JOURNAL OF CELLULAR PHYSIOLOGY 142255-260 (19901

Heat-Induced Cytotoxicity in H,O,-Resistant Chinese Hamster Fibroblasts DOUGLAS R. SPITZ* AND GLORIA C. LI Radidtion Oncology Rewarch Laboratory, CED-LOO, University of Ca/ifornia, San krancisco, California 94 143 Hydrogen-peroxide-resistant Chinese hamster fibroblasts, derived from the HA-1 cell line, were isolated following continuous culturing in the presence of progressively increasing concentrations of hydrogen peroxide. The hydrogen-peroxide-resistant phenotype has been stable for over 360 days following removal from H,O, stress. These ti,O,-resistant cell lines demonstrate increased resistance to hypertherniic cell killing mediated by continuous heating at 43°C but not 45°C. The relationship between rnarnirndlian cellular adaptation to oxidative stress mediated by H,O, and resistance to 43°C hyperthermia is discussed.

The mechanism(s) by which elevated temperature exerts its cytotoxic action is not well understood. However, many reports have demonstrated that living systems, from bacteria to man, express a highly conserved adaptive response to exposure to elevated temperature (Ashburner and Bonner, 1979; Lindquist, 1986). This adaptation renders the cells resistant to the cytotoxic actions of subsequent heat challenges (Ashburner and Bonner, 1979; Ashburner, 1982; Henle and Dethlefsen, 1978; Henle and Leeper, 1978; Li and Werb, 1482; Li et al., 1982a; Landry et al., 1982; Subjeck et al., 1982). Recently, several studies have shown that bacterial and mammalian systems also express an adaptive response to hydrogen peroxide (H,O,)-mediated oxidative stress. This adaptation results in the cells' becoming resistant to the cytotoxic actions of H202and other oxidizing agents (Christman et al., 1985; Demple and Halbrook, 1983; Winquist et al., 1984; Spitz et al., 1987). Several authors have shown that heat shock and oxidative stress responses share some overlapping characteristics and have suggested that this overlap might be indicative of a n underlying mechanism common to both phenomena (Christman et al., 1985; Morgan et al., 1986; Privalle and Fridovich, 1987; Spitz et al., 1987; Kapoor and Lewis, 1987). Our group has reported the isolation of stable (>360 days) H,O,-resistant variants (dose modifying factors, at 20% isosurvival, from 5 to 30) of the Chinese hamster HA-1 cell line following prolonged culture (>200 days) in progressively increasing concentrations (50800 pM) of H,O, (Spitz et al., 1988). The H202-resistant phenotype was accompanied by a n increase ( 5 40-fold) in expression of catalase activity (Spitz et al., 1988). An increase in chromosome number also occurred in cells adapted to 200-800 pM H,O,. Increases in aneuploidy and tetraploidy were not necessary for H,02 resistance, since they did not occur in cells adapted to 50-100 FM H,02. However, increases in chromosome number occurred in a greater percentage of cells adapted t o 800 FM H,O, (60%) as compared to cells adapted to 200 and 400 pM H,O, (20-30%). Also, when quasidiploid (21-22 chromosomes/cell) and qua0 1990 WILEY-1,TSS. INC

sitetraploid (40-44 chromosomesicell) cell lines were cloned from the 800 (*M H,O,-resistant population, the H,O, dose-modifying factors a t 20% isosurvival were 1 4 t 1 0 for the seven quasidiploid lines and 37210 for the six quasitetraploid lines (Spitz et al., 1 9 8 9 ~ )These . results indicate that on the average the quasitetraploid lines demonstrated greater stable resistance to H,O,mediated toxicity than the quasidiploid cell lines. In the present study, the relationship between cellular resistance to heat shock and oxidative stress was examined by comparing heat induced cytotoxicity in the HA-1 and H,O,-resistant variant cell lines. The primary goal of this work was to determine if the H,O,resistant phenotype was accompanied by increased resistance to heat shock. We felt that understanding the relationship between cellular resistance to these two stresses in mammalian cells might help us t o focus the efforts aimed a t determining what, if any, role oxidizing species might play in the mechanisms of heat induced lethality. We have also investigated the responses of quasidiploid and quasitetraploid cell lines cloned from the H,O,-resistant cell lines in order to determine what, if any, relationship exists between H,O,-induced changes in ploidy and heat shock responses.

