[CANCER RESEARCH 49, 4493-4498. August 15. 1989]

Modulation of Diamide Toxicity in Thermotolerant Cells by Inhibition ofProtein Synthesis1

Michael L. Freeman2 and Michael J. Meredith

Vanderbilt Center for Radiation Oncology, Vanderbiit University School of Medicine, Nashville, Tennessee 37232 [M. L. F.], and Department of Biochemistry, Schoolof Dentistry, Oregon Health Science University, Portland, Oregon 97201 [M. J. M.J

ABSTRACT

Chinese hamster ovary cells were exposed in vitro to various concentrations of diamide for l h at 37°C.This treatment resulted in a dosedependent increase in cytotoxicity. Cells were also heated at 43°Cfor 15min, incubated at 37°Cfor 3 h, and then exposed to various concentrations

of diamide. This heat shock has been shown previously to trigger thesynthesis of heat shock proteins and the development of thermotolerance.Further, under these experimental conditions both were inhibited ifprotein synthesis was inhibited by exposure to cycloheximide (M. L.Freeman et al., Radiât.Res., 112:195-203, 1987). Diamide toxicity wasdiminished in cells made thermotolerant by the 4.VT/15-inin heat shock.For example, at the highest dose used, 0.8 HIM,survival increased from0.93% to 6.1%. However, diamide toxicity was unaffected if the cellswere exposed to diamide 3 h after a 43°C/60min heat shock. This latter

heat shock produced significant inhibition of protein synthesis whereasthe 15-min heat shock did not (M. L. Freeman et al., Cancer Res., 48:7033-7037, 1988). Further, a 43°C/15-minheat shock did not confer

protection against diamide toxicity if the cells were simultaneously exposed to cycloheximide.

Exposure to 0.8 HIMdiamide was shown to oxidize specific cellularproteins as measured by 2-dimensional thiol blotting. However, thedegree of protein thiol modification was not affected by a prior heatshock. Nor did the heat shock increase the intracellular concentration ofglutathione or the activity of glutathione reducíase.The diamide treatment caused specific, as opposed to general, protein thiol oxidation andheat shock did not prevent this.

It is hypothesized that it was the oxidation of protein thiols which ledto cellular toxicity. Protein synthesis, triggered by heat shock, protectedcells from the diamide toxicity without preventing protein thiol modification. These results suggest that the proteins synthesized after heatshock can provide protection against the consequences of aberrant proteins produced by thiol oxidation.

INTRODUCTION

Thermotolerance denotes a reduction in the rate of thermalinactivation triggered by an initial heat shock (1). Once triggered it takes several hours to develop and decays slowly over24 to 72 h (2). There is some evidence that the synthesis ofHSP1 plays a role in the production of thermotolerance; how

ever, this point remains controversial in mammalian cells (3).In order to understand the molecular mechanisms responsible

for thermotolerance it is necessary to investigate the nature ofthe cellular damage responsible for thermal cytotoxicity. Basedon their initial work, from which they calculated the activationenergy, n, to be 140,800 cal/mol, Westra and Dewey (4) suggested that protein denaturation may be a primary cause of celldeath. This hypothesis has received support from the work of

Received 7/29/88; revised 2/15/89; accepted 5/17/89.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.

1This investigation was supported in part by USPHS/NIH Grants Ca 38079,

ES 03272, and ES 00267.2To whom requests for reprints should be addressed, at E-1200 MCN, Radia

tion Biology, Vanderbilt University Hospital, Nashville, TN 37232.3The abbreviations used are: HSP. heat shock protein; MPB, 3-(iV-maleimi-

dopropionyl)biocytin; CHO, Chinese hamster ovary; GSH, glutathione; PCA,perchloric acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electro-phoresis; DTNB, dithionitrobenzene; GSSG, oxidized glutathione.

Lepock et al. (5). They measured intrinsic protein fluorescenceand protein fluorophore to frans-paranaric acid energy transferin mitochondria! and plasma membranes and demonstrated theexistence of an irreversible transition in protein structure orarrangement above 40°C.Using reconstituted rabbit muscle

sarcoplasmic reticulum calcium ATPase, Cheng et al. (6) haveshown that inhibition of calcium uptake is due to thermaldenaturation of the protein. Cholesterol or glycerol could modify the temperature of denaturation by interacting with variousprotein domains. These transitions may also be affected by thelipids which surround membrane proteins inasmuch as Yatvin(7) and Guffy et al. (8) have provided evidence that alterationof membrane lipids will modify thermal sensitivity. Other workers have shown that heat shock causes nuclear protein tobecome insoluble and bind to the nuclear matrix (9, 10). In thisregard, it has been suggested that one of the functions of HSPsmay be to disaggregate denatured proteins (11, 12).

