INT. J. HYPERTHERMIA,
1992,
VOL.
8,
NO.
1, 121-130
Characteristic synthesis and redistribution of 70 kd heat shock protein in thermotolerant Chinese hamster V79 cells
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T. HATAYAMAS, Y. TANIGUCHIS, E. KANOS, M. FURUYAS, S. HAYASHIS, K. OHTSUKAS, T. WAKATSUKIS, T. KITAMURAS and H. IMAHARAS ?Department of Biochemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, Kyoto 607, Japan $Department of Experimental Radiology and Health Physics, Fukui Medical University School of Medicine, Fukui 910-1 1, Japan §Department of Experimental Radiology, Aichi Cancer Center Research Institute, Nagoya, Aichi 464, Japan (Received I3 February 1991; revised 20 May 1991; accepted 30 May 1991)
Upon exposure to heat shock the increased rate of hsp70 synthesis decreased more rapidly in thermotolerant V79 cells than in the non-thermotolerant cells. However, the levels of hsp70 in the thermotolerant cells at 12 h after a heat shock were almost the same as those in the non-thermotolerant cells. On the other hand, the migration of hsp70 from cytoplasm to nucleoli after a heat shock was very rapid in both thermotolerant and non-thermotolerant cells, but hsp70 in the nucleoli disappeared faster in the thermotolerant cells than in the non-thermotolerant cells, and this coincided with the faster decline of hsp70 synthesis in the thermotolerant cells. For the characteristic distribution of hsp70, protein synthesis was not required. Furthermore, the induction and expression of thermotolerance by the cells were little affected by the inhibition of protein synthesis. Thus, the synthesis of hsp70 itself seemed not to be essential for the induction and expression of thermotolerance of the cells, although hsp70 may be essential for thermoresistance of cells. The rapid decrease of hsp70 synthesis and the rapid disappearance of hsp70 from the nucleoli after a heat shock may be essential for the expression of thermotolerance of the cells. Key words: Hsp70, thermotolerance, cycloheximide, hyperthermia.
1. Introduction Cells of different organisms exposed to a sublethal heat shock acquire resistance to a subsequent heat shock that would normally be lethal, in a phenomenon called acquired thermotolerance (Gerner and Schneider 1975, Henle et al. 1978). Heat shock inhibits synthesis of protein, RNA, or DNA, and hnRNA processing (Henle and Leeper 1979, Yost and Lindquist 1986, Sadis et al. 1988), and causes the collapse of the structure of nucleolus and the intermediate cytoskeletal network (Collier and Schlesinger 1986, Shyy et al. 1989). In thermotolerant cells a variety of metabolic pathways are, however, protected following stress, or can be repaired faster (Mizzen and Welch 1988, Welch and Mizzen 1988, Lepock et al. 1990). Although no direct evidence has been provided to ascertain the role of heat shock proteins in rendering thermotolerance much evidence supports the idea that heat shock proteins, especially hsp70, are important in protecting cells against thermal damage or facilitating the recovery of cells from the damage caused by heat or other stresses (Pelham 1984, Lewis and Pelham 1985). A high level of hsp70 in cells is associated with their increased thermoresistance (Laszlo and Li 1985, Li and Werb 1982). Heat shock is lethal to fibroblasts microinjected with antibody against hsp70 (Riabowol et al. 1988), and a competitive inhibition of hsp70 gene expression causes thermo0265.6736192 $3.W 01992 Taylor & Francis Ltd
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sensitization of the cells (Johnston and Kucey 1988). Hsp70 has been classified into two forms depending on its expression in mammalian cells: a constitutive form that exists in large amounts in untreated cells and is induced at a low level by stress, and an inducible form that is not expressed, or else is expressed at a basal level in untreated cells and induced to a great extent by stress (Welch and Feramisco 1984). We have shown that thermotolerance can be induced in Chinese hamster V79 cells by various treatments (Hatayama et al. 1991). Heating of the cells at 42°C for 4 h induces thermotolerance immediately after the treatment. Heating of the cells at 44°C for 20 min, a treatment of the cells with 50 ~ L sodium M arsenite for ?$h, or a treatment of the cells with 20 pg/ml puromycin for 4 h induces thermotolerance at 4 h after the treatment. At 17 h after the heat treatments, cells heated at 42°C or 44°C are still thermotolerant, but thermotolerance induced by sodium arsenite or puromycin has diminished by this time. Upon exposure to the conditions under which thermotolerance is induced, the synthesis of a constitutive form of hsp70, hsp85, and hspl05 increases, but an inducible form of hsp70 is not synthesized (Hatayama et al. 1991). Here, to characterize the role of heat shock proteins in the expression of thermotolerance, we examined the synthesis and distribution of hsp70 in thermotolerant Chinese hamster V79 cells, and further determined whether the synthesis of hsp70 was essential for the induction and expression of thermotolerance in the cells. 2. Materials and methods 2.1. Cell culture and stress conditions Chinese hamster V79 cells were grown at 37°C as a monolayer in Eagle's minimal essential medium (Nissui, 7 - 3 g/l) containing NCTC-135 (Difco, 4.7 g/l), lactalbumin hydrolysate (Difco, 0.5 g/l), and heat-inactivated calf serum (Biken, 10%; this medium is called MLN-10) in a CO, incubator (5% C02 in air) at 37°C. Exponentially growing cells (6x 105/35-mmdish) were heated at 42°C for 4 h or at 44°C for 20 min in a water bath set at 42°C or at 44"C, respectively, or else were treated with 50 p~ sodium arsenite for 3 h or with 20 pg/ml puromycin for 4 h at 37°C (Hatayama et al. 1991). 2.2. Radioisotope labelling of cells Cells were incubated with MLN-10 containing 10-20 pCi/ml [''Slmethionine (1 100 Ci/mmol: New England Nuclear). After the labelling the radioactive medium was removed and the cells were washed twice with cold phosphate-buffered saline, solubilized in 0.1 % sodium dodecyl sulphate (SDS), boiled for 2 min, and stored at -20°C (Hatayama et al. 1986). The radioactivity incorporated into proteins was assayed by the count precipitated with hot trichloroacetic acid. 2.3. SDS-polyacrylamide gel electrophoresis Protein samples were electrophoresed on SDS- 10% polyacrylamide slab gels (Laemmli 1970). After electrophoresis, gels were stained with Coomassie brilliant blue R, destained, and dried. The dried gels containing labelled proteins were autoradiographed. The radioactivity incorporated into a protein was analysed with the use of a radioanalytic imaging system (AMBIS). The protein concentration was assayed by the Bradford method (Bradford 1976). 2.4. Immunoblot analysis Proteins separated on SDS-polyacrylamide slab gels were blotted onto a nitrocellulose membrane by an electrotransfer (Burnette 1981). Antigen-antibody complexes were stained
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with immunoperoxidase (Vectastatin ABC kit). The density of the stained bands was measured with a Shimadzu CS-910 Chromatoscanner. 2.5. Indirect immunojluorescence The intracellular distribution of hsp70 was studied with the use of rabbit anti-mouse hsp70 antibody diluted 60-fold (Ohtsuka and Laszlo 1989). Cells grown on 15-mm glass coverslips were fixed and made permeable by exposure to absolute methanol at -20°C for 10 min. Goat anti-mouse IgG antibody conjugated with fluorescein and diluted 50-fold was used to detect the primary antibody. The percentage of cells with brightly stained nucleoli was calculated by analysing more than 400 cells for each point. 2.6. Cell survival Cells were cultured at 37"C, so as to form colonies, in a CO, incubator. After 7-10 days of incubation the colonies were stained with crystal violet and counted.
