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Thermoresistant State: P Initial Damage or Better ANDREILASZLO' Section of Cancer Biology, Division of Radiation Oncology, Mallinckrodt Institute of Radiology, Washington University School of Medicine St. Louis. Missouri 63108

animal tumor models, and even n-r whole animals [4-71. in ~~he~motole~The biochemical mechanisms involve ante have been subject to intense ~~~~~t~g~tio~~ Although no consistent picture has emerged, the heat shock proteins (HSP) have been strongly implicated in this phenomenon. induce the elevate found to induce th dium arsenite, cadmium, and recovery from hypoxia [S]. A notable exception is the synthesis of elevat ~Q~st~lated that sensitization [9 J. amino acid analog e ~o~f~~ctio~a~ ~t~e~moto~era~ce 19, lo]. There have developing in tbe absence of elevated symhesis of the HSP and several states of th s5me of which are independent of the have been posturice obtained thus lated [11, 121. Nevertheless, th far strongly supports the notion that the HSP do play a key role in thermotolerance. Indeed, mutants expressing elevated levels of hsp 27, h 70, and. bsp 89 have been found to be beat resistant [13-151 an fected with hsp 27 and hsp 70 have been heat resistant [ 16,171 DTher for the involvement of the not unequivocal, it is nev what are the mechanisms thermotolerance on cells. the last few years that the resent in nonstressed cells, where they have imp nt functions. The HSP have been shown cellular protein metabolis protein trafficking, foFol elatiQns~i~ of these putative in [18, 191). However, HSP functions to the ~he~o~e~o~ of ~~o~oge~~~ thermotolerance and how t&e HSI? may pmtect cells from heat-induced cell death remains unclear. Perhaps the most significant obstacle to progress in answering the questions mentioned above is the paradoxical fact that we do not understand the mechanism of heat-induced cell killing [ZO, 213. Nevertheless, from a general conceptual view, the mechanism of thermotolerance may involve protection from initial: damage or

The induction of and recovery from heat-induced perturbations in several cellular parameters were examined in normal, transiently thermotolerant, and permanently heat-resistant HA-1 Chinese hamster fibrobeat-induced perturbations in total lasts. The initial cellular protein synthesis, RNA synthesis, vimentincontaining intermediate filaments, and nuclear protein mass were similar in the three different cell types which display various levels of thermal resistance as determined by clonogenic survival. The posthyperthermia recovery from the heat-induced perturbations in all of the cellular parameters was more rapid in both the permanently beat-resistant cells and in the transiently thermotolerant cells. This response was observed in cells in which transient thermotolerance was induced by either a mild heat shock or exposure to sodium arsenite. The development and decay of the capacity for more rapid recovery from the initial heat-induced perturbations in total cellular protein and RNA synthesis paralleled the development and decay of clonogenic thermotolerance. Overall, these results support the notion that more rapid recovery from similar levels of beat-induced perturbations in various cellular paramea salient feature of both the transiently and 0 1992 Academic Press, Inc. ntly beat-resistant state.

INTRODUCTION A variety of organisms in nature display a remarkable phenomenon in their response to elevated temperatures: following certain treatments they can become transiently thermotolerant, being able to survive otherwise lethal heat challenges. This phenomenon was originally described in the literature concerning phenocopy protection in DrosophiZa [I]. Thermotolerance as assayed by clonogenic survival was first established and characterized in mammalian cells in culture [2,3]. Clonogenic thermotolerance has also been reported in bacteria, yeasts, plants, a variety of animal cells in culture, 1 To whom reprint requests should be addressed at Section of Cancer Biology, 4511 Forest Park, Suite 419, St. Louis, MO 63108. 519

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0014-4827/92 $5.00 e opyright B 1992 by kcademic Press, Inc. rights of reproduction in ar,y form reserved.

