Correlation between redistribution of a 26 kDa protein and development of chronic thermotolerance in...

transcript

JOURNAL OF CELLULAR PHYSIOLOGY 1453324332 (1990)

Correlation Between Redistribution of a 26 kDa Protein and Development of Chronic

Thermotolerance in Various Mammalian Cell Lines

YONG J. LEE,* ZI-ZHENG HOU, LlNDALl CURETTY, MICHAEL J. BORRELLI, AND PETER M. CORRY

Department of Radiation Oncology, Research Laboratories, William Beaumont Hospital, Royal Oak, Michigan 48072

Previous studies suggested that a 26 kDa protein might play an important role in protein synthesis-independent thermotolerance development in CHO cells. To determine if this phenomenon was universal, four mammalian cell lines, viz., CHO, HA-1, murine Swiss 3T3, and human HeLa, were studied. Cells were heated at 42"C, and the level of 26 kDa protein in the nucleus was measured, together with clonogenic survival and protein synthesis. The results demonstrated that 1 ) the 26 kDa protein was present in the four different cell lines, and 2) the level of the 26 kDa protein in their nuclei was decreased by 30-70% after heating at 42°C for 1 hr. However, restoration of this protein occurred along with development of chronic thermotolerance. The protein synthesis inhibitor cycloheximide (10 pglml) neither inhibited the development of chronic thermotolerance nor affected the restoration of the 26 kDa protein in the nucleus. In fact, this drug protected cells from hypertherrnic killing and heat-induced reduction of 26 kDa protein in the nucleus. Heat sensitizers, quercetin (0.1 mM), 3,3'-dipentyloxacarbocyanine iodide (DiOC,[3]: 5 kg/ml), and stepdown heating (45°C-10 min -42"C), potentiated hyperthermic killing and inhibited or delayed the restoration of the 26 kDa protein to the nucleus. These results support a correlated, perhaps causal relationship between the restoration of the 26 kDa protein and chronic thermotolerance development in four different mammalian cell lines.

Hyperthermia exposures above 41°C have been used, intermittently, in cancer therapy for several decades (Meyer, 1984; Stewart and Gibbs, 1984). Due to tech- nical and biological advances during the past decade, interest in hyperthermia has expanded dramatically. However, several problems still remain to be solved to optimize the efficacy of hyperthermia in cancer therapy. Recent clinical studies demonstrated that heating nonsuperficial tumors above 42°C is technically diffi- cult and commonly leads to pain toxicity (Kapp et al., 1988; Sapozink et al., 1988; Shimm et al., 1988; Corry et al., 1988). These factors have directed researcher's attention to low temperature heating (~42°C). How- ever, it is well known that thermotolerance develops during prolonged, continuous exposures a t tempera- tures below 42.5"C (Urano, 1986). Therefore, the mechanism of chronic thermotolerance development should be clarified in order to elucidate and optimize better strategies for cancer therapy.

There appear to be two distinct types of thermotolerance (Lee and Dewey, 1987a,b, 1988; Laszlo, 1988). One type (Type I) is strongly dependent upon protein synthesis, especially that of the heat shock proteins (HSPs) (Yamamori and Yura, 1982; McAlister and Finkelstein, 1980; Hallberg et al., 1985; Li and Werb, 0 1990 WILEY-LISS, INC.

1982); the other type (Ty e 11) develops in the absence

1984; Watson et al., 1984; Widelitz et al., 1984, 1986; Lee and Dewey 1987c, 1988).

There is considerable evidence that Type I thermotolerance development is accompanied by preferential synthesis of HSPs (Subjeck et al., 1982; Landry et al., 1982; Li and Werb, 1982). As thermotolerance decays, the HSP levels, in particular those of HSP 70, return to control values (Landry et al., 1982; Li and Mak, 1985; Li, 1985). This observation is also supported by Lee and Dewey (1987a,b), who showed that thermotolerance induced by treatment with chemical agents, e.g., sodium arsenite, was suppressed by exposure to cycloheximide or puromycin. Thus, such chemical-induced thermotolerance appears to be Type I only.