MATERIALS AND METHODS Cells and culture conditions Chinese hamster fibroblasts designated HA-1 (Yang et al., 1966) and their H,O,-resistant variants were maintained in Eagle's Minimal Essential Medium supplemented with 15% fetal calf serum and gentamycin (50 pgiml). Cell cultures were kept a t 37°C in a humidified incubator with a mixture of 95% air and 5%' CO, and routinely checked for mycoplasma contamination. Received July 28, 1989; accepted September 26, 1989.

"To whom reprint rcquests/correspondence should be addressed a t Dept. of Pediatrics, MR-4 Room 3033, Univ. of Virginia Hospital, Charlottesville, VA 22908.

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Survival experiments Cells (1-2 x lo5) were plated in 60 mm petri dishes and grown for 48 hours a t 37°C. The experiments were performed when the cell density was 3-8 x l o 4 cells/ cm'. Following treatments, cells were trypsinized, counted, and plated after appropriate dilutions had been made. After 7-9 days of incubation at 37"C, colonies were fixed, stained with crystal violet, and counted. Control plating efficiencies of all cell lines were 60-90%. All survival results were normalized to the appropriate control plating efficiencies. Exposure to H,O, H,O, concentration was determined as previously described (Spitz et al., 1987), and H,O, was added to complete culture medium immediately before addition to the cell cultures. The cells were exposed to H,O, a t 37°C for 1 hr, rinsed 3 times with sterile phosphate buffered saline, trypsinized, counted, and plated for colony formation. Surviving fraction was normalized to control plating efficiency and was plotted as a function of pmoles H,O,/cell added at the beginning of the treatment interval in order to minimize cell density artifacts (Spitz et al., 1987). Heating of cells Heating of cell monolayers on 60 mm dishes was performed as previously described (Spitz et al., 1987) in specially designed, C0,-regulated circulating water baths (Li et al., 1982a). The temperature was maintained within +O.l"C. Cells were provided with fresh medium immediately before heating. Following heating cultures were rinsed, trypsinized, and plated for survival. Protocol for isolation of H,Oz-resistantvariants The H,O,-resistant variants of the HA-1 line were previously derived by culturing cells in progressively higher concentrations of HzOz(Spitz et al., 1988).H,O, resistance was tested periodically following removal from the adaptation protocol. The cells have maintained their resistant phenotype for over 1 year in the absence of H,O,. HA-1 cells cultured for the same

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Ring isolation and characterization of quasidiploid and quasitetraploid cell lines At the 60th passage following removal from the 800 pM H,O, stress, the H,O,-resistant cells were trypsinized, plated, and colonies were ring isolated a s previously described (Spitz et al., 1 9 8 9 ~ )The . isolated colonies were then grown into monolayers and were characterized by growth rates (doubling times), chromosome distributions (chromosomesicell), and responses to cytotoxic insults (H,O, and heat). Doubling times were obtained from plots of cell numbersi60 mm dish vs. time following plating of 1x los cells (Spitz et al., 1 9 8 9 ~ )Chromosomes . were prepared for counting on glass slides utilizing a modified air dried chromosome spreading technique described elsewhere (Spitz et al., 1 9 8 9 ~ )The . slides were mounted and counted using a phase contrast microscope. All cell lines (including HA-1) were either quasidiploid or quasitetraploid. For simplicity they will be referred to a s diploid or tetraploid in the rest of the text. RESULTS Survival response of HA-1 and H,O,-resistant cells to H,02 and heat The HA-1 and H,O,-resistant cells (200 and 800 pM adapted) were exposed to increasing concentrations of H,O, for 1hr a t 37°C and the survival results are shown in Figure 1. At this time, following removal from the adaptation protocol (140 days), these 2 cell