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better repair of similar levels of initial damage or some combination of both. (In this context, we use the term “damage” only as a concept, referring to perturbation in cellular parameters that may lead to or be associated with heat-induced cell death.) Several studies have been performed to examine the physiology of the thermotolerant cell. On the one hand, evidence has been presented that several functions associated with the plasma membrane, such as insulin binding, Con A binding and the Na+, K+, -ATPase, intermediary metabolism, cytoskeletal integrity, cell morphology, and protein synthesis are protected from heat-induced damage in thermotolerant cells [22-271. On the other hand, it has been reported that the heat-induced nuclear translocation of hsp 70, the heat-induced excess nuclear proteins and total cellular RNA and protein synthesis are not protected in thermotolerant cells [28-341. More rapid recovery of nucleolar morphology, vimentin cytoskeleton collapse, and protein synthesis were reported in rat cells recovering from an initial heat shock under certain experimental conditions [35, 361. However, in the latter studies, the relationship of such altered recovery to clonogenic thermotolerance was not examined. In the accompanying study 1331, the localization of hsp 70 in normal, thermotolerant, and permanently heat-resistant cells was examined. A more rapid recovery from similar initial levels of heat-induced increased nuclear association of hsp 70 was found in both thermotolerant and heat-resistant cells. The more rapid recovery in the thermotolerant cells was related to the development and decay of transient clonogenic thermotolerante which had been induced by several different agents. These results also indicated that the physiology of the permanently heat-resistant state may be very similar if not identical to that associated with the transiently thermotolerant state. To test the validity of the above two conclusions, heat-induced perturbations and the recovery from them in cellular parameters which have been postulated to play some significant role in heat-induced cell killing [20,21] were examined in three different cell types: normal HA-l Chinese hamster fibroblasts, HA-l cells in which transient thermotolerance was induced by a variety of agents including heat shock and sodium arsenite, and in the 3012 permanently heatresistant cell line derived from HA-l cells in which the cognate form of hsp 70, hsc 72, is overexpressed [14]. The parameters examined included total protein and RNA synthesis, intermediate filament organization, and nuclear protein content. MATERIALS

AND

METHODS

Cells and culture conditions. HA-1 Chinese hamster ovary fibroblast cells [37] and 3012 cells, a stable heat-resistant cell line derived from them [14], were grown in Earle’s MEM supplemented with 10% fetal calf serum (GIBCO), nonessential amino acids, and 50 pg/ml

LASZLO gentamycin in a humidified 95% air, 5% CO, atmosphere. Cells in the exponential phase of growth were used throughout these studies. For immunofluorescence experiments, the cells were grown on 9 X 9 mm coverslips (Bellco) which had been pretreated with a few drops of glacial acetic acid and were stored in 70% ethanol. They were flamed briefly before use. Heating. Before heating of cell monolayers growing in 35-mm tissue culture dishes, the growth medium was changed, the tissue culture plates were gassed briefly with sterile 5% CO,, 95% air, and were sealed with parafilm. The sealed plates were then immersed in a 37°C water bath, for 10 min. Following this incubation, the sealed plates were immersed in a precision controlled water bath at 45 or 44°C (*O.O5”C), for appropriate periods of time. Under these conditions, temperature elevation to 45°C was achieved in 2.5 min, (& = 45 s). For recovery at 37”C, the culture medium was changed after heating; the medium was changed again before the second heat challenge. Cells growing on coverslips in 60-mm tissue culture dishes were heated in the same manner. Induction of thermotolerance. Transient thermotolerance was induced in two different ways. Induction by heat was obtained by exposure of cells to 45°C for 15 min as described above, followed by recovery at 37°C for at least 12 h. In most experiments, cells which had recovered from the initial heat treatment for 24 h were used. The induction of thermotolerance by sodium arsenite involved a 55-min exposure to 100 git4 sodium arsenite (Sigma) in growth medium at 37°C followed by five rinses in fresh medium and recovery at 37°C for 12 or 24 h. Assay for total cellular protein and RNA synthesis. Immediately after various heat challenges or after various times of recovery, cell monolayers grown in 35-mm dishes (Falcon) were washed three times in warm (37’C) sterile PBS. They were then incubated either in methionine-free medium containing 10% dialyzed fetal calf serum, containing 0.1-0.2 &Wrnl [35S]methionine (Amersham) for 1 h for protein synthesis, or in regular medium containing 0.05 to 0.1 aCi/ml [3H]uridine (Amersham) for RNA synthesis. At the end of the labeling period, dishes were washed three times with ice-cold PBS and then the cells were fixed in ice-cold 5% TCA. The plates were then incubated at 4°C for at least 12 h. Plates were then washed with cold 5% TCA two times, and cold PBS three times. The cells were solubilized in 1 ml of 0.01 N NaOH, 0.1% SDS, and the extract was sheared by passage through a 23-gauge needle three times. Equal aliquots from the different samples were then counted by liquid scintillation spectrometry in a Packard Model Tri-Carb 4640 liquid scintillation spectrometer. The counting efficiency for [?S]methionine was 85% and for [3H]uridine was 29%. Sister plates were put through mock labeling protocols, washed with cold PBS three times, and then were trypsinized and counted with a Coulter counter. The counts from the labeling experiments were then normalized to account for cell loss due to the hyperthermic treatments. However, such cell loss was never greater than 20 to 25% of unheated controls, and was significant only after acute exposures, such as 45 and 60 min at 45°C. The results are expressed as relative synthesis, which represents the ratio of the dpm/cell between control and heated cells. All experiments were repeated at least three times, with consistent results. The values of the incorporation at various time points were within 15% of each other in different experiments. Cells grown on coverslips as deImmunofluorescence of vimentin. scribed above were subjected to various types of heat treatments. Immediately following such treatments or after various periods of recovery at 37”C, the cells were washed three times with PBS containing 0.02% sodium azide, were fixed in 3.7% formaldehyde, 0.2% Triton-X 100 in PBS for 10 min at room temperature, and were rinsed three times with PBS. The cells were then incubated in 10% normal goat serum in PBS for 30 min at 37°C followed by incubation with a 1:lOO dilution of a polyclonal serum against vimentin [38, 391 (gifts of Drs. Hynes and Cabral, respectively) in 0.2% BSA in PBS for 45 min at