Type I1 thermotolerance is independent of protein synthesis, particularly HSPs synthesis (Hall, 1983; Craig and Jacobsen, 1984; Watson et al., 1984; Widelitz et al., 1984,1986; Lee and Dewey, 1987c, 1988). Protein

of protein synthesis (Ha P 1, 1983; Craig and Jacobsen,

Received May 9, 1990; accepted August 3, 1990. "To whom reprint requests/correspondence should be addressed.

26 kDa PROTEIN IN VARIOUS CELL LINES 325

synthesis-inde endent thermotolerance may involve

Dewey, 1987b, 1988; Laszlo, 1988). Heat shock appears to induce both Type I and Type I1 thermotolerance.

Recently Lee et al. (1989) reported that heat prefer- entially reduced the amount of a 26 kDa protein in the nucleus. The reduction and subsequent restoration of the 26 kDa protein, after heating, correlated well with cell killing by hyperthermia. The goal of this study was to investigate further the correlation between the redistribution of this 26 kDa protein and chronic thermotolerance development. Four different mammalian cell lines (CHO, HA-1, 3T3, HeLa) were used to estab- lish that the 26 kDa protein redistribution was not limited to CHO cells. A heat protector (cycloheximide) (Lee and Dewey, 1986) or heat sensitizers, quercetin (Kim et al., 1984a), 3,3’-dipentyloxacarbocyanine iodide (DiOCJ31) (Borrelli et al., in press), or stepdown heating (Henle, 1980) were used to determine their effects in modulating redistribution of this protein and development of chronic thermotolerance.

the intracellu P ar redistribution of proteins (Lee and

MATERIALS AND METHODS Cell culture and survival determination

Ex onentially growing Chinese hamster ovary (CH&, Ch inese hamster fibroblasts (HA-11, murine Swiss 3T3, and human HeLa cells were cultured in McCoy’s 5a medium (Cellgro), Eagle’s minimal essential medium (Sigma), Dulbecco’s modification of Ea- gle’s medium (Cellgro), and Eagle’s minimal essential medium (Sigma), respectively. The media were supplemented with 26 mM sodium bicarbonate and 10% iron-supplemented calf serum (HyClone). T-75 flasks containin cells were kept in a 37°C humidified incu-

survival determination, cells were trypsinized, counted, and plated at appropriate dilutions. X-irradi- ated feeder CHO cells (25 Gy) were used to maintain the plated cell density a t 4,000 cells/cm2 (Highfield et al., 1984; Borrelli et al., 1989). After 1-2 weeks of incubation at 37”C, colonies were stained and counted.

Drug treatment Medium with cycloheximide (CHM; M.W. 281.3,

Sigma Chemical Co.), quercetin (M.W. 214.1, Sigma Chemical Co.), or 3,3’-dipentyloxacarbocyanine iodide (DiOC5[3]; M.W. 544, Molecular Probes, Inc.) was pre- pared 1 day prior to the experiment. Drug treatment was accomplished by aspirating the medium from the cells and replacing it with drug-containing medium. For CHM (10 pglml) treatment, cells were exposed to the drug for 2 hr before and during heating, or only during heating. For quercetin (21.4 pgiml) or DiOC5(3) (5 p Iml) treatment, cells were exposed to the drug for 1 hr % efore and during heating. To obtain the maximum effect, various preincubation times were tested previ- ousl for each drug (Lee and Dewey, 1986; Borrelli et a[, in press). Drug treatments were terminated by aspirating the medium containing drug, rinsing twice with Hanks’ balanced salt solution (HBSS), and re- placement with drug-free medium.

bator wit a a mixture of 95% air and 5% C 0 2 . For

Hyperthermic treatment T-75 flasks were heated by total immersion in a

circulating water bath (Heto) maintained within * 0.02”C of the desired temperature. For stepdown heat treatment, cells were first heated at 45°C for 10 min and then immediately challenged at 42°C.

Nuclear isolation The nuclear isolation procedure of Higashikubo et al.