3. Results 3.1. Synthesis of hsp 70 in thermotolerunt 1/79 cells To examine the role of synthesis of hsp70 during the expression of thermotolerance in V79 cells, the synthesis of hsp70 in the thermotolerant and non-thermotolerant cells after a challenging heat shock were analysed (Figure 1). When untreated V79 cells were treated with the challenging heat shock, the rate of synthesis of hsp70 increased gradually for 4-8 h. On the other hand, in the cells that had acquired thermotolerance immediately after being heated at 42°C for 4 h or at 4 h after being heated at 44°C for 20 min, the rate of synthesis of hsp70 which had been already enhanced did not increase, and rather decreased, after the heat shock. In the cells thermotolerant at 17 h after being heated at 44°C or at 42"C, the rate of synthesis of hsp70 increased immediately after the heat shock and decreased more rapidly than in non-thermotolerant cells. The rate of synthesis of actin was not different between the thermotolerant and non-thermotolerant cells. Furthermore, when the hsp70 level in V79 cells at 12 h after the challenging heat shock was estimated by immunostaining with anti-hsp70 serum, the level was not significantly different between the cells with thermotolerance and those without (Table 1). 3.2. lntrucellulur distribution of hsp70 during the induction and expression of thermotolerance Figure 2 shows the intracellular distribution of hsp70 in V79 cells during and after the induction of thermotolerance by various treatments, examined by an indirect immunofluorescence using anti-hsp70 serum. Hsp70 localized in the cytoplasm of unstressed cells, whereas hsp70 was found in the nucleoli as well as in the cytoplasm after a heat shock at 44°C for 20 min'. The proportion of cells with stained nucleoli gradually reduced by 4 or 17 h after the heat shock. When V79 cells were heated at 42°C for 1 h, hsp70 was found in the nucleoli of only 30% of the cells. During further incubation at 42°C the number of cells with the stained nucleoli decreased, and no cells had the stained nucleoli even after 4 h incubation at 42"C, at which the cell had acquired thermotolerance. During and after the induction of thermotolerance in the cells treated with sodium arsenite or puromycin, hsp70 was not found in any nucleoli. When unstressed V79 cells or the cells which had acquired thermotolerance after being heated at 44°C or 42°C were challenged by a heat shock at 44°C for 30 min, hsp70 was immediately found in the nucleoli of both the non-thermotolerant and thermotolerant cells (Figure 3). At 6 h after the challenging heat shock, hsp7O was still found in the nucleoli of almost all non-thermotolerant cells, and the proportion of the cells with the stained
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Time ( h ) or Figure I . Rate of synthesis of hsp70 in V79 cells after a heat shock. Untreated V79 cells (0) in the cells first treated at 44°C for 20 min (0)were incubated at 37°C for 4 h (A, C) or 17 h (B, D). The cells were then labelled with 20 pCi/ml [35S]methionine for 1 h (before HS), or challenged by a heat shock at 44°C for 30 min (HS) and then pulse-labelled with [%]methionine for 1 h every 2 h interval. When V79 cells were first heated at 42°C for 4 h (A),the cells were treated by the challenging heat shock immediately (A, C) or at 17 h (B, D) after the first heating. Ten micrograms of protein from these cells was analysed by SDS- 10% polyacrylamide gel electrophoresis. The radioactivity incorporated into the protein was analysed with the use of a raidoanalytic imaging system (AMBIS), and the rates of synthesis of hsp70 (A, B) and actin (C, D) were calculated as percentages of total protein synthesis.
nucleoli gradually decreased to about 50% and 20%, at 9 h and 12 h later, respectively. However, in the cells that acquired thermotolerance at 4 h after being heated at 44°C for 20 min o r immediately after being heated at 42°C for 4 h, the proportion of the cells with the stained nucleoli drastically decreased by 6 h after the challenging heat shock. In the cells that were still thermotolerant at 17 h after being heated at 44°C for 20 min, or at Table 1. Assay of the amount of hsp70 in V79 cells at 12 h after heat shock. Relative amount of hs1170 Interval between first and second heating Oh
4h 17 h
Control cells
('%)
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42°C
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Untreated V79 cells or the cells first treated at 44°C for 20 min were incubated at 37°C for 4 or 17 h. The cells were then treated with a heat shock at 44°C for 30 rnin. and further incubated at 37°C for 12 h. When the cells were first heated at 42°C for 4 h. the cells were heat-shocked at 44°C for 30 min immediately or at 17 h after the first heating. and then incubated at 37°C for 12 h. Hsp70 in these cells was detected irnrnunologically and assayed by densitometric scanning. The numbers represent the relative amount of hsp70 compared to control cells. Each value is the mean f SE of three determinations. The values of hsp70 in the thermotolertant cells were not significantly different from the control value ( p < O . O 5 ) .
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Figure 2. Intracellular distribution of hsp70 in V79 cells during the induction of thermotolerance. (A) Untreated V79 cells at 37°C. V79 cells were heated at 44°C for 20 min (B) and then incubated at 37°C for 4 h (C) or 17 h (D); or V79 cells were heated at 42°C for 1 h (E), 2 h (F), o r 4 h ( G ) ;after the 4 h of heating the cells were incubated at 37°C for 17 h (H). Some cells were treated with 50 W M sodium arsenite for 1 .5 h (I) or 3 h (J); after the 3 h of treatment the cells were incubated at 37°C for 4 h (K) or 17 h (L). Other cells were treated with 20 pgiml puromycin for 2 h (M) or 4 h (N); after the 4 h of treatment the cells were incubated at 37°C for 4 h (0)or 17 h (P). The distribution of hsp70 was analysed by indirect inimunofluorescence with anti-hsp70 serum.