37°C. The coverslips were then rinsed with PBS three times and were incubated with a I:100 dilution of FITC-tagged affinity-purified goat anti-rabbit serum (Sigma). Photography was performed using Kodak T-Max 400 film rated at an EI of 400 and developed in T-Max developer. The heat-induced cohapse of the vimentin filament network was scored as assigning cells to be in states 1, 2, or 3. State 1 represented cells with fully extended vimentin containing networks which went all the way to tbe membranes; state 2 represented cells which displayed significant retraction of the vimentin-containing network from the membrane; and state 3 represented the partial or total collapse of the vimentin-containing network onto the nucleus. Examples of each of these states can be seen in Figs. 6a and 6b. Excess m&ear proreeins. The ratio of protein to DNA in nuclei isolated from control and heated cells was determined by dual flow cytometric techniques [40]. Briefly, nuclei isolated by standard techniques from control and heated cells [41] were treated for 5 min at 37”C, with RNAase (h mg/ml, boiled for 3 min before use), were stained with 0.1 mg/ml fluorescein isothiocyanate (FITC) for 1 h at 4”C, and were stained with 35 pg/ml of propidium iodide (PI) overnight. The ratio of green FITC fluorescence (protein content) to red PI fluorescence (DNA content) was monitored with a Becton-Dickinson FACS IV equipped with a dual detection system. At least 10,000 cells were analyzed for each sample.

RESULTS Initial Heat-Induced Synthesis

Effects on Total Protein and RNA

Exponentially growing HA-1 Chinese hamster fibroblasts and a heat-resistant variant isolated from them, 3012, were exposed to 45°C for 15, 30, and 45 min and then were pulse labeled with medium containing [3H]uridine or [35S]methionine for 1 h. The effects of these beat challenges cn total RNA and protein synthesis was assayed by determining the TCA precipitable incorporation in heated cells relative to control, nonheated cells. As illustrated in ess of total RNA synthesis in wild-type quite sensitive to exposures to 45’C, leading to an 81, 94, and 98% inhibition after the 15-, 3O-, and 45-min challenge, respectively. In the 3012 heat-resistant cells, a 79, 92, and 97% inhibition was observed after the same heat challenges. The process of RNA synthesis was significantly more heat sensitive than protein synthesis in both wild-type HA-l and heat-resistant 3012 HR cells (data not shown and [31]). The heat-induced inhibition of RNA synthesis was next examined after various recovery times at 37°C from a 15-min 45°C heat shock, including 4, 8, 12, and 24 h. It has already been demonstrated that clonogenic thermotolerance develops and is maintained during this time period under similar experimental conditions [31]. The results of the experiments presented in Fig. 1 illustrate that at 8 and 12 h after the initial treatment to render the cells thermotolerant, the heat-induced inhibition of RNA synthesis for a given heat challenge at 45’C is significantly smaller than that found in control wildtype I-IA-1 and 3012 beat-resistant cells which were not preheated. This apparent resistance to the effects of hyperthermic exposure with respect to this parameter de-