One approach to the problem of determining the nature ofthermotolerance and the role of HSPs is to produce very specificdamage and then determine whether thermotolerant cells express resistance to it. We show here that in CHO cells thetriggering of thermotolerance by a nontoxic heat shock confersprotection against the toxicity caused by diamide induced protein thiol oxidation. This protection is lost when protein synthesis is inhibited.

MATERIALS AND METHODS

CHO cells growing exponentially in monolayer cultures were maintained at 37°Cand at pH 7.4 in McCoy's Medium 5A supplemented

with 10% fetal bovine serum, sodium bicarbonate (2.2 g/liter), penicillinG sodium (100 units/ml), and streptomycin sulfate (100 mg/ml).

Experiments were performed using T25 flasks containing 4 ml offresh growth medium and at a cell density of approximately 4xl()''

cells/flask. Both the diamide and the cycloheximide were made freshimmediately prior to use. At the end of the experimental protocol thecells were rinsed twice with Dulbecco's phosphate saline.

Cell survival was determined after trypsinizing the cells and theninoculating the appropriate number of cells needed to yield 100-200colonies into T25 flasks. The error in survival was calculated accordingto the method of Boag (13).

If glutathione was to be measured then the cells were lysed in 1 mlof ice cold 10% PCA immediately after the saline washes. Glutathioneconcentrations were determined using high performance liquid chro-matography (14). Cells were lysed in 3 ml of 10% PCA for proteinthiol measurements. The precipitated protein was washed once in 10%PCA and twice in ice cold Dulbecco's saline. Protein thiols were

quantitated using the method described by Habeeb (IS) or by labelingwith MPB in order to separate protein by SDS-PAGE and quantitateby thiol blotting (16).

The method described by Bayer et al. (17) and modified as followswas used for 2-dimensional thiol blots. The washed protein was solu-bilized in 9.5% urea, 2% Nonidet P-40, ampholines (1.6% pH 5-8;0.4% pH 3-10), 1 mM phenylmethylsulfonyl fluoride, and 0.0367 MgMPB/Mg of protein. The sample was incubated at 37°Cfor 1 h.

Two-dimensional electrophoresis was performed using the techniqueof O'Farrell (18) as described by Pollard (19). Cellular protein was

loaded into prefocused gels and run at 400 V, constant voltage, for 18

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HEAT SHOCK AND PROTEIN OXIDATION

h at 20°C.The pH was measured from slices obtained from a replicate

gel. Proteins were separated by molecular weight using 1.5-mm 9.5%polyacrylamide slab gels with a 4% stacking gel. The slabs were electro-phoresed at 30 mA/gel, constant current at 20°C.After electrophoresis,

proteins were electrophoretically transferred to nitrocellulose (20). Thenitrocellulose was treated in 3% bovine serum albumin overnight,washed, incubated at room temperature for 45 min with avidin conjugated alkaline phosphatase (3 ¿ig/ml),washed, and stained with 5-bromo-4-chloro-3-indolyl phosphate (/Moluidine salt) and nitro bluetetrazolium.

Protein synthesis was measured by labeling the cells (4 x 106/flask)in 3 ml of leucine free medium containing 25 ^Ci/ini of [3H]leucine

(specific activity, approximately 144 Ci/mol; New England Nuclear).After the labeling period, the cells were rinsed twice with 0.15 M NaCl-10 imi morpholinosulfonic acid (pH 7.4), scraped into 1 ml of thesame solution, and centrifugea at 65 x g. A replicate treated similarlywas used for protein determination. Cells were then dissolved in SOS -PAGE sample buffer [62 HIMTris-HCl (pH 6.8), 2% SDS, 10% glycerol,5% 2-mercaptoethanol, and 0.0025% bromophenol blue] and heated at95°Cfor 5 min. Gel lanes were loaded with approximately equal

amounts of protein. Proteins were electrophoretically separated using0.75-mm-thick 9.5% polyacrylamide gel slabs with a 4% acrylamidestacking gel in the discontinuous buffer system of Laemmli (21). Proteinmolecular weight was determined by comparison to molecular weightstandards (myosin, M, 205,000; /i-galactosidase, M, 116,000; phospho-rylase b, M, 97,400; bovine serum albumin, M, 66,000; egg albumin,M, 45,000; and carbonic anhydrase, M, 29,000). After electrophoresis,the gels were fixed (50% methanol-10% acetic acid), stained with