42°C for 4 h, the proportion of cells with the stained nucleoli also drastically decreased by 6 h or 9 h, respectively, after the challenging heat shock. To test newly synthesized proteins are required for the migration of hsp70, the protein synthesis of V79 cells was inhibited by cycloheximide. Cycloheximide at a concentration of 5 pg/ml inhibited more than 95 % of cellular protein synthesis of these cells, whether they were thermotolerant or not. Electrophoretic analysis of the proteins showed that heat shock proteins were not synthesized preferentially over other proteins in the presence of the drug (data not shown). When untreated cells were heat-shocked at 44°C for 30 min the proportion of the cells with the stained nucleoli gradually decreased after the heat shock, as shown in Figures 3 and 4, but the rate of the decrease was somewhat stimulated when the heated cells were incubated with cycloheximide (Figure 4). When the cells first heatshocked at 44°C for 20 min were incubated with or without cycloheximide for 4 h, and then given the second heat shock at 44°C for 30 min, hsp70 was immediately found in the nucleoli of almost all cells, whether treated with cycloheximide or not. The proportion of cells with the stained nucleoli decreased rapidly after the second heat shock, whether
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Time (hr) Figure 3. Nucleolar localization of hsp70 in V79 cells after a challenging heat shock. Untreated V79 cells (0)or the cells first treated at 44°C for 20 min (0)were incubated at 37°C for 4 h (A) or 17 h (B) (before HS), and then challenged by a heat shock (HS) at 44°C for 30 min. When V79 cells were first heated at 42°C for 4 h (A), the cells were treated by the challenging heat shock immediately (A) or at 17 h (B) after the first heating. After the challenging heat shock the cells were incubated at 37°C for 0, 3, 6. 9. or 12 h, and the distribution of hsp70 was analysed by indirect immunofluorescence with anti-hsp70 serum. The average percentage of cells with stained nucleoli from at least two independent experiments was plotted.
the cells were incubated with or without cycloheximide before and/or after the second heat shock (Figure 4). 3.3. Effects of cycloheximide on the induction and expression of thermotolerunce During the initial period of the induction of thermotolerance, V79 cells preferentially synthesize heat shock proteins, hsp70, hsp85, and hspl05, over other proteins (Hatayama et ul. 1991). Thermotolerant V79 cells synthesized hsp70 more rapidly after an exposure to heat shock with the synthesis decreasing rapidly thereafter than did the non-chermotolerant cells. To discover whether these characteristic syntheses of heat shock proteins in V79 cells are essential for the induction and expression of thermotolerance, the effect of inhibition of protein synthesis was analysed. By treating the cells with 5 pg/ml cycloheximide for 4 h, about 80% of the cells survived, and when the cells were incubated at 37°C with cycloheximide for 4 h after heat shock at 44°C for 0-60 min, the cell survivals of the cells were also about 80% of those of the cells heated and incubated without the drug. To induce thermotolerance, V79 cells were first heated at 44°C for 20 min and incubated at 37°C for 4 h. The cells were then challenged by a heat shock at 44°C for 0-60 min. Whether cycloheximide was added during the incubation of the cells between the first and second heat shocks, or during the 4 h of incubation after the second heat shock, or both, all of these cells became equally thermotolerant (Figure 5). 4. Discussion Hsp7O localizes in the cytoplasm of unstressed cells, but it is found in the nucleoli of various kinds of cells immediately after an exposure to heat shock (Welch and Feramisco 1984, Munro and Pelham 1984). Since the expression of high levels of hsp70 in COS cells results in a rapid recovery of normal nucleolar morphology after a heat shock, hsp70 localized to the nucleoli may catalyse the reassembly of preribosomes which were damaged by heat shock (Pelham 1984). In the present study the distribution of hsp70 was examined
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Time (hr)
Figure 4. Effects of cycloheximide on the intracellular distribution of hsp70. Untreated V79 cells were heated at 44°C for 30 min and then incubated at 37°C for 0, 3, 6, 9, or 12 h in the presence (0)or absence (0)of 5 &ml cycloheximide; or V79 cells first heated at 44°C for 20 min were incubated at 37°C for 4 h, then heat-shocked at 44°C for 30 min. and further incubated at 37°C for 0, 3. or 6 h in the presence (A)or absence (A) of cycloheximide. Some cells first heated at 44°C for 20 min were incubated at 37°C for 4 h in the presence of cycloheximide. then heat-shocked at 44°C for 30 min in the absence of cycloheximide, and further incubated at 37°C for 0 ,3, or 6 h in the presence (W) or absence (0) of cycloheximide. The distribution of hsp70 was analysed by indirect immunofluorescence with anti-hsp70 serum. The average percentage of cells with stained nucleoli from at least two independent experiments was plotted.
during the induction and expression of thermotolerance, since the level of hsp70 was not different between the thermotolerant and non-thermotolerant V79 cells. Hsp70 migrated to the nucleoli during the induction of thermotolerance of V79 cells by being heated at 42°C or 44"C, but not by treatment with sodium arsenite or puromycin, suggesting that the migration of hsp70 from cytoplasm to nucleoli was not essential for the cells to become thermotolerant. On the other hand, although hsp70 immediately migrated to the nucleoli in both thermotolerant and non-thermotolerant cells after a heat shock, hsp70 in the nucleoli of the non-thermotolerant cells disappeared more gradually than that of the thermotolerant cells during incubation at 37°C after a heat shock. Hsp70 disappears rapidly from the nucleoli in thermotolerant HA- 1 cells and the thermo-resistant variants as well (Ohtsuka and Laszlo 1989). So the phenomena seem to occur in thermotolerant cells in general. The rapid disappearance of hsp70 from the nucleoli of thermotolerant cells may mean that the damage of the nucleoli caused by heat shock is less in thermotolerant cells, or that the damage is repaired rapidly in thermotolerant cells. Since the level of hsp70 which might catalyse the reassembly of damaged preribosomes was not elevated in thermotolerant V79 cells, the damage of the nucleoli caused by heat shock may be less in the thermotolerant cells than in the non-thermotolerant cells. Inhibition of protein synthesis by cycloheximide in the thermotolerant and non-thermotolerant cells did not affect the immediate migration of hsp70 to the nucleoli after heat shock, suggesting that newly synthesized proteins were not required for the migration of hsp70 in V79 cells. The disappearance of hsp70 from the nucleoli of the non-thermotolerant cells was somewhat stimulated when the cells were incubated with cycloheximide. However, hsp70 in the nucleoli disappeared much faster in the thermotolerant cells than in the non-thermotolerant cells either in the presence or absence of cycloheximide. Cycloheximide may stimulate the disappearance of hsp70 from the nucleoli of the non-thermo-
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Figure 5 . Effects of cyclohexirnide on the induction and expression of therrnotolerance in V79 cells. V79 cells heated at 44°C for 20 min were incubated at 37°C for 4 h, then treated with a heat shock at 44°C for 0-60 min. and further incubated at 37°C for 4 h in the presence (C) or absence (A) o f 5 wg/ml cycloheximide (0). The cells heated at 44°C for 20 rnin were incubated at 37°C for 4 h in the presence of cycloheximide, then treated with a heat shock at 44°C for 0-60 niin in the absence of cycloheximide. and further incubated at 37°C for 4 h in the presence (D) or absence (B) of cycloheximide (0).As a control, untreated V79 cells were treated with a heat shock at 44°C for 0-60 min without cycloheximide (0). All cell5 were then incubated at 37°C without cycloheximide for 7-10 days for colony formation.