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lines were equally resistant to H202-mediatedcytotoxicity as measured by dose-modifying factor a t 20% isosurvival (DMFs= 29.3 and 29.4) relative to control HA-1 cells. The response of HA-1 and the H,O,-resistant lines (200 and 800 pM adapted) t o 43°C aqd 45°C is shown in Figure 2. Panel A shows that the H,O,-resistant lines were not resistant to 45"C, but panel B demonstrates that both these H,O,-resistant lines were resistant t o 43°C. This heat resistance became more pronounced a t longer heating intervals (4-6 hr). The shapes of the 43°C survival curves suggest that the H,O,-?esistant lines might be developing heat resistance similar to chronic thermotolerance. In the next series of experiments the HA-1 and the 800 pM H,O,-resistant variants were heated a t 42.5"C, 43"C, 44"C, and 45"C,and the survival results are presented in Figure 3. These results demonstrate that the H,O,-resistant variants were resistant to 42.5" and 43°C (panels A,B) heat killing, with less resistance to 44°C (panel C), and almost no resistance to 45°C (panel D). The results a t 43°C were reproduced with and without lethally irradiated (30 Gy) feeder cells (HA-1); the presence of feeder cells had no significant effect on the survival curves (data not shown). These results show that the heat resistance associated with the H202-resistant phenotype was most pronounced a t 43"C, and was almost non-existent when 45°C was used as the heat challenge.

Heat response of heat-selected clones The 800 FM adapted line was heated at 43°C for 10 h r (survival = 1x lop3) and five surviving clones were

ring isolated (after 10 days of growth a t 37°C) to obtain the C232,l-5 clonally isolated populations. A surviving clone from the HA-1 control line (E01) was isolated following selection with 43°C for 6 h r and was used for comparison. All six of the heat-selected clonally isolated cell lines had H,O, survival responses similar to the cell lines from which they were derived (data not shown). Figure 4A shows the 43°C heat response of the HA-1 and E01 control cell lines a s well a s the five heat-selected cell lines derived from the 800 pM adapted H,O,-resistant cells (C232,1-5). This figure shows that all the heat-selected cell lines derived from the H,O,-resistant cells were resistant to 43°C heat killing; but the clone that was heat-selected from the HA-1 cell line (E01) was not resistant to 43°C heat cytotoxicity. Figure 4B shows the 45°C survival response of the cell lines described above. None of the heat-selected clones were more resistant t o 45°C cytotoxicity than HA-1 cells. These results agree well with those obtained with the parental H,O,-resistant cell lines. Clearly, the H,O,-resistant phenotype is associated with resistance to 43°C heat killing, but not 45°C heat killing.

43°C Heat r e s p o n s e in diploid vs. tetraploid H,O,-resistant populations We have shown previously (Spitz et al., 1988; Spitz et al., 1 9 8 9 ~that ) the 800 FM adapted H,O,-resistant cell line is heterogeneous with respect to ploidy. We have also observed (from H20, survival curves) that the diploid cell lines, cloned from the heterogeneous population, have DMFs a t 20% isosurvival ranging from 3 to 27 with a mean of 14+10. The tetraploid cell lines

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cloned from the same heterogeneous population have DMFs ranging from 21 to 49 with a mean of 3 7 k 1 0 (Spitz et al., 1 9 8 9 ~ )Therefore, . the tetraploid cell lines demonstrated significantly greater stable H,O, resistance. In the next series of experiments the 43°C heat response of diploid and tetraploid cell lines were compared in order to determine if changes in ploidy were related to heat resistance. Figure 5A plots the 43°C heat survival response of three diploid (21-22 chromosomesicell) H,O,-resistant lines and HA-1 control and Figure 5B plots the 43°C heat survival response of three tetraploid (40-44 chromosomesicell) H,O,-resistant lines and HA-1 control. This figure shows that some diploid (OC12,OC14) a s well as some tetraploid cell lines (OC5,OC7) were resistant to 43°C heat killing, but there were no apparent differences between the diploid and tetraploid cell lines tested.

DISCUSSION This work demonstrates that mammalian cells adapted to oxidative stress mediated by H202 are resistant to 43°C cell killing but not to 45°C cytotoxicity. We have also shown that H,O,-induced increases in ploidy do not seem to be related to this heat resistance.