cayed by 24 h after the initial appears that there is a refractoriness to the beat-insynthesis duced effects on c thermot opment of clan refractoriness to the heat-induc synthesis during the same time the magnitude of this effect f smaller than that found for prot We also examined the initial min of exposure to 45°C of csntr01 II and in HA-1 and 3012 cells during t thermotolerance after an initial min (data not shown). The results were very similar, if not identical, to those already publisbed [31] and tberefore are not shown here. Recouery of Total Cellular Protein in Normal and T~ermoto~er~~t Exposure to Hyperthermia

and

The recovery of total RNA and protein synthesis after various hyperthermic challenges was compared in normal and thermotslerant cells Consi results of the experiments described above led to an experimental design in which cells were analyzed at 24 b after the triggering treatments for the thermotolerance. At this time sgenic tbermotokerance was still at its maxim the transient refractoriness to heat-induced m Mom otf both RNA and protein syntbesis bad g. 1 and Fig. 7 in Ref. [31]). Exponentially -1 cells were exposed to 45°C for 15 min an en were allowed to recover at 37°C for 24 b. Control, ~~~~~eated HA-1 cells and HA-1 cells which were thermQtQler~nt were then challenged witb heat treatments of 1.5, 30, 45, and 60 min at 45°C. Similar experiments were performed with cells in which transient t~errnQt~Iera~~e was induced by exposure of cells to 100 J;L~sodium arsenite for 1 h, followed by recovery for 24 h. (Under these conditions, the arsenite-induced clonogenic thermotoler velops somewhat faster than heat-induce ante, was still at its maximum [31]). T protein synthesis was determined imme heat challenge or at various times of recovery at 37”C, up to 15 h. The recovery of total protein and RNA synthesis after heat challenges of 15,30,45, and 60 min. at 45°C in the three types of cells (normal, heat-induced thermatolerant cells, and s arsemite~,i~d~ced tbermotolerant cells) are illust n Figs. 2 and 3, respectively. Several trends emerge from the consideration of these results. First, it appears that ery of protein synthesis and RNA ent in both normal and thermotole ery of total cellular protein synthesis occurs sooner than the recovery of total cellular R A synthesis; the rela-

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FIG. 1. Heat-induced inhibition of RNA synthesis in wild-type (HA-l) and heat-resistant (3012) Chinese hamster fibroblasts. Exponentially growing cells were exposed to 45°C for 15 min and were allowed to recover at 37°C for various lengths of time. Control, unheated cells (0, control), cells which have recovered at 37°C for 4 h ( (I, 4h TT), 8 h (m, 8h TT), 12 h (0,12h TT), and 24 h (A, 24h TT), respectively, were then exposed to 45°C for various lengths of time and the relative total RNA synthesis was determined as described under Materials and Methods. At each time point, the relative RNA synthesis as the percentage of control indicates the ratio of RNA synthesis in cells heated for various lengths of time to that observed in the unheated cells at that time point. The data represent one of three independent experiments.

tive delay in the recovery of RNA synthesis is larger in control cells than in thermotolerant cells. Second, both total cellular protein and RNA synthesis recover faster in cells which are thermotolerant than in normal cells. Complete recovery of protein synthesis within the time period examined occurs only after a 15min dose at 45°C in control cells and up to 30 min in the thermotolerant cells. At higher heat doses, 45 and 60 min at 45°C a partial recovery is observed, but only in the thermotolerant cells. Complete recovery of RNA synthesis was not seen in the control cells after all of the heat doses tested; at doses above 15 min at 45”C, significant recovery is only observed in the thermotolerant cells. Third, at doses including 30 min and above at 45°C there appears to be a difference between the response of cells in which thermotolerance was triggered by a mild heat treatment and exposure to the sodium arsenite: total cellular protein and RNA synthesis recovered sooner in cells in which thermotolerance was induced by a mild heat shock than in cells in which thermotolerance was induced by sodium arsenite. The ability of protein and RNA synthesis to recover faster than in control cells was observed in cells in which arsenite-induced thermotolerance had developed for only 12 h, with the relative difference between control and thermotolerant cells being similar to that observed in the experiments illustrated in Figs. 2 and 3, which used cells allowed to recover for 24 h after the initial exposure to sodium arse-