(1989) was adapted for these experiments. Heated and/or drug-treated, or control monolayer cells were trypsinized and pelleted by centrifugation at 4°C for 5 min at 9Og. Cell pellets were resuspended with spinner salt solution (5.36 mM KC1,0.83 mM MgS04, 116 mM NaC1, 26.2 mM NaHC03, 11.7 mM NaH2P04, 5 mM D-glucose, pH 7.0) and repelleted by centrifugation for 5 min at 300g. The spinner salt washes were repeated twice. Cells were resuspended in cold nuclear isolation buffer (50 mM NaC1,lO mM EDTA, 0.5% Nonidet P-40 in 50 mM Tris-base:Tris-HC1, pH 7.4) and set on ice for 4-7 min. Nuclei were pelleted by centrifugation for 10 min at l,OOOg, the supernatant was removed, and nuclei were resus ended in bicarbonate buffer (0.03 M NaC1, 0.03 M NafiCO,, 0.1 mM PMSF, pH 8.1). Bicar- bonate buffer washes were repeated twice by centrifugation.

Gel electrophoresis For one-dimensional polyacrylamide gel electro-

phoresis (PAGE), isolated nuclei were solubilized with lysis buffer (2.4 M glycerol, 0.14 M Tris, pH 6.8,0.21 M sodium dodecyl sulphate (6% SDS), 1.28 M mercaptoethanol, 0.3 mM bromphenol blue). Samples (3.4 x lo7 nucleiiml) were ipetted vigorously with a Pasteur

at - 70°C prior to electrophoresis. Samples with equal number of nuclei (6.8 X lo5) were analyzed on 7.520% linear gradient SDS polyacrylamide gels (Walker, 1984). After electrophoresis, gels were fixed in 30% trichloracetic acid (TCA) for 30 min, stained with Coomassie Brilliant Blue R in 3.5% perchloric acid overnight, destained 2~ in 7% acetic acid, and rinsed with water. Wet els were scanned with a computerized

Dynamics, Sunnyvale, CA). Quantitative measure- ment of each rotein band in the stained gels was performed wit R this instrument. Four parameters (smoothing, slope sensitivity, upward count, downward count) of the instrument were adjusted for analyses of gels.

For two-dimensional PAGE, isolated nuclei were treated with staphylococcal nuclease (50 pg/ml final concentration, Sigma Chemical Co.) and solubilized in 0.3% SDS and 1% P-mercaptoethanol, followed by a brief treatment with pancreatic DNase I (33 unitsiml final concentration, Promega Corp.) and RNase-A (50 pg/ml final concentration, Sigma Chemical Co.). The nuclear sample was lyophilized and then dissolved in sample buffer containing 8 M urea, 1.7% NP-40, and 4.3% P-mercaptoethanol. Proteins were first separated in isoelectric focusing gels (pH 3.5-10.5). These gels are then laid across the top of an SDS gradient slab gel, and the proteins were analyzed. After electrophoresis, gels

pipette and boile r! for 15 min. The samples were stored

laser scanning P ensitometer (Model 300A, Molecular

326 LEE ET AL.

were fixed and stained with the color-based silver stain. The procedure for autoradiography is described below.

Labeling of proteins, autoradiography, and fluorography

To study the restoration of extant proteins, cells were labeled continuously with 0.6 FCilml [l4C1 leucine (sp. act. 50 mCi/mmol, ICN) for 31 hr. After labelin , cells

in complete medium at 37°C before exposure to heat. After heating, nuclei were isolated, and lysed with sample buffer, as described above. For each sample, a volume of lysate giving an equal number of TCA- insoluble cpm (22,000 cpm) was applied to a gradient polyacrylamide gel. For autoradiography, destained

were rinsed 2 X with HBSS, and then incubated B or 1 hr

gels were dried in a slab gel dryer (Model 483, Bio-Rad, Richmond, CA) for 1.2 hr a t 80°C. The gels were autoradiographed on Kodak SB-5 X-ray film. After an exposure of 5 days, autoradiographic film was devel- o ed with Kodak GBX developer and fixed with Kodak ZBX fixer.

