glucocorticoid-induced heat resistance in mammalian cells

JOURNAL OF CELLULAR PHYSIOLOGY 128:127-132 (1986)

Glucocorticoid-Induced Heat Resistance in Mammalian Cells

GEORGE A. FISHER, ROBIN L. ANDERSON,* AND GEORGE M. H A H N Division of Radiobiology, Department of Radiology, Stanford University School of

Medicine, Stan ford Ca li forn ia 94305

Chinese hamster ovary cells were incubated for 24 h in a variety of steroid hormones (testosterone, progesterone, hydrocortisone, dexamethasone, and ecdysterone) to test their effect on the subsequent heat resistance of the cells. Only the glucocorticoids, hydrocortisone and dexamethasone, consistently induced heat resistance. Heat resistance induced by hydrocortisone at 10-6M developed after a lag of 2-3 h and was maximal by 20 h. Resistance was expressed in both asynchronous and plateau phase cells and was maintained for several days in medium without added hormone. Incubation of cells with hydrocortisone and a 100-fold excess of progesterone (a glucocorticoid antagonist) partially inhibited the development of resistance. Prior exposure to hydrocortisone did not inhibit the subsequent development of heat induced thermotolerance. However, cells made thermotolerant by prior heat shock did not display further heat resistance with hydrocortisone treatment. There was no evidence for the induction of heat shock proteins (HSP) by these steroid hormones although the 28 kDHSP was further enhanced by combined heat and hydrocortisone. Our results indicate that heat resistance in mammalian cells may be induced by physiological concentrations of glucocorticoids and that the characteristics of this resistance are consistent with a receptor mediated event.

Hyperthermia, which depresses protein synthesis in general, induces an increase in the rate of synthesis of a few specific proteins known as heat shock proteins (HSP) (Kelley and Schlesinger, 1978). This heat shock response is observed in all organisms from bacteria to man (Schlesinger et al., 1982). The presence and levels of HSP correlate well with the level of heat resistance or thermotolerance in mammalian cells (Li and Werb, 1982; Landry et al., 1982; Subjeck et al., 19821, but no function has yet been assigned to them.

Other agents, both physical and chemical, will also induce the HSP response (Ashburner and Bonner, 1979) and most of these agents also induce thermotolerance (Hahn and Li, 1982; Li and Werb, 1982). Several steroid hormones are amongst this group of agents. The insect steroid hormone a-ecdysterone, induces the synthesis of the low molecular weight HSP in Drosophila (Buzin and Bournais-Vardiabasis, 1982; Ireland et al., 1982; Berger and Woodward, 1983) with concomitant resistance to heat killing (Berger and Woodward, 1983). Also, in- creases in the hemolymph concentration of ecdysterone parallel the synthesis of HSP 23 in vivo (Cheney and Shearn, 1983) during a developmental stage that has been shown to be resistant to otherwise lethal tempera- ture (Berger and Woodward, 1983).

We began this study to see if an analogous hormone. and receptor-mediated heat resistance might occur in mammalian cells. We screened a variety of steroid hormones for their ability to induce heat resistance and alter the patterns of protein synthesis in Chinese hamster ovary cells. Since the ecdysterones are described as

0 1986 ALAN R. LISS, INC.

differentiation hormones in insects, we tested progesterone (prog) and testosterone (test) as they play a similar role in many mammalian systems. We also studied hydrocortisone (HC) because serum levels of this hormone are elevated in response to many types of stress in vivo, and because physiological stress may be a stimulus for the HSP response. To see if the ecdysterones could affect mammalian cell heat sensitivity we screened both a- ecdysterone and the plant-derived 0-ecdysterone. As an additional negative control we tested a non-steroid hormone, tri-iodothyronine (T3).

MATERIALS AND METHODS Cell culture

Chinese hamster ovary cells (HA-1) were routinely grown as a monolayer in Eagle’s minimum essential medium (MEM, GIBCO, Grand Island, NY) supple- mented with 10% fetal calf serum (Irvine Scientific, Santa Ana, CAI, streptomycin sulphate (200 mgA) and potassium penicillin (200,000 unitsA). The same serum lot was used in all experiments. The concentration of hydrocortisone in the serum was less than lO-’M, re- sulting in a final hydrocortisone level of less than lO-’M in the medium.