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Fig. 5. Heat survival curves of the HA-1 control and clonally isolated diploid (A) and tetraploid (B) H,O,-resistant cell lines following continuous heating of exponentially growing monolayers at 43°C. The diploid and tetraploid H,O,-resistant cell lines were isolated from the 800 pM adapted H,O,-resistant cell line which was heterogeneous with respect to ploidy (Spitz et al., 1 9 8 9 ~ )These . clones were isolated without any cytotoxic selection 60 passages (240 days) following removal of the 800 pM adapted line from the H,O, adaptation protocol. The arrow below the 43°C for 6 hr point, in panel B, indicates that at this time point no colonies survived (from 1.5x lo5 cells plated) in the O C l l cell line.

Another investigation has also shown that higher ploidy mammalian cells do not appear to be heat resistant (Rowley et al., 1987). This implies that mechanisms involved with cellular resistance to H,O, (and not higher ploidy) may also protect cells from heatinduced damage produced by 43°C but these protective mechanisms have little impact on lethal events initiated by 45°C heating. Several other investigators have shown that certain chemical or environmental factors protect or sensitize cells to heat cytotoxicity at 43"C, without dramatically effecting 45°C heat shock responses. Lee and Dewey (1986) have shown that cycloheximide given 2 h r before and during heating affords significant protection to the lethal effects of 43°C heating but has little impact on cell killing mediated by 45°C. Freeman et al. (1980) have shown that low pH heat sensitization is more dramatic at temperatures below 45°C. Mitchell and Russo (1983) have shown that depletion of the cellular antioxidant glutathione (GSH) using either buthionine sulfoximine (BSO) or diethyl maleate (DEM) potentiates heat damage produced by 42.5"C and 43°C to a greater extent than heat damage produced by 45°C. The sensitization to heat-induced kill-