RESULTS Effect of heat protector or heat sensitizers on

cell survival at 42°C Figure 1 shows the effect of cycloheximide (CHM),

quercetin, 3,3'-dipentyloxacarbocyanine iodide (DiOC5[31), or stepdown heating on cell survival following the 42°C exposure. None of the drug treatments showed signif-

> 0 2 4 6 0 100 2 4 6 8 10

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0 2 4 6 0 100 2 4 6 8 10

HOURS AT 4 2 ° C Fig. 1. The effect of cycloheximide (CHM, 10 pgiml), quercetin (21.4 pg/ml), DiOC,(3) (5 pg/ml), or stepdown heating on CHO, HA-1, 3T3, or HeLa cell survival at 42°C. Cycloheximide was added 2 hr before and left on during heating ( A ) or only during heating (v). Quercetin (0) or DiOC,(3) ( 0 ) was added 1 h! before and left on during heating. For stepdown heating cells were first heated at 45°C for 10 min and

then immediately challenged at 42°C (0). Cell survival was normal- ized for killing from the first heating (survival = 35-82%). t = Survival curve of control cells heated at 42°C. Error bars repre- sent one standard deviation of the data for each point. Error bars for high survival plots that are close together have been omitted for clarity.

Fig. 2. Coomassie blue-stained gradient SDS-polyacrylamide gel electrophoresis of nucleoproteins from CHO, HA-l,3T3, HeLa cells. A Nucleoproteins from unheated control cells. B Nucleoproteins from cells heated at 42°C for 4 hr. C: Nucleoproteins from cells heated at 45°C for 10 min. Molecular weight standards ( X are shown at the left. The location of 26 kDa protein is shown at the right.

icant cytotoxicity within 10 hr exposures for all four cell lines (data not shown).

All four mammalian cell lines developed thermotolerance, which was inferred from biphasic survival curves during chronic heating at 42°C (Fig. 1). Ther- motolerance development was modified by treatment with heat protectors or sensitizers. CHM treatment before and during heating reduced hyperthermic killing, and resulted in the early expression of thermotolerance. Figure 1 also shows that 2 hr of CHM pretreatment was not prerequisite to induce a protective effect when cells were heated at 42°C.

Quercetin increased heat killing in all cell lines. However, this drug did not inhibit chronic heat-induced thermotolerance. For instance, 5-20% of the cell population still developed thermotolerance.

DiC0,(3) or stepdown heating potentiated heat killing without a biphasic plateau. Interestingly, the effect of these agents was not identical in all cell lines. For exam le ste down heatin was a more effective sensi-

more effective in HA-1 and HeLa cells. tizer kr k H 8 and 3T3 cel k s. In contrast, DiC0,(3) was

Ubiquity of the 26 kDa protein To investigate the correlation between 26 kDa pro-

tein redistribution and chronic thermotolerance development in four different mammalian cell lines, we first examined whether this rotein was present ubiqui- tously. Figure 2 shows E oomassie blue-stained, 7.5- 20% linear gradient SDS-polyacrylamide gel electrophoresis of roteins from isolated nuclei. We observed

Nevertheless, the nuclei from each cell line contained the 26 kDa protein (lanes A in Fig. 2). Figure 2 also shows that the level of 26 kDa was reduced when cells were heated for 10 min at 45°C (lanes C). However, little or no reduction of this protein was observed following 42°C for 4 hr (lanes B). These phenomena were observed in all cell lines.

somewhat C f ifferent protein profiles in each cell line.

Reduction and restoration of 26 kDa protein during heating at 42°C

Figure 3 shows reduction and restoration of the 26 kDa protein during chronic heating at 42°C in CHO

328 LEE ET AL.

3 7. 42'-1H

0.5 I > I-

Q 0.5 0 I- LL 0

W - 0 - - - I-

Q, W cc 0 .5

0 4 3 2 6 4 3 2 6

M O L E C U L A R W E I G H T Fig. 3. Densitometer tracings from Coomassie blue-stained gradient SDS-polyacrylamide slab gel electro horesis of nucleoproteins. Nucleoproteins from CHO cells heated at 42°C. Molecular weight ( X 10- P . ) is shown at the bottom. Arrows indicate 26 kDa protein.

cells. When cells were heated at 42'C for 1 hr, the level of the 26 kDa protein in the nucleus was reduced. This reduction was reversed when the cells were either continuously heated at 42°C (Fig. 3) or incubated at 37°C following heat shock (data not shown). The restoration of this protein occurred along with development of thermotolerance (Fig. 1). Kinetically, 4-6 hr at 42°C were required for the restoration of the protein and the development of thermotolerance.