Plateau phase cells were obtained by seeding 2-3 x lo5 cells per 60 mm petri dish (Nunc, Thousand Oaks, CA) and changing medium daily until a density of 1-2

Received November 18, 1985; accepted February 13, 1986. *To whom reprint requestskorrespondence should be addressed.

128 FISHER. ANDERSON. AND HAHN

x lo7 cells per dish was attained. Exponentially growing cells were treated 2-3 days after seeding. Heating was done in specially designed water baths (+O.l°C) in an atmosphere of 5% C02. Media pH was maintained within a range of 7.2-7.5. Cell survival was assayed by the cloning technique of Puck and Marcus (1956).

Hormone treatment HC, prog, test, T3, a- and 0-ecdysterones (Sigma, St.

Louis, MO), and dexamethasone (dex) (Sigma or Merck, Sharpe and Dohme, Los Angeles, CA) were dissolved in saline or ethanol and diluted in Eagle's MEM. The final ethanol concentration did not exceed 0.1%. Cells treated with 0.1% ethanol alone exhibited growth rates and heat sensitivities indistinguishable from those of saline- treated controls.

Gel electrophoresis For analysis of the patterns of protein synthesis, cells

growing as monolayers were incubated with various concentrations of steroid hormones and with either 35S- methionine (Amersham, Arlington Heights, IL, specific activity 1,065 Ci/mmol) 3H-amino acid mix (ICN, Costa Mesa, CA, specific activity 225 mCi/mg). After the appropriate incubation time, cells were placed on ice and rinsed four times in phosphate buffered saline. The cells were transferred by scraping into gel sample buffer (Laemmli, 1970) for analysis by one-dimensional SDS- polyacrylamide gel electrophoresis (Laemmli, 1970) using an acrylamide concentration of 13%. Aliquots were taken for measurement of trichloracetic acid (TCA) pre- cipitable radioactivity and protein (Peterson, 1979) to measure the effect of the drug treatment on the rate of protein synthesis. The gels were stained with Coomassie blue, destained, dried, and exposed to Kodak XAR-2 film. Tritium labelled gels were treated with Enhance WEN, Boston, MA) before drying to generate a fluorograph instead of a radiograph.

Lipid analysis For membrane lipid analysis after HC treatment, cel-

lular lipids were extracted into chlorofordmethanol as described earlier (Anderson and Parker, 1982). Free cholesterol levels in the lipid extract were measured using the cholesterol oxidase assay procedure of Heider and Boyett (1978). Phospholipid was quantitated by measur- ing the amount of phosphorus in the lipid extract, as described by Anderson and Davis (1982). Protein was assayed by the method of Lowry et al., (1951) using, the cell pellet remaining after chlorofordmethanol extraction.

RESULTS Induction of heat resistance

Cells were exposed to varying concentrations of hormones (10-9-10-5M) at 37°C for 24h. The cells were then rinsed, heated at 45°C for 45 min, and plated for clonogenic survival. The results for plateau phase cells given lop6 M drug are shown in Figure 1. The cells treated with the glucocorticoids HC, or dex showed increased resistance to the lethal effects of heating. The degree of resistance varied from experiment to experiment but, in all cases, surviving fractions increased by a factor between 10 and 100. The survival of cells given test, prog a or P-ecdysterone, and T3 was either moder- ately elevated (less than two-fold) or not significantly different from controls. Similar results were obtained

L

- D E i kl!J PROG TEST ECDYSTERONE

Fig. 1. Effect of various hormones on the heat sensitivity of HA-1 cells. Cells were incubated at 37°C for 24 h in the presence of 10-6M drug and were then rinsed, heated at 45°C for 45 min, and assayed for survival. Error bars in this and subsequent figures represent f SE of the mean of triplicate assays.

1o-lI

0 ,0-9 10-8 ,0-7 10-6 10-5 10-4

HC CONCENTRATION (MI

Fig. 2. Concentration dependence of HC-induced heat resistance. Cells were treated with various concentrations of HC for 24 h at 37°C and then were rinsed, heated at 45°C for 45 min and assayed for survival.

when concentrations as high as were used (data not shown).