Coomassie Brilliant Blue R250, destained, treated for fluorographyusing Kn 'Hanee (New England Nuclear) dried, and exposed to KodakSB-5 X-ray film at -70"C.

Hyperthermic treatment was performed by immersion into a waterbath in which the temperature was controlled ±0.05°C.Temperature

was monitored with a mercury in glass thermometer traceable toNational Bureau of Standards and was ±0.05°C.

Irradiation was carried out using a '37Cs irradiator and a dose rate

of 4.29 Gy/min.Glutathione reducíasewas measured in 4 x 10'' cells which were

lysed by freezing and thawing 3 times. The activity was measured in 1ml of a buffer containing 3 HIMmorpholinosulfonic acid (pH 7.2), 12HIMMgCh, 54 mM KC1, 28 mM NaCl, 4.5 mM EDTA, 1.3 mM GSSG,and 0.1 mM NADPH. The decrease in absorbance at 340 nm wasmeasured at 22°C.

Experiments were repeated a minimum of two times.

RESULTS

Previous work from this laboratory (22) has shown thatprotein synthesis was not affected by a 15-min/43°Cheat shockas assessed by incorporation of [35S]methionine into eithertrichloroacetic acid precipitable proteins or SDS-PAGE fluo-rographs quantitated by scanning densitometry. The pattern ofprotein synthesis observed during and up to 6 h after the 15-min heat shock was indistinguishable from controls with theexception of enhanced rates of synthesis for proteins withapproximate molecular weights of 110,000, 90,000, 70,000,60,000, and 28,000 (HSP). A 60-min/43°C heat shock, on the

other hand, inhibited protein synthesis by 74%. Protein synthesis still had not returned to control levels 6 h after the heatshock. Enhanced synthesis of proteins with approximate molecular weights of 90,000 and 70,000 was only observed 4 h ormore after the 60-min heat shock.

The fluorograph shown in Fig. 1 shows the pattern of proteinsynthesis obtained 3 h after a 15- or 60-min 43°Cheat shock

performed under the present experimental conditions. The resulting fluorograph obtained after ['Hjleucine incorporation

illustrates that 3 h after the 15-min heat shock protein synthesiswas indistinguishable from control with exception of the ele-

Co

Fig. 1. Fluorograph of cellular protein obtained from cells heat shocked foreither 15 (Lane 2) or 60 (Lane I) min. After the heat shock the cells wereincubated for 3 h at 37"C and then labeled with [3H]leucine for 1 h. Lane C0,

protein from unheated cells, left ordinate, molecular weight marker positions;right ordinate, location of the major HSPs.

vated synthesis of the M, 110,000, 90,000, 70,000, and 28,000HSPs. However, very little incorporation was observed 3 h afterthe 60-min heat shock (Fig. 1, Lane 1). These results agree withthose discussed above. Further, we have shown that thermo-tolérancewas fully developed 3 h after the 15-min heat shock(22, 23). Both the synthesis of HSP and the development ofthermotolerance were inhibited by exposure to 20 /¿g/mlofcycloheximide which reduced protein synthesis by 93%.

After a 43°C/15-min heat treatment followed by a 3-h incubation at 37°C,CHO cells were protected against diamide

toxicity (Fig. 2). This occurred concomitant with the development of thermotolerance (22) and the synthesis of HSP (Fig.1) (22, 23). The cells were exposed to various concentrations ofdiamide 3 h after the 43°C/15-min heat shock (compare open

triangles to open circles). At all concentrations tested the heatshock resulted in a statistically significant increase in survivalas determined by a paired Student's i test (P < 0.05). For

example, a concentration of 0.4 mM diamide reduced survivalto 0.51 ±0.04. Three h after the heat shock this concentrationof diamide reduced survival to only 0.78 ±0.05. A concentration of 0.8 mM diamide reduced survival to 0.0093 ±0.0012.If the cells were treated with the heat shock prior to the diamidetreatment then survival was 0.061 ±0.004. A protection factorof 1.32 was obtained when the concentrations necessary toreduce survival to 10% were compared for heat shocked andnon-heat shocked cells. These results, combined with thosepresented in Ref. 22, indicate that inhibition and recovery ofprotein synthesis were not necessary for protection to occur;i.e., the 43°C/15-min heat shock did not result in protein

synthesis inhibition (22). Rather, this heat shock triggered HSPsynthesis.