tolerant cells, probably as a result of its protective effects such as the stabilization of polysomes (McCormick and Penman 1969) and the suppression of heat-induced DNA repair inhibition (Armour et al. 1988). After a heat shock the increased rate of hsp70 synthesis diminished faster in thermotolerant V79 cells than in the non-thermotolerant cells. On the other hand, the rate of synthesis of actin, one of main cellular proteins, was not significantly different from these cells. Furthermore, since the rates of synthesis of hsp85 and hspl05 were also not significantly different between these cells (data not shown), the phenomenon seemed to be specific to hsp70. The rapid decline of hsp70 synthesis coincided with the faster disappearance of hsp70 from the nucleoli of the thermotolerant cells. However, the levels of hsp70 in the thermotolerant cells at 12 h after the heat shock were almost the same as those in the non-thermotolerant cells. The rapid decrease of hsp70 synthesis after heat shock may indicate that the production of hsp70 is self-regulated in stressed cells, as has been suggested by others (Mizzen and Welch 1988, DiDomenico er al. 1982). Cycloheximide, an inhibition of protein synthesis, inhibits the induction of thermotolerance in some cells (Henle and Leeper 1982, Freeman et al. 1987, Lee and Dewey 1987a) but not in others (Ohtsuka et a/.1986, Widelitz et al. 1986). However, in these studies it is not clear how effectively this drug blocks synthesis of proteins, heat shock proteins in particular, in various kinds of cells (Lee and Dewey 198713). In the present study, protein synthesis was inhibited more than 95% by 5 pg/ml cycloheximide in both non-thermotolerant and thermotolerant V79 cells, and heat shock proteins were not preferentially synthesized compared to other proteins under the conditions. V79 cells synthesize
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hsp70 preferentially during the initial period of the induction of thermotolerance (Hatayama et al. 1991). However, the inhibition of the characteristic synthesis of hsp70 by cycloheximide did not affect the induction and expression of thermotolerance in V79 cells. Thus, the synthesis of hsp70 itself seemed not to be directly related to the induction and expression of thermotolerance in the cells. The rapid decrease of hsp70 synthesis and the rapid disappearance of hsp70 from the nucleoli after a heat shock may be essential for the expression of thermotolerance. However, these phenomena may simply represent characteristics of thermotolerant cells. The molecular mechanisms underlying the phenomena remain to be elucidated.
Acknowledgements This study was supported in part by Grant-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture, Japan. References ARMOUR, E. P., LEE, Y. J., CORRY,P. M., and BORELLI,M. J., 1988, Protection from heatinduced protein migration and DNA repair inhibition by cycloheximide. Biochemical and Biophysical Research Communications, 157, 61 1-617. BRADFORD, M. M., 1976, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72,248-254. BURNETTE, W. N., 1981, ‘Western blotting’: electrophoretic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry, 112, 195-203. COLLIER, N. C., and SCHLESINGER, M. J., 1986, The dynamic state of heat shock proteins in chicken embryo fibroblasts. Journal of Cell Biology, 103, 1495-1507. DIDOMENICO, B. J., BUGAISKY, G. E., and LINDQUIST, S., 1982, The heat shock response is selfregulated at both the transcriptional and posttranscriptional levels. Cell, 31, 593-603. FREEMAN, M. L., SCIDMORE, N. C., and MEREDITH, M. J., 1987, Inhibition of heat shock protein synthesis and thermotolerance by cycloheximide. Radiation Research, 112, 564-574. GERNER,E. W . , and SCHNEIDER, M. J., 1975, Induced thermal resistance in HeLa cells. Nature, 256, 500-502. T., HONDA,K., and YUKIOKA, M., 1986, HeLa cells synthesize a specific heat shock HATAYAMA, protein at 42°C but not at 45°C. Biochemical and Biophysical Research Communications, 137, 957-963. T., KANO,E., TANIGUCHI, Y., NITTA,K., WAKATSUKI, T., KITAMURA, T., and HATAYAMA, IMAHARA, H., 1991, Role of heat-shock proteins in the induction of thermotolerance in Chinese hamster V79 cells by heat and chemical agents. International Journal of Hyperrhemzia, 7, 61-74. HENLE,K. J., KARAMUZ, J. E., and LEEPER,D. B., 1978, Induction of thermotolerance in Chinese hamster ovary cells by high (45°C) or low (40°C) hyperthermia. Cancer Research, 38, 570-574. HENLE,K. J., and LEEPER,D. B., 1979, Effects of hyperthermia (45°C) on macromolecular synthesis in Chinese hamster ovary cells. Cancer Research, 39, 2665-2674. HENLE,K. J., and LEEPER,D. B., 1982, Modification of the heat response and thermotolerance by cycloheximide, hydroxyurea, and lucanthone in CHO cells. Radiation Research, 90, 339-347. JOHNSTON, R. N., and KUCEY,B. L., 1988, Competitive inhibition of hsp70 gene expression causes thermosensitivity. Science, 242, 1551-1554. LAEMMLI, U. K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. LASZLO,A,, and LI, G. C., 1985, Heat-resistant variants of Chinese hamster fibroblasts altered in expression of heat shock protein. Proceedings of the National Academy of Sciences, USA, 82, 8029-8033. LEE, Y. J., and DEWEY,W . C., 1987a, Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins. Journal of Cellular Physiology, 132, 41-48. LEE, Y. J., and DEWEY,W . C., 1987b, Effect of cycloheximide or puromycin on induction of