HEAT KILLING IN H,O,-RESISTANT CELLS

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These data support the idea that adaptation to oxiing seen with GSH depletion has also been confirmed by Freeman et al. (1985) and Shrieve et al. (1986). The dative stress is involved with a subset of those events studies with GSH depletion may be particularly perti- which contribute to resistance to 43°C heat-induced lenent to the effects we have reported, since GSH and thality. This may be indicative of common protective some of the enzymes which cycle GSH (glutathione per- mechanisms acting upon common damaging species. oxidase and glutathione-S-transferase) are also in- We have shown that the H202-resistantcells have dravolved in the metabolism and detoxification of hydro- matically increased expression of catalase activity peroxides. These data suggest that the effects that (Spitz et al., 1988; Spitz e t al., 1989c) which correlates modifiers of heat damage have on heat induced lethal- with H202resistance. We have also measured signifiity may depend on the temperature a t which the chal- cant increases in the levels of superoxide dismutase lenge is delivered. There may also be differences in the activity, total GSH, GSH-peroxidase activity, and mechanisms by which 43°C and 45°C hyperthermia kill GSH-transferase activity in H,O,-resistant variants (Spitz et a]., 1989a,b) Future studies will hopefully recells. Several investigators have suggested that oxidative veal which (if any) of these changes in cellular antioxstress may contribute to heat-induced lethality (Mitch- idants may be contributing to resistance to 43°C inell and Russo, 1983; Issels et al., 1986; Christman et duced cytotoxicity. al., 1985; Spitz et al., 1987; Privalle and Fridovich, 1987). This suggestion has been made based on the ACKNOWLEDGMENTS following evidence: This work was supported by NIH grants CA90215 1. The alterations in protein synthesis profiles inand CA31397. duced by heat shock (termed heat-shock proteins) which correlate in time with the induction of resistance LITERATURE CITED to heat damage can also be induced by treatment with presumptive oxidizing agents including H,O, (Ash- Ashburner, M. (1982) The effects of heat shock and other stress on gene activity. In: Heat Shock from Bacteria to Man. M.J. Schlcsburner and Bonner, 1979; Christman et al., 1985; Moringer, M. Ashburner, and A. Tissieres, ed. Cold Spring Harbor Labgan e t al., 1986; Spitz et al., 1987; Lindquist, 1986). oratory, Cold Spring Harbor, New York, pp. 1-10. 2. Pretreatment with presumptive oxidizing agents Ashburner, M. and Bonner, J. (1979)The induction of gene activity in (including H,O,) has been shown to render cells someThosophila by heat shock: a review. Cell, 17t241-254. what resistant to further treatments with heat shock Christman, M.F., Morgan, R.W., Jacobson, F.S., and Ames, B.N. (1985) Positive control of a regulon for defenses against oxidative (Spitz et al., 1987; Li and Shrieve, 1982; Li et al., stress and some heat shock proteins in Salmonella typhimurium. 1982b; Freeman et al., 1987). Cell, 41:753-762. 3. Heat shock has been shown to induce increases in Demple, B., and Halbrook, J. (1983)Inducible repair of oxidative damage in Escherichia coli. Nature, 304r466-468. cellular antioxidants such as glutathione, superoxide dismutase activity, and peroxidase activity (Mitchell Freeman, M.L., Malcolm, A.W., and Meredith, M.J. (1985) Role of glutathione in cell survival after hyperthermic treatment of Chiand RUSSO,1983; Loven et al., 1985; Christman et al., nese hamster ovary cells. Cancer Res., 45t6308-6313. 1985; Morgan et al., 1986; Privalle and Fridovich, Freeman, M.L., Raaphorst, G.P., Hopwood, L.E., and Dewey, W.C. (1980) The effect of pH on cell lethality induced by hyperthermic 1987; Kapoor and Lewis, 1987). treatment. Cancer, 45;2291-2300. 4.Heat shock has been shown to induce resistance to M.L., Scidmore, N.C., Malcolm, A.W., and Meredith, M.J. further treatment with presumptive oxidizing agents Freeman, (1987) Diamide exposure, thermal resistance, and syntheais; of including H,O, (Spitz et al., 1987; Kapoor and Lewis, stress (heat shock) proteins. Biochem. Pharmacol., 36/1).2-29. Henle, K.J., and Dethlefsen, L.A. (1978)Heat fractionation and ther1987; Li e t al., 1982b) motolerance: a review. Cancer Res., 38:1843-1851. 5. Hyperthermic conditions have been shown to inK.J., and Leeper, D.B. (1978) Induction of thermotolerance in crease the reactivity of oxidizing species, the enzymatic Henle, Chinese hamster ovary cells by high (45") or low (40") hyperthergeneration of those species in vitro, and cyanide resismia. Cancer Res., 381570-574. tant respiration in Escherichia coli, which was believed Issels, R.D., Fink, R.M., and Lengfelder, E. (1986) Effects of hyperthermic conditions on the reactivity of oxygen radicals. Free Rad. to indicate a n increase in superoxide production (Issels Kes. Comm., 2121:7-18. et al., 1986; Privalle and Fridovich, 1987). Kapoor, M., and Lewis, J . (1987) Heat shock induces peroxidase acAll these observations provide indirect evidence that tivity in Neurospora crassa and confers tolerance toward oxidative some overlap or common underlying mechanism of acstress. Biochem. Biophys. Ree. Comm., 14713/:904-910. tion does exist between adaptation t o oxidative stress Landry, J., Chretien, P., Bernier, D., Nicole, L.M., Marceau, N., and Tanguay, R.M. (1982) Thermotolerance and heat shock proteins and heat-induced stress. induced by hyperthermia in rat liver cells. Int. J. Radiat. Oncol. Our data with H,O,-resistant mammalian cells, deBiol. Phys.. 8t59-62. rived either by acute exposure to H,O, (Spitz et al., Lee, Y.J., and Dewey, W.C. (1986) Protection of Chinese hamster ovary cells from hyperthermic killing by cycloheximide or puromy1987) or by chronic exposure to H,O, (this paper), inRadiat. Res., 106:98-110. dicate that H,O, resistance is accompanied by some Li,cin. G.C., Petersen, N.S., and Mitchell, H.K. (1982a) Induced thermal resistance to the cytotoxicity of hyperthermia. Howtolerance and heat shock protein synthesis in Chinese hamster ever, the magnitude of the heat resistance seen in ovary cells. Int. J . Kadiat. Oncol. Biol. Phys., 8:63-69. H,O,-resistant cells is not as great as the heat resis- Li, G.C., and Shrieve, D.C. (1982) Thermal tolerance and specific protein synthesis in Chinese hamster fibroblasts exposed to prolonged tance seen in thermotolerant cells. Furthermore, the hypoxia. Exp. Cell Res., 142t464-468. H,O,-resistant phenotype is accompanied by greater Li, G.C., Shrieve, D.C., and Werb, Z. (1982b) Correlations between resistance to 43°C heat-induced lethality than 45°C synthesis of heat-shock proteins and development of tolerance to heat and to adriamycin in Chinese hamster fibroblasts: heat shock heat-induced lethality. One explanation for the 43°C and other inducers. In: Heat Shnck from Bacteria to Man. M.J. heat resistance seen in H202-resistantcell lines might Schlesinger, M. Ashburner, and A. Tissieres, eds. Cold Spring Harinvolve a n increase in the permissive temperature at bor Laboratory, Cold Spring Harbor, New York, pp. 395-404. which chronic thermotolerance develops. Li, G.C., and Werb, Z. (1982) Correlation between synthesis of heat