These results are in direct contrast to those obtained whenthe cells were exposed to a 60-min/43°C heat shock 3 h prior

to the diamide treatments. Survival for this combined protocolis illustrated by the open diamonds. The 60-min heat shockkilled 85 ±10% of the cells when administered by itself. The15-min heat shock did not produce any cell killing. When

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HEAT SHOCK AND PROTEIN OXIDATION

IOV

10"

io-2

43V 15min -diamine

43V60min,.— diamide

diamide alone

43°/60min—-diamide

0.2 0.4 0.6 0.8mM diamide

1.0

Fig. 2. Diamide toxicity in CHO cells. Cells were incubated at 43'C for 0 (O),

15 (A), or 60 (O) min, 3 h prior to being exposed to the indicated diamideconcentrations. Survival after 15 min at 43'C was 1.00 ±0.04 while after a 60-

min heat shock it was 0.15 ±0.015. When survival obtained after the combined60 min-diamide treatment was corrected for the heat alone toxicity, the curvedesignated by * resulted. Error bars are shown when larger than the diameter ofthe symbol.

survival obtained after the combined diamide-60-min heatshock is corrected for the heat alone toxicity then the curveshown in Fig. 2 (closed diamonds) results. This curve illustratesthat in the absence of appreciable levels of protein synthesis aheat shock did not protect cells from diamide toxicity andinhibition of protein synthesis did not increase cell sensitivityto the diamide treatment.

Cycloheximide inhibition of protein synthesis inhibited heatshock induced protection against diamide toxicity. As diagramed in Fig. 3, CHO cells were subjected to one of sevenprotocols. Fig. 4 represents the average of 3 experiments conducted as described in Fig. 3. Protocols A, B, and C producedsurvival levels of 1.09 ±0.04, 1.00 ±0.04, and 1.00 ±0.05,respectively, compared to untreated controls. The heat shockprotected cells against either a 0.4 or 0.8 HIMdiamide treatment(P < 0.05) as determined by Student's / test (compare Curve D

to Curve F). For example, exposure to 0.4 mividiamide reducedsurvival to 0.59 ±0.02. Survival after protocol F was 0.79 ±0.02. The protection factor determined at a level of 10% survivalwas 1.30. The protection afforded by the heat shock was inhibited if the cells were treated with cycloheximide which inhibitedprotein synthesis by 93% and therefore by extension HSPsynthesis (see Ref. 23, Fig. 1). This is illustrated in Fig. 4 byCurves G and E. At a survival level of 10% the protection factorwas 1.10. Previous work has shown that cycloheximide did notinhibit the development of thermotolerance by 100% (23). Asimilar result was observed here. Heat shock protection was notcompletely inhibited by the addition of cycloheximide if thecells were exposed to 0.8 HIMdiamide. However, at a concentration of 0.4 mivi survival was 0.63 ±0.2 and 0.58 ±0.02 (P^ 0.05) for protocols E and G, respectively. Taken together theresults shown in Figs. 2 and 4 show that protein synthesisbefore, during, and after heat shock is necessary for protection

—30--H5-1

1L1

1 II"kL1

kHS-i'Ur

(r

!• 1ii-i i"-H5—|U

__—•

180 :*

*—

r wY-60-,diomidc-min

ProtocolrK

Fig. 3. Diagram of experimental protocols. In protocol A, cells were exposedto 20 jig/ml of cycloheximide (CHX) for 285 min at 37'C. In protocol B. theywere incubated at 43'C for 15 min. In protocol C, they were exposed to cycloheximide for 30 min at 37'C, for 15 min at 43'C, and for 240 min at 37'C after the

heat shock. In protocol D, they were exposed to 0.1, 0.4, or 0.8 min diamide forl h at 37°C.In protocol E, they were exposed to cycloheximide for 225 min priorto and during the diamide treatment. In protocol F, they were incubated at 43'Cfor 15 min and at 37°Cfor 3 h and then exposed to diamide. Finally in protocolG, they were exposed to cycloheximide for 30 min at 37°C,15 min at 43°C.and3 h at 37'C and during the 1-h diamide treatment. Cells were also subjected to

an 8th protocol which is not shown, that of untreated controls a, 20 ¿ig/mlcycloheximide; b, heat shock (HS) at 43°Cfor 15 min.