nite (data not shown). Thus, the difference between the cells in which thermotolerance was induced by heat and sodium arsenite was not due to the more rapid decay of the thermotolerant state induced by sodium arsenite treatment. The Relationship of Clonogenic Thermotolerance and the Ameliorated Ability to Recover from Heat-Induced Perturbations in Protein and RNA Synthesis The series of experiments described above established that in thermotolerant cells, the recovery of total cellular protein and RNA synthesis from similar levels of initial inhibition was more rapid. An important question that arises from the consideration of these results is what, if any, relationship exists between clonogenic thermotolerance and this ameliorated ability of two basic metabolic processes to recover after heat-induced inhibition. This notion was examined by considering the temporal aspects of clonogenic thermotolerance which have been defined operationally [42], namely triggering, development, maintenance, and decay. With respect to triggering, the data obtained thus far demonstrate that the ability of total cellular protein and RNA synthesis to recover faster in thermotolerant cells than in normal cells is independent of the mode of triggering of thermotolerance by either heat or sodium arsenite. The question of whether clonogenic thermotolerance and ability to recover faster from heat-induced pertur-

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FIG. 2. Recovery of total cellular protein synthesis after exposure to various lengths of time at 45°C in normal and transiently thermotolerant HA-1 cells. Exponentially growing cells exposed to either 45°C for 15 min or 100 PM sodium arsenite for 55 min were allowed to recover at 3’7°C for 24 h. Control, untreated ceils (0, con.), heat-induced thermotolerant cells (a, beat), and arsenite-induced tbermotolerant ceils (A, am.) were all exposed to 45°C for 15 min (15 min), 30 min (30 mm), 45 min (45 mm), and 60 min (60 mm), respectively, and were allowed to recover at 37°C for 15 h. The relative rate of total protein synthesis was determined every 3 h during this recovery period as described under Materials and Methods. The results are expressed as “Relative Synthesis” which is the ratio of the protein synthesis observed at a particular time point to that observed in the respective control, nonheated cells. The figure represents one of three independent experiments.

bations in total cellular protein and RNA synthesis develop in parahel is difficult to answer because of the transient refractoriness of both protein synthesis [31] and RNA synthesis (Fig. 1) to heat-induced perturbations during the time period of the elevated synthesis of the HSP, which coincides with the time period during which thermotolerance develops in the cells used in this study under our experimental conditions [43,44]. The status of the ameliorated ability to recover from heat-induced perturbations in total cellular protein and RNA synthesis during the time period of the maintenance and decay of clonogenic thermotolerance was examined in the following set of experiments. Exponentially growing HA-l cells were exposed to 45°C for 15 min and then were allowed to recover at 37*C for

various lengths of time. After various time intervals of recovery from the triggering treatment, the cells were challenged with a 27-min treatment at 45’C and total RNA and protein synthesis was deter ately after this heat challenge or at v recovery at 37°C. The results are presented in Figs. 4a and 4b. The data indicate that the capacity1 for the ameliorated recovery of RNA and protein synthesis is developed by 12 h after the triggering t~eat~e~~t and that it starts decaying somewhere between 48 and 60 h after the triggering treatment. The kinetic ameliorated ability for the recovery of synthesis from heat-induced perturb allel the kinetics of decay of ~bermo~o~era~ce as meain the accompanysured by clonogenicity as presente

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FIG. 3. Recovery of total cellular RNA synthesis after exposure to various lengths of time at 45°C in normal and transiently thermotolerant HA-l cells. The experimental conditions and the plotting of the data are identical to that described in the legend for Fig. 2, except that the rate of total cellular RNA synthesis was determined as described under Materials and Methods.

ing paper (Fig. 12 in [33]). These results indicate that two cellular parameters, clonogenic thermotolerance and ameliorated ability to recovery from heat-induced perturbations of total protein and RNA synthesis, decay in narallel and thus mav be closelv related. I