The concentration and time dependence of HC-induced heat resistance are shown in Figures 2 and 3. Cells were exposed to varying concentrations of HC for 24 h, heated at 45°C for 45 min and then assayed for survival (Fig. 2). Maximal heat resistance was evident at doses as low as lO-'M. Exposure to concentrations as high as 10-4M had no additional effect on the heat resistance of the cells. When cells were exposed to 1OP6M HC for varying lengths of time and then assayed for heat survival, resistance began to develop after a lag of 2-3 h and was

GLUCOCORTICOID-INDUCED HEAT RESISTANCE 129

TABLE 1. Inhibition of HC-induced heat resistance bv uroe

Surviving fraction after Pretreatment 45°C for 45 min

None HC M) Prog ( ~ o - ~ M ) HC + uroe

2.4 f 0.5 X 8.5 f 0.7 X 5.5 f 0.5 x 10-4 4.3 + 0.8 x lo-*

Cells were exposed to HC M), or to both for 24 h at 37°C and then were heated (45°C for 45 min) and assayed for survival. Survival values represent the mean (+ SE) of six samples.

M), prog

maximal by 20 h (Fig. 3). The resistance was expressed in both asynchronous exponentially growing cells (data not shown) as well as in plateau phase cells and remained for up to several days. If, after a 24-h incubation with HC, cells were rinsed thoroughly and returned to 37°C for 1-3 days in regular growth medium (without added HC), there was no loss of the HC-induced heat resistance and survival levels remained approximately ten-fold higher than heated controls.

To test the possibility that the HC-induced heat resistance is mediated by the glucocorticoid receptor, we incubated plateau phase cells for 24 h with HC (lOP7M) and a 100-fold greater concentration of progesterone. Progesterone is a partial glucocorticoid antagonist known to inhibit allosterically HC-receptor binding (Svec and Rudis, 1981). As shown in Table I, prog effectively inhibited the development of HC-induced heat resistance. This result was also observed in exponentially growing cells (data not shown).

Hydrocortisone-induced resistance and thermotolerance

To compare the HC-induced heat resistance with the phenomenon of thermotolerance (heat-induced heat resistance), we performed the following experiments. Thermotolerance was induced by exposing cells to 45°C for 10 min followed by a 37°C incubation for 24 h. As shown in Figure 4, this treatment rendered the cells much more resistant to the lethal effects of a second heating. Figure 4 also shows the effect of a 24-h HC treatment (1OP6M) on cell survival at 45°C. Significant protection was evident but not to the extent of that induced by prior heating. When the two treatments were combined (i.e., 45"C/10 min followed by 24 h at 37°C with HC, there was no increase in heat resistance compared to the heat-induced thermotolerance alone.

Membrane lipid changes Glucocorticoids have been shown to alter membrane

cholesterol content and fluidity (Boullier et al., 1982). Since the cholesterol content has been proposed as a determinant of cellular heat sensitivity (Cress et al., 19821, we assayed cholesterol in cells exposed to 10-6M HC for 24 h (Table 2). Whether expressed on a protein basis or as the molar ratio of cholesterol to phospholipid, we observed no significant differences from controls.

Protein synthesis changes Since it has been shown by others (Kasambalides and

Lanks, 1983; Maytin and Young, 1983) that HC and

t 1641 . . . . . . . . . . . .:. ~

4 8 12 16 20 24 48 72

Time (hours) Fig. 3. Time dependence of HC-induced heat resistance. Cells were treated with 10-6M HC for varying lengths of time and then were rinsed, heated at 45°C for 45 min, and assayed for survival. The data presented are from one of three experiments which gave similar results.

16

C

+ U

0 .-

2 - 1 0

0-l C > > L 3 u,

.-

.-

ld 20 40 60 80 100

Time at 45'C(rnin) Fig. 4. Effect of HC on the heat survival of control and thermotolerant cells. Survival values of control cells (0) and thermotolerant cells (A) are lotted as a function of time at 45°C. A 24 h pretreatment with

HC 37"C/24 h (0); 45"C/lOmin followed by HC 37"C/24 h (A). Data points are the means of triplicate assays. Variations between replicates were less than 25% of the means. The combined data from three separate experiments are shown. Plating. efficiencies were between

10- r M HC yields the survival values indicated by the closed symbols.

dexamethasone can modify the pattern of protein synthesis, HA-1 cell proteins were analyzed by SDS-poly- 70% and 100%

I

130 FISHER, ANDERSON, AND HAHN

TABLE 2. Cholesterol content of HC-treated cells

ChoUprotein Choliphospholipid (Pdmgf (molar ratio)

Control 63.5 f 3.5 0.22 + 0.01 HC W24 h) 62.2 + 1.3 0.20 + 0.01

Cholesterol, phospholipid, and protein content were determined as described in Materials and Methods. Values represent the mean of three replicate samples (* SE).