IO.00.4 0.6 0.8

mM diamide

Fig. 4. Heat shock protection against diamide toxicity. Cells were treated asdescribed in Fig. 3. Error bars are shown if they exceed the diameter of thesymbol. Protocol D (O); Protocol F (A); Protocol E (•);Protocol G (A).

against diamide toxicity produced by thiol oxidation.Diamide toxicity was enhanced if the cells were additionally

exposed to cycloheximide (Fig. 4). Because diamide toxicity isdirectly proportional to GSH levels (data not shown) the GSHlevels in cells treated with cycloheximide were measured. GSHlevels fell by 26% after a 4-h incubation in 20 Mg/ml of cycloheximide. Similar results were obtained with 100 Mg/ml ofpuromycin (data not shown).

To determine whether heat shock could protect CHO cellsfrom the oxidative damage produced by ionizing radiation, cellswere incubated at 43°Cfor 15 min, incubated at 37°Cfor 3 h,

and then irradiated (Fig. 5). These results demonstrate that4495

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HEAT SHOCK AND PROTEIN OXIDATION

10°

10'

10-

43V 15min

10

Gy

Fig. 5. Dose-response curve for "y-irradiation. Cells were incubated at 43" foreither 0 (O) or IS (A) min 3 h prior to -y-irradiation. Error bars are shown iflarger than the diameter of the symbols.

Table 1 Modification of protein sulfliydryls by heat shock diamide"

ProtocolABCDEFGCommentUntreatedCHX*

alone43°C/15min43°C/15min

+CHXDiamide

aloneDiamide+CHX43-C/15min

+diamide43"C/15min

+diamide

+CHXnmol

sulfhydryl/mg pro

tein1111191299287497560a101122314985

" Determined by the method of Habeeb (15). There were 6 flasks/point and 1

of 2 experiments.* CHX, cycloheximide.

thermotolerance and the synthesis of HSP did not protect cellsfrom the DNA damage caused by 7-irradiation. These resultsagree with earlier work (24).

The 43°C/15-min heat shock did not confer protection

against diamide induced inhibition of protein synthesis. Proteinsynthesis was inhibited by 99.2% in cells exposed to 0.8 mMdiamide for l h at 37°C,as determined by ['H]leucine incor

porated into trichloroacetic acid precipitable protein. If the cellswere given a 43°C/15-min heat shock 3 h prior to the diamide

treatment, inhibition was still 99.2% of control values. Theheat shock by itself did not affect incorporation (data notshown). These data indicate that not all types of damage causedby diamide exposure are protected by a prior heat shock.

In order to identify which, if any, proteins were modified bythe diamide treatment, proteins isolated from CHO cells afterexposure to 0.8 mM diamide were analyzed by thiol blotting orby reaction with DTNB. CHO cells contained approximately111 nmol of sulfhydryl/mg of protein (Table 1). Neither cycloheximide or heat shock or the combination affected proteinsulfhydryls (Table 1, Lines A, B, and C; Fig. 6, Lanes A, B, andC). After a 1-h exposure to 0.8 mM diamide, 87 ±14 nmol of

2 o s—

1 16

97.4

29—

I

A D E F G B C

Fig. 6. Thiol blot of cells exposed to the protocols described in Fig. 3. Leftordinate, molecular weight marker positions. Lane C0, protein from control cells.

reduced sulfhydryls/mg of protein were recovered as measuredby the DTNB assay. This represents a 22% difference relativeto control. However, this was not a statistically significantchange (compare Lanes C0 and D in Fig. 6). Thiol blots werealso obtained from cells exposed to 0.8 HIMdiamide for 5, 10,15, 30, and 60 min. MPB labeling in the blots was the same ascontrol (data not shown). The same degree of protein thioloxidation occurred when the cells were given a 43°Cheat shock

3 h prior to the diamide treatment (protocol F versus D; 75versus 87, P > 0.05; Table 1 and Fig. 6, Lanes F and D.) Asshown in Fig. 4, diamide sensitivity was enhanced by exposureto cycloheximide. This was reflected in the data obtained fromthe sulfhydryl assay. Protocol E of Table 1 resulted in recoveryof 49 ±9 nmol of sulfhydryl/mg protein. This represents achange of 56% and is statistically significant (P < 0.05). Asimilar result was found in Fig. 6. These observations were notdue to changes in the amount of protein recovered which stayedconstant at about 0.4 mg.