Recovery from Heat-Induced Perturbations in Total Cellular RNA and Protein Synthesis in Permanently Heat-Resistant Cells The response of total cellular protein and RNA synthesis in the 3012 heat-resistant cells to hyperthermic challenges was examined in order to determine whether or not their response was similar to that of transiently thermotolerant cells. The initial heat-induced inhibition of protein and RNA synthesis in heat-resistant 3012 cells was similar to that found in normal and thermotolerant HA-l cells (Fig. 1 and Fig. 7 in [31]). Exponentially growing wild-type HA-l and heat-resistant 3012 cells were exposed to 45°C for 15 and 30 min, and total RNA and protein synthesis was determined at

various times of recovery at 37°C after the heat challenges for up to 15 h. The results of this series of experiments are illustrated in Fig. 5. The magnitude of the initial inhibition of the two macromolecular processes under these conditions is similar. The data clearly demonstrate that after both heat challenges, total RNA and protein synthesis recovers faster in the 3012 heat-resistant cells than in their parental wild-type HA-l counterparts. Thus, the state of permanent heat resistance that is mediated by the overexpression of the cognate form of hsp 70, hsc 72 [ 141, is associated with an ameliorated recovery in these two processes as well, consistent with the notion that there is a similarity of the physiological state of permanently heat-resistant and transiently thermotolerant cells. Heat-Induced Perturbations Intermediate Filaments

in the Organization

of

Exposure to elevated temperatures also perturbs cytoskeletal organization. The exact effects are cell type and

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FIG. 4. Recovery of total cellular protein and RNA synthesis during the decay of heat-induced clonogenic tbermotolerance. Exponentially growing HA-1 cells were treated at 45°C for 15 min and were allowed to recover at 37°C for various lengths of time. Control, nonpreheated cells (0, C), and heated cells which have recovered at 37°C for 12 h (m, 12 hT), 24 h (e, 24 hT), 48 b (A, 46 hT), 72 h ( ,72 hT), aad 96 h (v, 96 ha‘) were all exposed to 45°C for 27’ min and were allowed to recover at 37°C for 16 h. The relative total cellular protein synthesis (a) and RNA synthesis (b) were determined every 4 h and the results plotted as described under Materials and Methods. The data represent one of two independent experiments.

species specific (reviewed in Refs. [6] and [21]). In CHO cells, heat-induced destruction of the actin-containing stress filaments and the collapse of the vimentin-containing IF network have been reported [45, 461. In the HA-1 cells studied in this report, exposure to elevated

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temperatures also results in the disr mentin-containing intermediate fiIame~t$ (Figs. 6a and 6b), leading to a retraction of the ~~a~e~ito~~ network from the periphery of the cell, resultin collapse around the nucleus. The initial heat-induced

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FIG. 5. Recovery of total cellular protein and RNA synthesis in wild-type HA-1 (HA-l) and 3012 (20) heat-resistant cells after exposure to 45°C. Exponentially growing wild-type HA-l cells (0, 0) and 3012 heat-resistant cells (A, .a) were exposed to 45OC for 15 min (0, A) or 39 min (0, A) and were allowed to recover at 37°C for 15 h. Every 3 h, the relative total cellular protein (Prot.) and RNA (RNA) synthesis was determined and plotted as described under Materials and Methods. The data represent one of three independent experiments.

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FIG. 6. The appearance of the vimentin-containing intermediate filaments in control and heated wild-type HA-l cells. Exponentially growing control HA-1 cells (a) were exposed to 45°C for 15 min (b); both control and heated cells were then processed for immunofluorescence with vimentin-specific antibodies as described under Materials and Methods. In (a), the upward pointing arrow points to two cells which are scored as state 1, while in (b) the upward pointing arrow points to two cells scored as state 2 and the downward pointing smaller arrows point to two cells in state 3: states 1, 2, and 3 are defined under Materials and Methods. Bar, 10 pm.

retraction and eventual collapse of the vimentin-containing network and the posthyperthermia return of its original organization was monitored in normal, permanently heat-resistant, and transiently thermotolerant cells. Exposure to 45°C for 15 min led to similar levels of destruction of the vimentin-containing network in all three cell types: about 75 to 80% of cells displayed a collapse of the vimentin-containing network. The return of the initial organization of the vimentin-containing intermediate filament network after such a hyperthermic challenge was more rapid in thermotolerant wild-type HA-l cells in which thermotolerance was induced both by mild heat shock and sodium arsenite

treatment and in the control permanently heat-resistant 3012 cells than in the control HA-l cells (Fig. 7). Heat-Induced

Excess Nuclear Proteins

One of the earliest heat-induced perturbations in cellular structure that can be detected in mammalian cells is the increase in the protein to DNA ratio in isolated nuclei and chromatin (reviewed in [20, 211). This phenomenon has been studied most extensively in HeLa cells, where a time/temperature dose response was observed at all temperatures studied [20]. Furthermore, dose-modifying treatments with heat sensitizers, heat

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that hsp 70 and hsc 72 are the major c~rn~o~~e~ts of the heat-induced excess rmclear proteins 14’71.