Effect of heat protector or heat sensitizers on reduction and restoration of 26 kDa protein in

the nucleus at 42°C Experiments were performed to strengthen the cor-

relation between the restoration of the 26 kDa nucleoprotein and development of chronic thermotolerance. Figure 4 shows the effect of quercetin (21.4 pg/ml) on the reduction and restoration of the 26 kDa protein content in the nucleus of CHO cells, during heating at 42°C. Pretreatment with quercetin for 1 hr at 37°C did not affect the nuclear content of the 26 kDa protein (lane B). When cells were heated at 42°C for 1 hr in the presence of quercetin, the 26 kDa protein in the nucleus was reduced to 28% of the control level (lane C in Fig. 4 and 0 in Fi . 5) . Although treatment with quercetin

reduction of the 26 kDa protein in the nucleus of CHO cells, the rate of restoration of this protein was slower in the drug-treated cells.

Figure 5 shows the effect of a heat rotector or heat

protein during heating at 42°C (Fig. 5). The amount of 26 kDa rotein in treated cells relative to that of untreate a control cells was measured and plotted in

before and d uring heating had little effect on the

sensitizers on restoration kinetics F or the 26 kDa

Figure 5 . The reduction of this protein following 42°C for 1 hr varied from 25 to 75% of control. This variation may reflect an intrinsic difference in heat sensitivity in various cell lines. Nevertheless, restoration occurred and was completed during continuous 42°C treatment for 4-6 hr in each cell line. CHM added 2 hr before and during heating, protected from heat-induced reduction of 26 kDa protein in the nucleus of every cell line ( a 1. As shown in Figure 5 , two hours of CHM pretreatment was not required to induce protection when cells were heated at 42°C ( v ). Quercetin potentiated heat-induced 26 kDa rotein reduction in every cell line, except CHO

protein. The level of the protein in the nucleus gradu- ally increased to 80% of control levels during continuous heating at 42°C. DiC0,(3) or stepdown heat treatment sensitized heat-induced reduction of the 26 kDa rotein in every cell line. In this case, the level of 26 K Da was reduced to 3-18% of untreated control follow-

ing 42°C for 1 hr. Furthermore, these latter treatments greatly inhibited 26 kDa protein restoration. As shown in Figure 5 , no restoration was observed during heating at 42°C.

Restoration of extant 26 kDa protein during heating at 42°C

To investigate whether the restored 26 kDa protein represented a return to the nucleus of extant rotein,

dium, with [l4C1 leucine for 31 hr. After cells were labeled, they were washed and incubated at 37°C for 1 hr in nonradioactive growth medium, and then heated at 42°C. Autoradiographs of these prelabeled nucleoproteins were analyzed and compared to prior

cells. T R e drug also affected the restoration of this

CHO cells were labeled continuously, in comp f ete me-

Coomassie blue-stained gels. The autoradiograph in Figure 6 shows reduction of extant 26 kDa protein following 42°C for 1 hr and the restoration of this extant protein into the nucleus during continuous heating. The results from the autoradiograph were similar to that from Coomassie blue-stained gels (data not shown). Thus the restored 26 kDa protein was mainly synthesized before the heat shock. Figure 6 also shows that preferential accumulation of extant 70 kDa protein in the nucleus following 42°C for 1 hr was reversed when the cells were heated continuously at 42°C. Observations from two-dimensional gel electrophoresis of [l4C1 leucine-prelabeled proteins (Fig. 7) and immunoblotting study (data not shown) suggested that this rotein was constitutive heat shock 70 kDa protein ( c h P 70).