94-

6 7-

43-

30-

20-

1 2 3 4 5 6 7

Fig. 5. Radiograph prepared from a gel of 35S-methionine-labelled proteins from cells treated with heat shock (lane 2); IO-'M a-ecdysterone (lane 3); 10-7M HC (lane 4): the combined treatment of 10-7M HC and 10-5M progesterone (lane 5) or with 10-5M progesterone alone (lane 6). Lanes 1 and 7 represent cells that received no pretreatment. Cells were incubated in the appropriate steroid hormone for 24 h, with 35S-methionine present during the last 8 h of treatment. Samples containing equal radioactivity but less than 100 pg protein were loaded on the gel. The numbers down the left-hand side denote the molecular weights of known standards while the bars on the right-hand side mark the positions of the major heat-induced proteins.

acrylamide gel electrophoresis following exposure to these hormones in the presence of 35S-methionine. The pattern obtained was compared to that in control or heat-shocked cells (Fig 5). Tke promjnent heat-induced proteins seen after S-methionine incorporation have molecular weights of 110, 89, 70, and 60 kD. No alterations in protein patterns were seen after treatments with HC, a-ecdysterone, prog, or with the combined treatment of HC and prog. In addition, dex did not alter the pattern of protein synthesis (data not shown).

Some of the HSPs, especially those with smaller subunit molecular weights, are methionine-poor proteins

se 67-

7 2 3 4 Fig. 6. Fluorograph prepared from a gel of 3H-labelled proteins from cells treated with 10-6M HC (lane 1): 1O-'M HC followed by a heat shock of 10 min at 45°C (lane 2); control cells (lane 3); 10 min at 45°C (lane 4). Cells were incubated in 1O-'M HC, where appropriate, for 16 h followed by a heat shock of 10 min at 45°C (also only where appropriate). They were then rinsed free of the added HC and incubated for the next 8 h in regular medium containing a 3H-amino acid mix. The numbers down the left-hand side denote the molecular weights of known standards while the bars on the right point to the locations of HSPs. Samples containing equal radioactivity were loaded in each well. The rate of total protein synthesis was unaffected by HC but was reduced by approximately 20% when the cells were heated. The combined treatment of HC followed by heat caused no further reduction in protein synthesis than that from heat alone. Hence, lanes 2 and 4 also contain an equal total protein loading whilst lanes 1 and 3 have an equal but lower amount (20% less protein).

(Kim et al., 1983). For this reason, we also used 3H- labelled amino acid incorporation instead of 35S-methionine, to look for alterations in the pattern of protein synthesis after HC treatment. Figure 6 shows a fluorograph of cells treated with either heat or HC or with a combination of both. Heat alone induces two additional HSP not seen using methionine incorporation, at 22 and 28 kD. HC alone does not obviously induce any of these proteins. However, after the combined treatment, the 28 kD protein is markedly more prominent than after heat alone. Densimetric scanning of the radiograph revealed that in addition to HSP 28, the synthesis of HSP 89 and a protein with a subunit molecular weight of 49kD were also increased by the combined treatment.

The protein analysis experiments described so far in- volved incubation of cells in radioactive amino acids for 6-8 h either during or after the HC treatment. The possibility still remained that the altered protein syn-

GLUCOCORTICOID-INDUCED HEAT RESISTANCE 131

thesis pattern occurred over a short period, possibly just after the addition of HC. To check this possibility, an additional experiment was performed in which cells were pulse-labelled for 1 h, either a t the beginning, during or after HC exposure, and with either 3H or 35s-labelled amino acids. Under no conditions tested, were HSPs or any other marked alterations in protein synthesis observed.

DISCUSSION Many theories have been proposed for the molecular

mechanism of thermal cell killing and heat resistance though none to date have gained wide acceptance. There are undoubtedly several biochemical pathways that could lead to alterations in heat sensitivity. We started this study to see if there exists a receptor-mediated pathway to heat resistance in mammalian cells.

