and rhTNF concentration.

The substantial hydroxyl radical production by cells in theabsence of rhTNF, as observed in this and the subsequentexperiments of the present study, may be attributed to mitochondria! respiration and the other redox known to occur incells and thus generate hydroxyl radicals, and is in accord withsimilar findings by other investigators for phagocytes and Erlichtumor cells in vitro in the presence of DMSO alone (19, 24).

The next series of experiments were performed to determinewhether TNF is directly involved in the reaction yielding hydroxyl radical in the manner of anticancer quinone and mena-

dione, which are known to function as electron acceptors andthus produce hydroxyl radicals by reduction of oxygen molecules (24, 30). The results tended to preclude this possibility,and show rather that TNF promotes hydroxyl radical production through stimulation of intracellular production, as: (a) nosuppression of rhTNF cytotoxicity occurred in L-M cell incubations with extracellular reactive oxygen suppressed by SOD,catalase, or DETAPAC; (b) no observable increase of hydroxylradical production during incubation of cell-free medium waseffected by the addition of rhTNF; and (c) the rhTNF stimulation of hydroxyl radical production was apparently suppressedcompletely by the addition of the monoclonal antibody IH 2F3,which was shown to block rhTNF receptor binding and completely neutralize rhTNF cytotoxicity.

The correlation between TNF cytotoxic effect and hydroxylradical production was further shown by the L-M cell incubation in the presence of rhTNF and bipyridine. In the presenceof bipyridine, with the resulting suppression of rhTNF-stimu-

lated hydroxyl radical production, the cytotoxic effect of rhTNFon the L-M cells was inhibited significantly, suggesting that thestimulation of hydroxyl radical production is an important partof the mechanism of rhTNF cytotoxicity.

Comparison of the observed time course of rhTNF cytotoxicity in the absence of hydroxyl radical scavenger with that ofthe hydroxyl radical production induced by rhTNF indicatesthat the cytotoxic effect and the increased hydroxyl radicalproduction are approximately concurrent. The observed inhibition of rhTNF cytotoxicity by DMSO and bipyridine, furthermore, suggests that hydroxyl radical production is rather acause than a result of cell death.

The investigation with KYM and HEL cell lines also showeda correlation between rhTNF sensitivity and hydroxyl radicalproduction. In incubation of TNF-sensitive KYM cells, theaddition of rhTNF resulted in an increase in hydroxyl radical

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HYDROXYL RADICAL PRODUCTION BY TNF

production levels similar to that found for L-M cells. In theincubation of TNF-insensitive HEL cells under the same conditions, however, no observable increase in hydroxyl radicalproduction resulted from the addition of rhTNF.

The results of this study thus clearly indicate that rhTNFstimulates production of the hydroxyl radical by rhTNF-sensi-tive cells, and that this radical plays an important role in theoverall mechanism of rhTNF cytotoxicity. It is not yet possibleto provide a definitive description of the mechanism of thestimulated production or the precise role of the radical in theoverall mechanism of rhTNF cytotoxicity, but a partial explanation may be attempted in the light of the present study andrelated findings.

Various recent reports have shown that TNF cytotoxicity iseffectively suppressed by a mitochondrial inhibitor (11, 12) andby inhibitors of arachide nie acid metabolism (13, 14). Moreover, both the mitochondrial electron-transport chain (31) andthe arachidonic acid cascade (32) are known to generate thehydroxyl radical. It is therefore possible that the rhTNF-stim-ulated increase in hydroxyl radical production as observed inthe present study may be caused by activation of either or bothof these metabolic systems.

The hydroxyl radical, on the other hand, is known to inducelipid peroxidation (33) and to cause activation of lysosomalenzyme by its induction of lipid peroxidation in lysosomalmembrane (34), and also to directly effect DNA chain fragmentation (35). Lipid peroxidation (13), lysosomal enzyme activation (12), and DNA fragmentation (36) have all been reportedto occur in cells under stimulation by TNF. It would thusappear reasonable to suggest that the hydroxyl radical inducedby TNF could be the actual mediator of these intracellularreactions.

ACKNOWLEDGMENTS

We wish to thank Orville M. Stever for his help in preparation ofthis manuscript.

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1989;49:1671-1675. Cancer Res   Naofumi Yamauchi, Hiroshi Kuriyama, Naoki Watanabe, et al.  

in Vitroin the Killing of Tumor Cells Recombinant Human Tumor Necrosis Factor and Its Implication Intracellular Hydroxyl Radical Production Induced by

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