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FIG. ‘7. The recovery of the vimentin-containing intermediate filament organization in normal, thermotolerant, and heat-resistant cells exposed to 45°C. Tbermotolerance in exponentially growing wild-type HA-l cells growing on coverslips was induced by a brief heat treatment or exposure to sodium arsenite as described in the legend for Fig. 2. Control, nontreated wild-type HA-l cells (@, C), heat-induced thermotolerant wild-type HA-1 cells (B, H), sodium arsenite-induced thermotolerant wild-type HA-l cells (A, A), and control, nontreated 3012 heat-resistant cells (0, HR) were all exposed to 45°C for 15 min and were allowed to recover at 37°C for 12 h. Every 3 h, coverslips with each different celi type were fixed and processed for immunofluorescence with a vimentin-specific antibody as described under Materials and Methods. For each time paint, at least 300 cells were observed under a microscope equipped with fluorescent optics, in a blind fashion, and the number of cells displaying the three different states of organization of the vimentin-containing intermediate filaments were determined. This scoring system is described under Materials and Methods. The percentage of cells displaying state 2 and state 3 type of organization is plotted at each time point. The data represent one of two independent experiments which gave essentially the same pattern of results.

protectors, and thermotolerance have been shown to alter the magnitude of this response [20]. The ratio of protein to DNA in nuclei isolated from cells which had been exposed to elevated temperatures and allowed to recover for various lengths of time was examined in normal, transiently thermotolerant, and permanently beat-resistant cells (Fig. 8). These results indicate that the initial increase in the protein/DNA ratio as a consequence of exposure to 44°C for various lengths of time was similar in all three cell types, similar to what has been reported for thermotolerant HeLa cells [20, 291. However, the increase in the protein to DNA ratio resulting from an exposure to 44°C for 30 min decays to t&at observed in control, nonheated cells with faster kinetics in both the transiently thermotolerant wild type HA-1 cells and the permanently heat-resistant 3012 cells. These results are reminiscent of those obtained when the appearance and decay of heat-induced association of hsp 70 with the nucleus was examined in these three cell types [33]. Indeed, we have recently demonstrated

The work described in this report was u~~~ertake~ as part of a continuing investigation oftbe molecular mecbanisms associated with the beat-resistant state and had two related but distinct aims. The rst aim was to determine if one of the main conclusions reached in the accompanying paper 1331, namely that similar degrees of heat-induced perturbations in the ~~ca~~zat~~~~ of hsp 70 are repaired more rapidly in heat-resistant cells, applies rations in other cellular parameters to heat-induced aim was to examine the ~oss~b~~~~y as well. The seco cells that the physiology of ermanently heat-resistant ly tbe~rn~tQ~e~a~t cells is similar to that of e~u~batio~~~ in cellular with respect to heat-induce parameters other than tbe 1Qca~~zati~m of hsp 70. Hn order to accomplish these aims, the magnitude of,

a role in the process beat-i was determined an transi manently heat-resistant -1 CRinese hamster blasts. The cellular cellular RNA and protein synthe zation, and heat-induced excess

fibro-

RNA synthesis was examined during of the thermoto~era~t state after a 45”C, conditions under which cionog ante reaches its maximum after 8 h, essentially at the same level fQp”at least 24 h [31, 331. When compared to control ce of total cellular RNA synthe which thermotolerance was deve ery at 37°C after the initial he thermotolerance. Such prot duced inhibition of RNA sy and 12 h of recovery, but not after 24 h of recovery (Fig. 1). This transient pattern of resistance is very similar that reported for total cellular protein symlhesis [31] _ the latter report, a good correlation between tbe protection from the init of beat on protein synt and the time pe vated synthesis of the was demonstrate ut the association of such protection with the therm~t~le~a~t state as assayed by c1o genie survival broke down between 12 to 24 b after initial treatment [31]. Recently, similar results were found in a comparison of tbe b~a~-i~~~ced pertur tions in protein synthesis in two rat cell lines, one of which has lost its ability to induce hsp 70 upon beating. The development and decay of the ~ef~~ct~~iness of protein synthesis to heat-induce to correlate exactly with the time