Although a direct assay of the glucocorticoid receptor activity in these cells is not presented here, we report a number of results that are consistent with the hypothe- sis that glucocorticoid induced heat resistance is receptor mediated. First, the effect is specific for glucocorticoids; other steroid hormones tested do not induce significant heat resistance (Fig. 1). Second, the effect is observed with physiologically relevant concentrations of glucocorticoid. Third, the heat resistance reaches a plateau value at concentrations consistent with estimates of those required to saturate the glucocorticoid receptor (Sibley and Tomkins, 1974). Fourth, the kinetics of the development of the heat resistance are consistent with a receptor-mediated event. The heat resistance develops after a lag of 2-3 h and presumably involves steroid- receptor binding and activation, translocation to the nucleus, and intiation of the transcription of specific sequences followed by their translation into effector proteins whose functions are responsible for the observed resistance to heat. The development of resistance in the next 20 h might then correspond to the accumulation or secondary function of these proteins. Similar reasoning has been used to correlate thermotolerance with heat shock proteins (Li and Werb, 1982; Landry et al., 1982; Subjeck et al., 1982) although the induction of these appears not to be receptor mediated and heat-induced tolerance has kinetics different from those of steroid- induced tolerance. An exception to this statement is the slow development of thermotolerance following a very severe priming heat dose (Sciandra and Subjeck, 1984). In this case, the kinetics of development are similar to those seen in this study after HC treatment. Fifth, the heat resistance is maintained for 2-3 days following removal of added steroid (by multiple washes with regular growth medium). This is consistent with the classi- cal steroid-receptor pathway; the longevity of the effect should depend on the synthesis and degradation of the effector proteins and not on the continued presence of the steroid. Sixth, and perhaps most compelling, is the fact that prog, which allosterically inhibits the binding of glucocorticoids to their cytoplasmic receptor (Svec and Rudis, 1981), effectively inhibits the HC-induced heat resistance.

Drosophila cells respond to heat shock with the synthesis of at least seven HSPs designated by their molecular weights: HSP 82, 70, 68, 27, 26, 23, and 22 kD (Ashburner and Bonner, 1979). Since ecdysterone ren-

blue exclusion, yet induces the synthesis of only the four smaller HSP (HSP 27,26,23, and 221, Berger and Wood- ward (1983) have argued that it is the small molecular weight proteins that are responsible for thermotolerance in Drosophila. Li (19851, in contrast suggests that it is the 70 kD HSP which correlates best with heat resistance in Chinese hamster cells, although the low molecular weight methionine-poor proteins were not evaluated in that study. The synthesis of HSP 27 was measured by Landry et al. (1982). The induction by heat was minor compared to the higher molecular weight HSPs, possibly due to the use of radiolabelled methionine as a marker. Our study failed to detect any HSPs induced by either ecdysterone or by HC alone (20-h exposure to concentrations of 10-7-10-5M), but the synthesis of HSP 28 and some higher molecular weight HSPs was further enhanced by the addition of HC (Fig. 6). The significance of this finding is not clear a t the present time.

It is of interest to make a comparison between the heat survival data in this study and the altered patterns of protein synthesis following hormone exposure observed by others (Kasambalides and Lanks, 1983; May- tin and Young, 1983). Of the hormones tested in this study, only the glucocorticoids dex and HC increased heat resistance. Prog and test had either minimal or no effect. Using mouse L cells, Kasambalides and Lanks (1983) found altered patterns of protein synthesis caused by the two glucocorticoids but no alterations with test or prog. These authors also report that under conditions of glucose deprivation, glucocorticoids induce the synthesis of two proteins with molecular weights of 85 kD and 69 kD. They suggest that these polypeptides are identical to the 85 kD and 69 kD proteins induced by heat shock in mouse L cells. However, in rat thymocytes, dex also induces increased synthesis of six proteins, all of which were judged by two-dimensional giant gel electrophoresis to be different from the proteins induced by heat shock (Maytin and Young, 1983).