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shock proteins and development of thermotolerance in Chinese hamster fibroblasts. Proc. Natl. Acad. Sci. USA, 79t3218-3222. Lindquist, S. (1986) The heat-shock response. Ann. Rev. Biochem., 55;1151-1 191. Loven, D.P., Leeper, D.B., and Oberley, L.W. (1985) Superoxide dismutase levels in Chinese hamster ovary cells and ovarian carcinoma cells after hyperthermia or exposure to cycloheximide. Cancer Res., 45t3029-3034. Mitchell, J.B., and Russo, A. (1983) Thiols, thiol depletion, and thermosensitivity. Radiat. Res., 95r471-485. Morgan, R.W., Christman, M.F., Jacobson, F.S., Storz, G., and Ames, B.N. (1986) Hydrogen peroxide inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc. Natl. Acad. Sci. USA, 83t8059-8063. Privalle, C.T., and Fridovich, I. (1987) Induction of superoxide dismutase in Escherichia coli by heat shock. Proc. Natl. Acad. Sci. USA, 84:2723-2726. Rowley, R., Joyner, D.E., and Stewart, J.R. (1987)In vitro response to hyperthermia or x-irradiation of diploid and tetraploid RIF-1 cells separated by centrifugal elutriation. Int. J. Hyperthermia, 3131: 235-244. Shrieve, D.C., Li, G.C., Astromoff, A., and Harris, J.W. (1986) Cellular glutathione, thermal sensitivity, and thermotolerance in Chinese hamster fibroblasts and their heat resistant variants. Cancer Res., 46:1684-1687. Spitz, D.R., Dewey, W.C., and Li, G.C. (1987) Hydrogen peroxide or

heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts. J . Cell. Physiol., 131r364-373. Spitz, D.R., Li, G.C., McCormick, M.L., Sun, Y., and Oberley, L.W. (1988) Stable H,O,-resistant variants of Chinese hamster fibroblasts demonstrate increases in catalase activity. Radiat. Res., 114: 114-124. Spitz, D.R., Elwell, J.H., Sun, Y., Oberley, L.W., Oberley, T.D., Sullivan, S.J., and Roberts, R.J. (1989a) Oxygen toxicity in control and H,O,-resistant Chinese hamster fibroblast cell lines, (submitted). Spitz, D.R., Malcolm, R.R., and Roberts, R.J. (1989b) Cytotoxicity and metabolism of 4-hydroxynonenal and 2-nonenal in H,O, resistant cell lines, (submitted). Spitz, D.R., Mackey, M.A., Li, G.C., Elwell, J.H., McCormick, M.L., and Oberley, L.W. (1989~)The relationship between changes in ploidy and stable cellular resistance to hydrogen peroxide. J. Cell. Physiol., 139t592-598. Subieck. J.R.. Sciandra. J.J.. and Johnson. R.J. (1982) Heat shock proteins and thermotolerance; a comparison of induction kinetics. Br. J. Radiol., 55t579-584. Winquist, L., Rannug, U., Rannug, A,, and Ramel, C. (1984) Protection from toxic and mutagenic Gffects of hydrogen peroxide by catalase induction in Salmonella typhimurium. Mutat. Res., 141t145147. Yang, S.J., Hahn, G.M., and Bagshaw, M.A. (1966) Chromosome aberrations induced by thymidine. Exp. Cell Res., 42:130-135.