Unfortunately, two dimensional gel electrophoresis failed to show 26 kDa protein. This may be due to low solubility in the isoelectric focusing lysis buffer or that the isoelectric oint of this protein may be out of the pH

ment of the techniques in order to study this protein with two-dimensional gel electrophoresis.

DISCUSSION Our experiments (Figs. 1, 5) illustrate a correlated,

perhaps causal relationship between the restoration of a 26 kDa protein in the nucleus and development of chronic thermotolerance during continuous heating at 42°C in four different mammalian cell lines (CHO, HA-1, 3T3, HeLa). Figure 6 shows that this restored protein was mainly extant 26 kDa protein. These results were consistent with reports of 26 kDa rotein

43°C or 455°C (Lee et al., 1989; Kim et al., 1990). However, Figure 3 and Lee et al. (1989) clearly show the differences between chronic heating and acute heating. The restoration of the 26 kDa protein into the nucleus occurred during chronic heating. In contrast, the restoration of this protein occurred during recovery at 37°C after acute heating.

The restoration of the 26 kDa protein was not inhibited by treatment with the protein synthesis inhibitor, cycloheximide (Fig. 5 and Lee et al., 1989). Although the restoration of the 26 kDa protein occurred indepen- dently of metabolic synthetic processes, this process is likely dependent on energy state. When CHO cells were exposed to cold temperature (4°C) after initial heating (45.5"C-10 min), the restoration of 26 kDa protein did not occur (Kim et al., 1990). Furthermore, the development of thermotolerance was not observed (Henle and Dethlefsen, 1978; Kim et al., 1990) in these later conditions. Our data from Figures 1 and 5 show that the restoration of the 26 kDa protein, and the development of chronic thermotolerance, were prevented by treatment with heat sensitizers, DiOC5(3), or stepdown heating in four different mammalian cell lines. These results suggest that the presence of 26 kDa protein in the nucleus moderates the development of thermotolerance both by its function and its abilities to redistrib-

range 3.5-10. fF his problem will require further refine-

restoration in the nucleus following acute heat s R ock at

ute within the cell. Lee et al. (1989) showed that the 26 kDa protein was

not a member of the HSP 28 familv as described for HeLa cells (Arrig.0 and Welch. 1987) br HSP 27 in CHO

cells (Landr et al., 1988; Crete and Landry, 1990). It has been we P 1 known that HSP 28 contains very little or no methionine (Kim et al., 1984b). In contrast, the 26 kDa protein contains a substantial amount of methionine compared to HSP 28 (unpublished data). The 26 kDa protein which is highly localized in the nucleus is a phosphorylated protein (unpublished data).

At this time, only speculations can be made concern- ing the function of the 26 kDa rotein. The present data

its stabilization in the nucleus may have an important role in thermotolerance development. Based on these data we propose that the development of some types of thermotolerance occur without the necessity of rotein

ations, the 26 kDa protein itself may be altered or the binding affinity of this protein to nuclear components may be changed during heat treatment a t 42°C for 1 hr. This alteration may trigger the reduction of 26 kDa protein in the nucleus. Cycloheximide may stabilize the 26 kDa protein binding to the nuclear components. This

suggest that the restoration o r! this 26 kDa protein and

synthesis. Since heat causes many biochemica Y alter-

Fig. 4. Nucleoproteins from unheated untreated cells (A) or cells exposed to quercetin for 1 hr a t 37°C (B). Nucleoproteins from cells exposed to quercetin for 1 hr before and during heating at 42°C for 1 hr (C), 2 hr (D), 4 hr (E), 6 hr (F), or 8 hr (G). Molecular weight standards ( X are shown at the left. The location of 26 kDa protein is shown at the right.