Although our data suggest that the HC effect occurs via a pathway involving the glucocorticoid receptor, we still have no clear indication of the critical cellular change that results in heat resistance. The fluidity of the plasma membrane and its modulation by cholesterol have been postulated to be of importance in determining a cell’s sensitivity to heat (Gerner et al., 1980; Cress et al., 1982), a finding not corroborated in two other studies (Anderson and Parker, 1982; Gonzalez-Mendez et al., 1982). Furthermore, glucocorticoids have been shown to produce changes in the cholesterol content of many cell lines (Boullier et al., 1982). However, when we determined the cholesterol content normalized to cell protein, or to the cholesterol/phospholipid molar ratio in HA-1 cells, we observed no difference between HC-treated and control cells (Table 2).

Glucocorticoids have been shown to influence cell cycle parameters as well as saturation densities of mammalian cells grown in culture (Ponec et al., 1979; Bakke and R4nning, 1982). Both cell cycle distribution and cell density can alter the heat sensitivity of cells (reviewed in Hahn, 1982). In our experiments with exponentially growing cells, we found that HC consistently induced a 12-24 h delay in cell division followed by a doubling time which was identical to that of controls (16-18 h). Although doubling times recovered, the saturation den-

ders Drosophila cells heat resistant as assayed by trypan sity of cells grown in more than 10-7M hydrocortisone

132 FISHER, ANDERSON, AND HAHN

was consistently 20-30% of that of control cells (data not tion between amounts of cellular membrane components and sensi- shown). m e n we heated cells at equivalent cell densi- tivity to hyperthermia in a variety of mammalian cell lines in culture.

Cancer Res., 42r1716-1721. ties we found that the HC-treated (lP6 days Of Gerner, E.W., Cress, A.E., Stickney, D.G., Holmes, D.K., and Culver, growth in HC were consistently more resistant P.S. (1980) Factors regulating membrane premeability alter thermal than control cells. To minimize potential artifacts with resistance. Annals N.Y. Acad. Sci., 335:215-233. cell cycle alterations we performed most of our experi- Gonzalez-Mendez, R., Minton, K.W., and Hahn, G.M. (1982) Lack of

correlation between membrane lipid composition and thermotolerance in Chinese hamster ovary cells. Biochim. Biophys. Acta, ments with plateau phase cells (> 90% in Gl).

HC-induced heat resistance appears to be distinct from 692:168-170, heat-induced heat resistance (thermotolerance) in Hahn, G.M. (1982) Hyperthermia and Cancer. Plenum Press, New Chinese hamster cells. Thermotolerant cells can be ap- York, pp 23-25 preciably resistant to heat than HC-treated cells Hahn,G.M., and Li, G.C. (1982) Thermotolerance and heat shock pro-

teins in mammalian cells. Radiat. Res., 92452-457. (Fig. 4). HC appears not to induce the major HSPs that Heider, J.G., and Boyett, R.L. (19781 The picomole determination of accompany the development of thermotolerance in HA- free and total cholesterol in cells in culture. J. Lipid Res., 19:514- 1 cells (Fig. 5). Heat-induced tolerance in HA-1 cells 518. develops rapidly than does tolerance induced by Ireland, R.C., Ek-ger, E., Sirotkin, K., Yund, M.A., Osterbur, D., and

Fristrom, J. (1982) Ecdysterone induces the transcription of four glucocorticoids ai et al., 1982), except where lethal heat-shock genes in Drosophila S3 cells and imaginal discs, Dev. priming doses of heat are used (Sciandra and Subjeck, Biol. 93:498-507. 1984). Treatment with HC does not inhibit the subse- Kasambalides, E.J., and Lanks, K.W. (1983) Dexamethasone can mod- quen+, development of heat-induced heat resistance (data ulate glucose-regulated and heat shock protein synthesis. J . Cell.

Physiol., 114:93-98. not snown), while heat shock) cannot be made more heat resistant by HC (Fig. analogues and heat shock on gene expression in chicken embryo 4). This latter observation suggests that thermotolerant fibroblasts. Cell, 15:1277-1286. cells are either resistant to the steroid or are already Kim, Y.J., Shuman, M., Setto, M., and Przybyla, A. (B83) Arsenite

induces stress proteins in cultured rat myoblasts. J. Cell Biol., 96:393- maximally heat resistant. A recent study reports that 400. heat shock can temporarilv inhibit a cell’s resDonse to Laemmli. U.K. (1970) Cleavage of structural Droteins durinv the RS-

made thermotolerant Kelley, P.M., and Schlesinger, M.J. (1978) The effect of amino acid