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thermotolerance by heat in Chinese hamster ovary cells: dose fractionation at 4 5 ~ 5 ° CCancer . Research, 47, 5960-5966. LEPOCK,J . R., FREY,H. E., HEYNEN, M. P., NISHIO,J., WATERS,B., RITCHIE,K. P., and KRUUV, J., 1990, Increased thermostability of thermotolerant CHL V79 cells as determined by different scanning calorimetry. Journal of Cellular Physiology, 142, 628-634. LEWIS,M. J . , and PELHAM,H. R. B., 1985, Involvement of ATP in the nuclear and nucleolar functions of the 70 kd heat shock protein. EMBO Journal, 4, 3137-3143. LI, G. C., and WERE, Z . , 1982, Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts. Proceedings of the National Academy of Sciences, USA, 79, 3218-3222. MCCORMICK, W., and PENMAN, S . , 1969, Regulation of protein synthesis in HeLa cells: translation at elevated temperatures. Journal of Molecular Biology, 39, 315-333. MIZZEN,L. A,, and WELCH,W. J . , 1988, Characterization of the thermotolerant cell. I. Effect on protein synthesis activity and the regulation of heat-shock protein 70 expression. Journal of Cell Biology, 106, 1105-1 116. MUNRO,S . , and PELHAM, H. R. B . , 1984, Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp70. EMBO Journal, 3, 3087-3093. OHTSUKA, K., FURUYA, M., NITTA,K., and KANO,E., 1986, Effect of cycloheximide on the development of thermotolerance and the synthesis of 68-kilodalton heat shock protein in Chinese hamster V79 and mouse L cells in vitro. Journal of Radiation Research, 27, 291-299. OHTSUKA, K., and LASZLO,A,, 1989, Intracellular localization of hsp70 in normal and thermotolerant HA-1 Chinese hamster fibroblasts and in their heat resistant variants. Hyperthermic Oncology 1988, Vol. I , Summary Papers, edited by T. Sugahara and M. Saito (London, New York, Philadelphia: Taylor & Francis), pp. 56-58. PELHAM, H. R. B., 1984, Hsp70 accelerates the recovery of nucleolar morphology after heat shock. EMBO Journal, 3, 3095-3100. RIABOWOL, K., MIZZEN,L. A., and WELCH,W. J . , 1988, Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70. Science, 242, 433-436. E., and WEBER,L. A,, 1988, Effect of heat shock on RNA metabolism in HeLa SADIS,S., HICKEY, cells. Journal of Cellular Physiology, 135, 377-386. SHYY,T-T., ASCH, B. B, and ASCH, H. L., 1989, Concurrent collapse of keratin filaments, aggregation of organelles, and inhibition of protein synthasis during the heat shock response in mammary epithelial cells. Journal of Cell Biology, 108, 997-1008. J. R., 1984, Nuclear and nucleolar localization of 72,000-dalton WELCH,W. J . , and FERAMISCO, heat shock protein in heat-shocked mammalian cells. Journal of Biological Chemistry, 259, 4501 -45 13. WELCH,W. J . , and MIZZEN,L. A,, 1988, Characterization of the thermotolerant cell. 11. Effect on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes. Journal of Cell Biology, 106, I 1 17-1 130. R. B., MAGUN,B . , and GERNER, E. W., 1986, Effect of cycloheximide on thermoWIDELITZ, tolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts. Molecular and Cellular Biology, 6, 1088-1094. YOST,H. J . , and LINDQUIST, S . , 1986, RNA splicing is interrupted by heat shock and rescued by heat shock protein synthesis. Cell, 45, 185-193.
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Characteristic synthesis and redistribution of 70 kd heat shock protein in thermotolerant Chinese hamster V79 cells
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T. HATAYAMAS, Y. TANIGUCHIS, E. KANOS, M. FURUYAS, S. HAYASHIS, K. OHTSUKAS, T. WAKATSUKIS, T. KITAMURAS and H. IMAHARAS ?Department of Biochemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto, Kyoto 607, Japan $Department of Experimental Radiology and Health Physics, Fukui Medical University School of Medicine, Fukui 910-1 1, Japan §Department of Experimental Radiology, Aichi Cancer Center Research Institute, Nagoya, Aichi 464, Japan (Received I3 February 1991; revised 20 May 1991; accepted 30 May 1991)
Upon exposure to heat shock the increased rate of hsp70 synthesis decreased more rapidly in thermotolerant V79 cells than in the non-thermotolerant cells. However, the levels of hsp70 in the thermotolerant cells at 12 h after a heat shock were almost the same as those in the non-thermotolerant cells. On the other hand, the migration of hsp70 from cytoplasm to nucleoli after a heat shock was very rapid in both thermotolerant and non-thermotolerant cells, but hsp70 in the nucleoli disappeared faster in the thermotolerant cells than in the non-thermotolerant cells, and this coincided with the faster decline of hsp70 synthesis in the thermotolerant cells. For the characteristic distribution of hsp70, protein synthesis was not required. Furthermore, the induction and expression of thermotolerance by the cells were little affected by the inhibition of protein synthesis. Thus, the synthesis of hsp70 itself seemed not to be essential for the induction and expression of thermotolerance of the cells, although hsp70 may be essential for thermoresistance of cells. The rapid decrease of hsp70 synthesis and the rapid disappearance of hsp70 from the nucleoli after a heat shock may be essential for the expression of thermotolerance of the cells. Key words: Hsp70, thermotolerance, cycloheximide, hyperthermia.