330 LEE ET AL.

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HOURS AT 42" C Fig. 5. The effect of cycloheximide (CHM, 10 pgiml), quercetin (21.4 pgiml), DiOC,(3) (5 ygiml), or stepdown heating on the level of 26 kDa protein in the nucleus during heating at 42°C. CHM was added 2 hr before and left on during heating ( A ) or only durin heatin (0). Quercetin (0) or DiOC,(3) ( 0 was added 1 hr before an2 left on furing heating. For stepdown heatin cells were first heated at 45°C for 10 min and then immediately chjlenged at 42°C (0). + = Survival curve

stabilization would then prevent heat-induced reduction of 26 kDa protein level in the nucleus. Since protein synthesis is almost totally inhibited by 10 p,g/ml cycloheximide, this process is protein synthesis- independent, especially heat shock protein synthesis- independent (Lee et al., 1987; Crete and Landry, 1990).

Our data also show that the 26 kDa protein was restored while cells were heated continuous1 at 42°C

et al., 1989). As mentioned previously, this restoration process may require ATP or be affected by the cellular environment, e.g., ionic stren h, pH, temperature, etc.

kDa protein restoration in various ways. Quercetin is known as a mutagenic agent (Bjeldanes and Chang, 1977). It induces topoisomerase I1 dependent DNA cleavage (Yamashita et al., 1990) and inhibits many

(Fig. 3) or incubated at 37°C following heat s hy ock (Lee

Heat sensitizers probably a P ect the process of the 26

of control cells heated at 42°C. The nuclei were isolated from the various cells (CHO, HA-1, 3T3, HeLa) and their nucleoproteins were analyzed on a gradient SDS-polyacrylamide gel. The gel was analyzed with a densitometer. The amount of protein is plotted as a function of heat dose. The value plotted is the amount of nucleoprotein in treated cells relative to that of untreated control cell.

cellular enzymes such as Na', K+- (Lang and Racker, 1974) and Ca2+, Mg2+-ATPase (Shoshan and MacLen- nan, 1981). It is also known as an inhibitor of lactate transport, which causes intracelluar acidification and inhibition of glycolysis (Belt et al., 1984). DiOCJ3) inhibits ATP generation (Howard and Wilson, 1979; Borrelli et al. in press), Ca2+-ATPase (Russell et al., 1979) and enhances heat-induced DNA unwinding (Borrelli et al., in press). The latter effect of DiOC6(3) may disrupt the binding site of the 26 kDa protein in the nucleus, thus preventing its restoration. Stepdown heating results in a sensitization to the lower temper- atures (Henle, 1980). It probably results from inhibition of repair of cellular damage and the development of thermotolerance. Henle et al. (1989) suggested that stepdown heating inhibits protein glycosylation, which may have an important role in development of thermo-

C H O Fig. 6. Autoradiograph of a gradient SDS polyacrylamide slab gel of [l4C1 leucine-labeled nucleoproteins. CHO cell were prelabeled for 31 hr before the experiment. After cells were labeled, they were rinsed twice with Hanks' balanced salt solution and then incubated for 1 hr in complete medium at 37°C before being exposed to heat. The nuclei were isolated and analyzed immediately after various heat doses (1-8 hr) at 42°C. Gel lane C: Nucleoproteins from unheated cells. Molec- ular weights (x are shown at the left. Note that the restored 26 kDa protein mainly represented a return to the nucleus of old protein.

tolerance. Obviously, further studies at the biochemical level are necessary to understand the mechanism of reduction and restoration of the 26 kDa protein during heating at 42"C, and how thermomodifiers affect these processes.

ACKNOWLEDGMENTS This research was supported by NCI grants

CA48000, CA44550, CA49715, and William Beaumont Hospital Research Institute grants 89-02 and 89-06.

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N U C L E I

3 7 OC 4 2 ' C - l h

Fig. I . Autoradiographs of two-dimensional SDS-polyacrylamide gels. Cells were prelabeled, rinsed, and then heated at 42°C for 1 hr. The nuclei were isolated and analyzed on a two-dimensional gel. Arrows indicate the location of cHSP-70. Actin (Mr 43,000) is indicated by A. Left panel: Nucleoproteins from unheated control cells. Right panel: Nucleoproteins from heated cells. See legend of Figure 6 for further details.

332 LEE ET AL.

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Correlation between redistribution of a 26 kDa protein and development of chronic thermotolerance in...

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