The data provided in Table 1 suggest that diamide exposureresulted in a small degree of thiol oxidation. To determinewhether diamide exposure produced specific oxidation ratherthan nonspecific or general protein oxidation, proteins weresubjected to 2-dimensional thiol blotting. General or nonspe

cific oxidation would result if the rate of protein oxidation wasthe same for all proteins. Specific oxidation would be the resultof different proteins having different rates of sulfhydryl oxidation. Fig. 1A represents protein thiols from untreated controlcells. Fig. IB represents protein thiols from cells exposed toprotocol D. A comparison of the thiol blots reveals that certainspecific proteins are modified by the diamide treatment. Theseare identified as proteins 1-5. Other proteins are also modifiedbut they are not as prominent. The important point is that thioloxidation or thiolation resulting from the diamide exposureappears to oxidize certain proteins to a greater extent thanothers.

The data obtained using DTNB as a probe for thiol modification revealed that heat shock prior to the diamide treatmentdid not significantly increase protein thiol modification. Thesame qualitative results were obtained when the correspondingthiol blots were examined. Fig. 1C represents protein thiolsfrom cells given the 43°Cheat shock prior to the diamide

treatment (protocol F). In summary, these results show thatheat shock protected cells from the toxic effects of diamide but

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HEAT SHOCK AND PROTEIN OXIDATION

DS

r~

r---u T * -

234 5

Fig. 7. Thiol blot of untreated cells (/)), diamidetreated cells (/?), and cells given a heat shock priorto the diamide exposure (C).

2 3

not by preventing thiol modification as assessed using thistechnique of measurement.

The protection triggered by the heat shock treatment was notdue to an increase in either intracellular GSH or an increasedactivity of glutathione reducíase. The activity of glutathionereducíasein untreated CHO cells was 2.3 ±0.2 milliunits/106cells. Three h after a 15-min 43°Cheat shock the measuredactivity was 1.9 ±0.2 milliunits/106 cells. Similarly, untreated

cells contained 55 nmol of GSH/mg protein (Table 2). Immediately after treatment with 0.8 HIMdiamide the cells contained18 nmol/mg (protocol D). This level of depletion (64%) byitself does not cause toxicity in CHO cells (25). Heat shockalone or administered 3 h prior to the diamide treatment didnot affect GSH levels.

DISCUSSION

The object of this investigation was to produce a specific typeof cellular damage and then determine whether resistance wouldbe observed as cells developed thermotolerance and synthesizedHSP. This was accomplished by incubating CHO cells at 43°C

for 15 min and then exposing the cells to diamide 3 h later.Three h after this heat shock thermotolerance has been shownto be fully developed and dependent on protein synthesis (22,23). This heat shock also triggered the development of diamideresistance. Resistance occurred to only a specific type of damage. Resistance was not observed to diamide inhibition of

Table 2 GSH levels in CHO cells

ProtocolDBFCommentsUntreatedDiamide

alone43"C/15min43°C/15

min + diamidenmolGSH/mgprotein55184320a1671

protein synthesis or DNA damage produced by 7-irradiation.The data presented in this current investigation have shown

that diamide produces thiol oxidation of specific proteins asmeasured by 2-dimensional thiol blot analysis. These observations agree with those obtained by Grimm et al. (26) wholooked at protein mixed disunities in rat cardiac cells. Theyfound that addition of 0.5 HIMdiamide to the cultured rat cellsproduced S'-thiolation of at least 21 proteins. However, not all