1. Introduction Cells of different organisms exposed to a sublethal heat shock acquire resistance to a subsequent heat shock that would normally be lethal, in a phenomenon called acquired thermotolerance (Gerner and Schneider 1975, Henle et al. 1978). Heat shock inhibits synthesis of protein, RNA, or DNA, and hnRNA processing (Henle and Leeper 1979, Yost and Lindquist 1986, Sadis et al. 1988), and causes the collapse of the structure of nucleolus and the intermediate cytoskeletal network (Collier and Schlesinger 1986, Shyy et al. 1989). In thermotolerant cells a variety of metabolic pathways are, however, protected following stress, or can be repaired faster (Mizzen and Welch 1988, Welch and Mizzen 1988, Lepock et al. 1990). Although no direct evidence has been provided to ascertain the role of heat shock proteins in rendering thermotolerance much evidence supports the idea that heat shock proteins, especially hsp70, are important in protecting cells against thermal damage or facilitating the recovery of cells from the damage caused by heat or other stresses (Pelham 1984, Lewis and Pelham 1985). A high level of hsp70 in cells is associated with their increased thermoresistance (Laszlo and Li 1985, Li and Werb 1982). Heat shock is lethal to fibroblasts microinjected with antibody against hsp70 (Riabowol et al. 1988), and a competitive inhibition of hsp70 gene expression causes thermo0265.6736192 $3.W 01992 Taylor & Francis Ltd
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sensitization of the cells (Johnston and Kucey 1988). Hsp70 has been classified into two forms depending on its expression in mammalian cells: a constitutive form that exists in large amounts in untreated cells and is induced at a low level by stress, and an inducible form that is not expressed, or else is expressed at a basal level in untreated cells and induced to a great extent by stress (Welch and Feramisco 1984). We have shown that thermotolerance can be induced in Chinese hamster V79 cells by various treatments (Hatayama et al. 1991). Heating of the cells at 42°C for 4 h induces thermotolerance immediately after the treatment. Heating of the cells at 44°C for 20 min, a treatment of the cells with 50 ~ L sodium M arsenite for ?$h, or a treatment of the cells with 20 pg/ml puromycin for 4 h induces thermotolerance at 4 h after the treatment. At 17 h after the heat treatments, cells heated at 42°C or 44°C are still thermotolerant, but thermotolerance induced by sodium arsenite or puromycin has diminished by this time. Upon exposure to the conditions under which thermotolerance is induced, the synthesis of a constitutive form of hsp70, hsp85, and hspl05 increases, but an inducible form of hsp70 is not synthesized (Hatayama et al. 1991). Here, to characterize the role of heat shock proteins in the expression of thermotolerance, we examined the synthesis and distribution of hsp70 in thermotolerant Chinese hamster V79 cells, and further determined whether the synthesis of hsp70 was essential for the induction and expression of thermotolerance in the cells. 2. Materials and methods 2.1. Cell culture and stress conditions Chinese hamster V79 cells were grown at 37°C as a monolayer in Eagle's minimal essential medium (Nissui, 7 - 3 g/l) containing NCTC-135 (Difco, 4.7 g/l), lactalbumin hydrolysate (Difco, 0.5 g/l), and heat-inactivated calf serum (Biken, 10%; this medium is called MLN-10) in a CO, incubator (5% C02 in air) at 37°C. Exponentially growing cells (6x 105/35-mmdish) were heated at 42°C for 4 h or at 44°C for 20 min in a water bath set at 42°C or at 44"C, respectively, or else were treated with 50 p~ sodium arsenite for 3 h or with 20 pg/ml puromycin for 4 h at 37°C (Hatayama et al. 1991). 2.2. Radioisotope labelling of cells Cells were incubated with MLN-10 containing 10-20 pCi/ml [''Slmethionine (1 100 Ci/mmol: New England Nuclear). After the labelling the radioactive medium was removed and the cells were washed twice with cold phosphate-buffered saline, solubilized in 0.1 % sodium dodecyl sulphate (SDS), boiled for 2 min, and stored at -20°C (Hatayama et al. 1986). The radioactivity incorporated into proteins was assayed by the count precipitated with hot trichloroacetic acid. 2.3. SDS-polyacrylamide gel electrophoresis Protein samples were electrophoresed on SDS- 10% polyacrylamide slab gels (Laemmli 1970). After electrophoresis, gels were stained with Coomassie brilliant blue R, destained, and dried. The dried gels containing labelled proteins were autoradiographed. The radioactivity incorporated into a protein was analysed with the use of a radioanalytic imaging system (AMBIS). The protein concentration was assayed by the Bradford method (Bradford 1976). 2.4. Immunoblot analysis Proteins separated on SDS-polyacrylamide slab gels were blotted onto a nitrocellulose membrane by an electrotransfer (Burnette 1981). Antigen-antibody complexes were stained
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with immunoperoxidase (Vectastatin ABC kit). The density of the stained bands was measured with a Shimadzu CS-910 Chromatoscanner. 2.5. Indirect immunojluorescence The intracellular distribution of hsp70 was studied with the use of rabbit anti-mouse hsp70 antibody diluted 60-fold (Ohtsuka and Laszlo 1989). Cells grown on 15-mm glass coverslips were fixed and made permeable by exposure to absolute methanol at -20°C for 10 min. Goat anti-mouse IgG antibody conjugated with fluorescein and diluted 50-fold was used to detect the primary antibody. The percentage of cells with brightly stained nucleoli was calculated by analysing more than 400 cells for each point. 2.6. Cell survival Cells were cultured at 37"C, so as to form colonies, in a CO, incubator. After 7-10 days of incubation the colonies were stained with crystal violet and counted.
3. Results 3.1. Synthesis of hsp 70 in thermotolerunt 1/79 cells To examine the role of synthesis of hsp70 during the expression of thermotolerance in V79 cells, the synthesis of hsp70 in the thermotolerant and non-thermotolerant cells after a challenging heat shock were analysed (Figure 1). When untreated V79 cells were treated with the challenging heat shock, the rate of synthesis of hsp70 increased gradually for 4-8 h. On the other hand, in the cells that had acquired thermotolerance immediately after being heated at 42°C for 4 h or at 4 h after being heated at 44°C for 20 min, the rate of synthesis of hsp70 which had been already enhanced did not increase, and rather decreased, after the heat shock. In the cells thermotolerant at 17 h after being heated at 44°C or at 42"C, the rate of synthesis of hsp70 increased immediately after the heat shock and decreased more rapidly than in non-thermotolerant cells. The rate of synthesis of actin was not different between the thermotolerant and non-thermotolerant cells. Furthermore, when the hsp70 level in V79 cells at 12 h after the challenging heat shock was estimated by immunostaining with anti-hsp70 serum, the level was not significantly different between the cells with thermotolerance and those without (Table 1). 3.2. lntrucellulur distribution of hsp70 during the induction and expression of thermotolerance Figure 2 shows the intracellular distribution of hsp70 in V79 cells during and after the induction of thermotolerance by various treatments, examined by an indirect immunofluorescence using anti-hsp70 serum. Hsp70 localized in the cytoplasm of unstressed cells, whereas hsp70 was found in the nucleoli as well as in the cytoplasm after a heat shock at 44°C for 20 min'. The proportion of cells with stained nucleoli gradually reduced by 4 or 17 h after the heat shock. When V79 cells were heated at 42°C for 1 h, hsp70 was found in the nucleoli of only 30% of the cells. During further incubation at 42°C the number of cells with the stained nucleoli decreased, and no cells had the stained nucleoli even after 4 h incubation at 42"C, at which the cell had acquired thermotolerance. During and after the induction of thermotolerance in the cells treated with sodium arsenite or puromycin, hsp70 was not found in any nucleoli. When unstressed V79 cells or the cells which had acquired thermotolerance after being heated at 44°C or 42°C were challenged by a heat shock at 44°C for 30 min, hsp70 was immediately found in the nucleoli of both the non-thermotolerant and thermotolerant cells (Figure 3). At 6 h after the challenging heat shock, hsp7O was still found in the nucleoli of almost all non-thermotolerant cells, and the proportion of the cells with the stained
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Time ( h ) or Figure I . Rate of synthesis of hsp70 in V79 cells after a heat shock. Untreated V79 cells (0) in the cells first treated at 44°C for 20 min (0)were incubated at 37°C for 4 h (A, C) or 17 h (B, D). The cells were then labelled with 20 pCi/ml [35S]methionine for 1 h (before HS), or challenged by a heat shock at 44°C for 30 min (HS) and then pulse-labelled with [%]methionine for 1 h every 2 h interval. When V79 cells were first heated at 42°C for 4 h (A),the cells were treated by the challenging heat shock immediately (A, C) or at 17 h (B, D) after the first heating. Ten micrograms of protein from these cells was analysed by SDS- 10% polyacrylamide gel electrophoresis. The radioactivity incorporated into the protein was analysed with the use of a raidoanalytic imaging system (AMBIS), and the rates of synthesis of hsp70 (A, B) and actin (C, D) were calculated as percentages of total protein synthesis.