D ---- -- ~~~~~ ~~~.....

steroids (wolffe et Ll., 19;84), possibly explai&g the sembly ‘of the head of bacteryophaae T4. Natdre, 227r680-685. inability of HC to increase the heat resistance of heat Landry, J., Bernier, D., Chretien, P., Nicole, L.M., Tanguay, R.M. and Marceau, N. (1982) Synthesis and degradation of heat shock proteins

during development and decay of thermotolerance. Cancer Res., shocked thermotolerant cells. 422457-2461. The finding that phvsiolopical doses of glucocorticoids

are capable of indicihg heat resistance & mammalian Li, G.C. (1985) Elevated levels of’ 70,000 dalton heat shock protein in cells may be of importance in the clinical application of transiently thermotolerant Chinese hamster fibroblasts and in their

stable heat resistant variants. Int. J. Radiat. Oncol. Bio. Phys., hyperthermia. It may prove valuable to measure serum Ilr165-178. HC (cortisol) levels and to delete dex from the drug Li, G.C., Fisher, G.A., and Hahn, G.M. (1982) Induction of thermotoler- regimen of any patient considered for hyperthermia ance and evidence for a well-defined thermotropic cooperative pro- treatments. cess. Radiat. Res., 89:361-368.

Li, G.C., and Werb, 2. (1982) Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts. Proc. Natl. Acad. Sci., USA, 79r3218-3222. ACKNOWLEDGMENTS

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Anderson, R.L., and Davis, S. (1982) An organic phosphorus assay 258:12718-E722. which avoids the use of hazardous perchloric acid. Clin. Chim. Acts, Peterson, G.L. (1979) Review of the Folin phenol protein quantitation 121 ;111-116. method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem.,

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Bakke, O., and Rbnning, O.W. (1982) The role of protein metabolism in 227. glucocorticoid-induced prolongation of G1 phase in human NHIK Puck, T.T., and Marcus, P.I. (1956) Action of x-rays on mammalian 3025 cells. J. Cell. Physiol., 113:459-464. cells. J. Exp. Med., 103:653-666.

Berger, E.M., and Woodward, M.P. (1983) Small heat shock proteins in Schlesinger, M., Ashburner, M., and Tissieres, A. (eds.) (1982) Heat Drosophila may confer thermal tolerance. Exp. Cell. Res., 147r437- Shock: From Bacteria to Man. Cold Spring Harbor, New York. 442. Sciandra, J.J., and Subjeck, J.R. (1984) Heat shock proteins and protec-

Boullier, J.A., Melnykovych, G., and Barisas, G. (1982) A photobleach- tion of proliferation and translation in mammalian cells. Cancer Res. ing recovery study of glucocorticoid effects on lateral mobilities of a 44:5188-5194. lipid analog in S3G HeLa cell membranes. Biochim. Biophys. Acta, Sibley, C.H., and Tomkins, G.M. (1974) Mechanisms of steroid resis- 692:278-286. tance. Cell, 2r221-227.

Buzin, C.H., and Bournias-Vardiabasis, N. (1982) The induction of a Subjeck, J.R., Sciandra, J.J., and Johnson, R.J. (1982) Heat shock subset of heat-shock proteins by drugs that inhibit differentiation in proteins and thermotolerance: A comparison of induction kinetics. Drosophila embryonic cell cultures: In: Heat Shock From Bacteria Br. J . Radiol., 55.579-584. to Man. M. Schlesinger, M. Ashburner, and A. Tissieres, eds. Cold Svec, F., and Rudis, M. (1981) Progestin-induced enhancement of dex- Spring Harbor, New York, pp. 387-394. methasone dissociation from glucocorticoid hormone receptors. Arch.

Cheney, C.M., and Shearn A. (1983). Developmental regulation of Biochem. Biophys., 212:417423. Drosophila imaginal disc proteins: Synthesis of a heat shock protein wolffe, A.P., Perlman, A.J., and Tats, J.R. (1984) Transient paralysis under non-heat-shock conditions. Dev. Biol., 95r325-330. by heat shock of hormonal regulation of gene expression. EMBO J.,

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and CA-19386 from the National Cancer Institute.

LITERATURE CITED

glucocorticoid-induced heat resistance in mammalian cells

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