proteins were S-thiolated equally and some proteins were notmodified at all. Diamide reacts with acidic, small molecularweight thiols such as glutathione. In general, protein thiolswould not be expected to be directly oxidized because they aregenerally less acidic and sterically less accessible. This does notpreclude specific proteins from reacting directly with diamide.Most protein oxidation, however, will be due to the formationof mixed disulfides. Kosower et al. (27) have carefully calculatedthe rate constants for reaction of diamide with compoundsother than glutathione (e.g., NADH, NADPH, or lipoic acid).They found that the fastest reaction occurred with glutathione;it was approximately 87 times faster than that which occurredwith NADH or NADPH. Only after glutathione depletion hasoccurred would diamide be expected to react with other cellularcompounds. The data shown in Table 2 of the present studyindicate that the diamide exposure depleted GSH levels by only64%. GSH levels were also assayed 5, 10, 15, or 30 min intothe diamide exposure interval and depletion never exceeded64% (data not shown). Thus, diamide would be expected toreact only with GSH to form GSSG and possibly certain specificprotein thiols. Formation of GSSG could result in increasedprotein thiol oxidation via glutathione protein mixed disulfides.In this regard, Kosower and Kosower (28) have shown that inhuman RBC formation of intra- and interchain disulfide bondsin proteins is directly related to the rate of oxidation of GSHto GSSG. One further consideration is that diamide exposurewill result indirectly in the reversible oxidation of NADPH via

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HEAT SHOCK AND PROTEIN OXIDATION

glutathione reducíase,potentially altering the pyridine nucleo-tide redox position. However, the major consequence of diam-ide treatment must be considered to be an increase in theformation of GSSG.

The mechanism responsible for the diamide toxicity is notknown. Toxicity is directly related to redox potential in CHOcells. For example, when cells were exposed to 0.1 HIMdiamidefor l h at 37°Cin Hanks' balanced salt solution in the absence

of glucose, survival was 0.02 ±0.005. Addition of 2 HIMglutamine or 10 mivi glucose, as a source for NADPH regeneration, increased survival to 0.3 ±0.02 and 1.00 ±0.03, respectively (data not shown). It is reasonable to speculate that toxicityis a consequence of thiol oxidation but other possibilities cannotbe excluded. The data shown in Fig. 7 and Table 1 show thatexposure to diamide produced specific thiol oxidation. Exposure to 0.8 HIMdiamide killed more than 90% of the cells yetoxidized only 22% of the proteins. The most prominent oxidized proteins are identified in Fig. 7. It would be expected thatoxidation of only some of these proteins results in disruptionof the tertiary structure. It is the distortion of protein structurethat would allow these proteins to be candidates for the repairby HSP synthesis in accordance with the model of HSP function(12). Thus it may be expected that thermotolerance wouldprotect only a small number of proteins.

Previous work has shown that thiol oxidation upon exposureto diamide triggers both the development of thermotoleranceand the synthesis of HSP in CHO cells (29, 30). Diamide isnot the only oxidant to do this. Exposure to H2O2 also triggersHSP and tolerance in Salmonella typhimurium and CHO cells(31, 32).

When CHO cells were triggered to develop thermotoleranceand synthesize HSP by administration of a nontoxic heat shock,they also developed resistance to diamide. The development ofdiamide resistance was inhibited if the cells were treated withcycloheximide or a heat shock which inhibited protein synthesis. Cycloheximide has been shown to inhibit the developmentof thermotolerance in CHO cells. Further, resistance could notbe attributed to an increase in the activity of glutathione reducíaseor to an increase in the glutathione concentration. Thus,cells exhibited resistance to diamide toxicity when they developed thermotolerance and synthesized HSP, and resistancefailed to develop when tolerance and protein synthesis wereinhibited. These results suggest that it is the development ofthermotolerance and, by extension, the synthesis of HSP whichconfers resistance to the toxicity caused by oxidation of specificprotein thiols.

ACKNOWLEDGMENTS

It is a pleasure to thank Sarah Coode and De'Rita Darden-Small-

wood for their expert technical assistance.

REFERENCES

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4. Westra, A., and Dewey, W. C. Variation in sensitivity to heat shock during

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6. Cheng, K. H., Hui, S. W., and Lepock, J. C. Protection of the membranecalcium adenosine triphosphatase by cholesterol from thermal inactivation.Cancer Res. 47: 1255-1262, 1987.

7. Yatvin, M. B. The influence of membrane lipid composition and procaine inhyperthermic death of cells. Int. J. Radiât.Biol. 32: 513-521, 1977.

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