nucleoli gradually decreased to about 50% and 20%, at 9 h and 12 h later, respectively. However, in the cells that acquired thermotolerance at 4 h after being heated at 44°C for 20 min o r immediately after being heated at 42°C for 4 h, the proportion of the cells with the stained nucleoli drastically decreased by 6 h after the challenging heat shock. In the cells that were still thermotolerant at 17 h after being heated at 44°C for 20 min, or at Table 1. Assay of the amount of hsp70 in V79 cells at 12 h after heat shock. Relative amount of hs1170 Interval between first and second heating Oh
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('%)
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Untreated V79 cells or the cells first treated at 44°C for 20 min were incubated at 37°C for 4 or 17 h. The cells were then treated with a heat shock at 44°C for 30 rnin. and further incubated at 37°C for 12 h. When the cells were first heated at 42°C for 4 h. the cells were heat-shocked at 44°C for 30 min immediately or at 17 h after the first heating. and then incubated at 37°C for 12 h. Hsp70 in these cells was detected irnrnunologically and assayed by densitometric scanning. The numbers represent the relative amount of hsp70 compared to control cells. Each value is the mean f SE of three determinations. The values of hsp70 in the thermotolertant cells were not significantly different from the control value ( p < O . O 5 ) .
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Figure 2. Intracellular distribution of hsp70 in V79 cells during the induction of thermotolerance. (A) Untreated V79 cells at 37°C. V79 cells were heated at 44°C for 20 min (B) and then incubated at 37°C for 4 h (C) or 17 h (D); or V79 cells were heated at 42°C for 1 h (E), 2 h (F), o r 4 h ( G ) ;after the 4 h of heating the cells were incubated at 37°C for 17 h (H). Some cells were treated with 50 W M sodium arsenite for 1 .5 h (I) or 3 h (J); after the 3 h of treatment the cells were incubated at 37°C for 4 h (K) or 17 h (L). Other cells were treated with 20 pgiml puromycin for 2 h (M) or 4 h (N); after the 4 h of treatment the cells were incubated at 37°C for 4 h (0)or 17 h (P). The distribution of hsp70 was analysed by indirect inimunofluorescence with anti-hsp70 serum.
42°C for 4 h, the proportion of cells with the stained nucleoli also drastically decreased by 6 h or 9 h, respectively, after the challenging heat shock. To test newly synthesized proteins are required for the migration of hsp70, the protein synthesis of V79 cells was inhibited by cycloheximide. Cycloheximide at a concentration of 5 pg/ml inhibited more than 95 % of cellular protein synthesis of these cells, whether they were thermotolerant or not. Electrophoretic analysis of the proteins showed that heat shock proteins were not synthesized preferentially over other proteins in the presence of the drug (data not shown). When untreated cells were heat-shocked at 44°C for 30 min the proportion of the cells with the stained nucleoli gradually decreased after the heat shock, as shown in Figures 3 and 4, but the rate of the decrease was somewhat stimulated when the heated cells were incubated with cycloheximide (Figure 4). When the cells first heatshocked at 44°C for 20 min were incubated with or without cycloheximide for 4 h, and then given the second heat shock at 44°C for 30 min, hsp70 was immediately found in the nucleoli of almost all cells, whether treated with cycloheximide or not. The proportion of cells with the stained nucleoli decreased rapidly after the second heat shock, whether
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Time (hr) Figure 3. Nucleolar localization of hsp70 in V79 cells after a challenging heat shock. Untreated V79 cells (0)or the cells first treated at 44°C for 20 min (0)were incubated at 37°C for 4 h (A) or 17 h (B) (before HS), and then challenged by a heat shock (HS) at 44°C for 30 min. When V79 cells were first heated at 42°C for 4 h (A), the cells were treated by the challenging heat shock immediately (A) or at 17 h (B) after the first heating. After the challenging heat shock the cells were incubated at 37°C for 0, 3, 6. 9. or 12 h, and the distribution of hsp70 was analysed by indirect immunofluorescence with anti-hsp70 serum. The average percentage of cells with stained nucleoli from at least two independent experiments was plotted.
the cells were incubated with or without cycloheximide before and/or after the second heat shock (Figure 4). 3.3. Effects of cycloheximide on the induction and expression of thermotolerunce During the initial period of the induction of thermotolerance, V79 cells preferentially synthesize heat shock proteins, hsp70, hsp85, and hspl05, over other proteins (Hatayama et ul. 1991). Thermotolerant V79 cells synthesized hsp70 more rapidly after an exposure to heat shock with the synthesis decreasing rapidly thereafter than did the non-chermotolerant cells. To discover whether these characteristic syntheses of heat shock proteins in V79 cells are essential for the induction and expression of thermotolerance, the effect of inhibition of protein synthesis was analysed. By treating the cells with 5 pg/ml cycloheximide for 4 h, about 80% of the cells survived, and when the cells were incubated at 37°C with cycloheximide for 4 h after heat shock at 44°C for 0-60 min, the cell survivals of the cells were also about 80% of those of the cells heated and incubated without the drug. To induce thermotolerance, V79 cells were first heated at 44°C for 20 min and incubated at 37°C for 4 h. The cells were then challenged by a heat shock at 44°C for 0-60 min. Whether cycloheximide was added during the incubation of the cells between the first and second heat shocks, or during the 4 h of incubation after the second heat shock, or both, all of these cells became equally thermotolerant (Figure 5). 4. Discussion Hsp7O localizes in the cytoplasm of unstressed cells, but it is found in the nucleoli of various kinds of cells immediately after an exposure to heat shock (Welch and Feramisco 1984, Munro and Pelham 1984). Since the expression of high levels of hsp70 in COS cells results in a rapid recovery of normal nucleolar morphology after a heat shock, hsp70 localized to the nucleoli may catalyse the reassembly of preribosomes which were damaged by heat shock (Pelham 1984). In the present study the distribution of hsp70 was examined
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Time (hr)
Figure 4. Effects of cycloheximide on the intracellular distribution of hsp70. Untreated V79 cells were heated at 44°C for 30 min and then incubated at 37°C for 0, 3, 6, 9, or 12 h in the presence (0)or absence (0)of 5 &ml cycloheximide; or V79 cells first heated at 44°C for 20 min were incubated at 37°C for 4 h, then heat-shocked at 44°C for 30 min. and further incubated at 37°C for 0, 3. or 6 h in the presence (A)or absence (A) of cycloheximide. Some cells first heated at 44°C for 20 min were incubated at 37°C for 4 h in the presence of cycloheximide. then heat-shocked at 44°C for 30 min in the absence of cycloheximide, and further incubated at 37°C for 0 ,3, or 6 h in the presence (W) or absence (0) of cycloheximide. The distribution of hsp70 was analysed by indirect immunofluorescence with anti-hsp70 serum. The average percentage of cells with stained nucleoli from at least two independent experiments was plotted.
during the induction and expression of thermotolerance, since the level of hsp70 was not different between the thermotolerant and non-thermotolerant V79 cells. Hsp70 migrated to the nucleoli during the induction of thermotolerance of V79 cells by being heated at 42°C or 44"C, but not by treatment with sodium arsenite or puromycin, suggesting that the migration of hsp70 from cytoplasm to nucleoli was not essential for the cells to become thermotolerant. On the other hand, although hsp70 immediately migrated to the nucleoli in both thermotolerant and non-thermotolerant cells after a heat shock, hsp70 in the nucleoli of the non-thermotolerant cells disappeared more gradually than that of the thermotolerant cells during incubation at 37°C after a heat shock. Hsp70 disappears rapidly from the nucleoli in thermotolerant HA- 1 cells and the thermo-resistant variants as well (Ohtsuka and Laszlo 1989). So the phenomena seem to occur in thermotolerant cells in general. The rapid disappearance of hsp70 from the nucleoli of thermotolerant cells may mean that the damage of the nucleoli caused by heat shock is less in thermotolerant cells, or that the damage is repaired rapidly in thermotolerant cells. Since the level of hsp70 which might catalyse the reassembly of damaged preribosomes was not elevated in thermotolerant V79 cells, the damage of the nucleoli caused by heat shock may be less in the thermotolerant cells than in the non-thermotolerant cells. Inhibition of protein synthesis by cycloheximide in the thermotolerant and non-thermotolerant cells did not affect the immediate migration of hsp70 to the nucleoli after heat shock, suggesting that newly synthesized proteins were not required for the migration of hsp70 in V79 cells. The disappearance of hsp70 from the nucleoli of the non-thermotolerant cells was somewhat stimulated when the cells were incubated with cycloheximide. However, hsp70 in the nucleoli disappeared much faster in the thermotolerant cells than in the non-thermotolerant cells either in the presence or absence of cycloheximide. Cycloheximide may stimulate the disappearance of hsp70 from the nucleoli of the non-thermo-
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Figure 5 . Effects of cyclohexirnide on the induction and expression of therrnotolerance in V79 cells. V79 cells heated at 44°C for 20 min were incubated at 37°C for 4 h, then treated with a heat shock at 44°C for 0-60 min. and further incubated at 37°C for 4 h in the presence (C) or absence (A) o f 5 wg/ml cycloheximide (0). The cells heated at 44°C for 20 rnin were incubated at 37°C for 4 h in the presence of cycloheximide, then treated with a heat shock at 44°C for 0-60 niin in the absence of cycloheximide. and further incubated at 37°C for 4 h in the presence (D) or absence (B) of cycloheximide (0).As a control, untreated V79 cells were treated with a heat shock at 44°C for 0-60 min without cycloheximide (0). All cell5 were then incubated at 37°C without cycloheximide for 7-10 days for colony formation.
tolerant cells, probably as a result of its protective effects such as the stabilization of polysomes (McCormick and Penman 1969) and the suppression of heat-induced DNA repair inhibition (Armour et al. 1988). After a heat shock the increased rate of hsp70 synthesis diminished faster in thermotolerant V79 cells than in the non-thermotolerant cells. On the other hand, the rate of synthesis of actin, one of main cellular proteins, was not significantly different from these cells. Furthermore, since the rates of synthesis of hsp85 and hspl05 were also not significantly different between these cells (data not shown), the phenomenon seemed to be specific to hsp70. The rapid decline of hsp70 synthesis coincided with the faster disappearance of hsp70 from the nucleoli of the thermotolerant cells. However, the levels of hsp70 in the thermotolerant cells at 12 h after the heat shock were almost the same as those in the non-thermotolerant cells. The rapid decrease of hsp70 synthesis after heat shock may indicate that the production of hsp70 is self-regulated in stressed cells, as has been suggested by others (Mizzen and Welch 1988, DiDomenico er al. 1982). Cycloheximide, an inhibition of protein synthesis, inhibits the induction of thermotolerance in some cells (Henle and Leeper 1982, Freeman et al. 1987, Lee and Dewey 1987a) but not in others (Ohtsuka et a/.1986, Widelitz et al. 1986). However, in these studies it is not clear how effectively this drug blocks synthesis of proteins, heat shock proteins in particular, in various kinds of cells (Lee and Dewey 198713). In the present study, protein synthesis was inhibited more than 95% by 5 pg/ml cycloheximide in both non-thermotolerant and thermotolerant V79 cells, and heat shock proteins were not preferentially synthesized compared to other proteins under the conditions. V79 cells synthesize
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hsp70 preferentially during the initial period of the induction of thermotolerance (Hatayama et al. 1991). However, the inhibition of the characteristic synthesis of hsp70 by cycloheximide did not affect the induction and expression of thermotolerance in V79 cells. Thus, the synthesis of hsp70 itself seemed not to be directly related to the induction and expression of thermotolerance in the cells. The rapid decrease of hsp70 synthesis and the rapid disappearance of hsp70 from the nucleoli after a heat shock may be essential for the expression of thermotolerance. However, these phenomena may simply represent characteristics of thermotolerant cells. The molecular mechanisms underlying the phenomena remain to be elucidated.
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