528

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0

10

20 Time

I 11

I

30 al 44OC lminl I

I

bb

I

OHA-1,

l HA+ A 3012,

1.8

I 0

I

I

1

I

1

2

4

6 Recovery

8

10

I hrsl

FIG. 8. The effect of exposure to hyperthermia on the relative nuclear protein content of normal, thermotolerant, and heat-resistant cells. Thermotolerance in exponentially growing wild-type HA-l cells was induced by exposure to 45”C, 15 min, as described in the legend for Fig. 2. In (a), control wild-type HA-1 cells (0, HA-l,), thermotolerant wild-type HA-l cells (m, HA-l,), and control 3012 heat-resistant cells (A, 3012,) were exposed to 44°C for various lengths of time. Nuclei were isolated from control and heated cells and their protein to DNA ratio was determined as described under Materials and Methods. In (b), the same three different cell types were exposed to 44°C for 30 min and allowed to recover at 37°C. After 0,2,5,7, and 9 h of recovery, nuclei were isolated from the three different cell types and their protein to DNA ratio was determined as described under Materials and Methods. The figure represents one of two independent experiments.

synthesis of the HSP [48]. A similar correlation between the initial protection of RNA synthesis from heat-induced perturbations and the elevated synthesis of the HSP at 8 and 12 h after the initial treatment was also observed (Laszlo, unpublished results). The correlation

*LAYiX9.l ’ “--

-

between the protection from heat-induced inhibition of RNA synthesis and clonogenic thermotolerance was broken at 12 to 24 h after the initial treatment, as reported for protein synthesis [31]. The refractoriness to the initial effects of hyperthermia-induced effects on protein and RNA synthesis, which decays rapidly, may or may not be an important physiological aspect associated with this state. Nevertheless, clonogenic thermotolerance can still be observed under conditions when there is no protection from the initial heat-induced perturbations of protein or RNA synthesis. Therefore, all experiments examining the magnitude of and recovery from heat-induced perturbations in various cellular parameters were performed 24 h after the initial treatment. At this point in time, the initial effect of a hyperthermic challenge on both protein and RNA synthesis was not significantly different in cells displaying clonogenie thermotolerance than in control cells. The assumption that there may also be transient protection from heat-induced perturbations in cytoskeletal organization and heat-induced excess nuclear proteins during the development of thermotolerance was based on preliminary experiments that indicated the presence of such protection at early times during the development of thermotolerance (Laszlo, unpublished observations). Some of the controversies in the literature concerning the physiology of thermotolerant cells, as reviewed briefly in the Introduction, may be based on the fact that there is great variation in the experiments performed in various laboratories both with respect to the magnitude of three important parameters: the temperature and duration of the challenging heat treatment and the time after recovery from the initial triggering treatments at which experiments are performed. Under the experimental conditions used in the work performed in this report, namely at 24 h of recovery from the initial 15 min at 45°C that was used to induce thermotolerance, no significant differences in the initial heat-induced perturbations in cytoskeletal organization (Fig. 7) and excess nuclear proteins (Fig. 8) were found in control and thermotolerant cells. With respect to the heat-induced excess nuclear proteins, although the initial increase in the mass of protein associated with the nucleus is similar in the three cell types tested, the possibility exists that the population of proteins comprising this increase may be different in control and heat-resistant cells. A comparison of heat-induced excess nuclear proteins in control and thermotolerant HeLa cells has indicated that this situation may in fact be the case (J. L. Roti Roti and N. Turkel, personal communication). Recently, we have identified hsp 70 as one of the excess nuclear proteins [47]. Thus, the bulk of the heatinduced ENP displays characteristics similar to that observed with the heat-induced increase in the association of hsp 70 with the nucleus [33]: faster recovery in heat-

INITIAL

DAMAGE

AND

REPAIR

IN THE

resistant cells from similar levels of initial perturbations. The recovery from such heat-induced perturbations in total cellular RNA and protein synthesis (Figs. 2 and 3), cytoskeletal organization (Fig. 7), and excess nuclear proteins (Fig. 8) was always more rapid in thermoresistant than in normal cells For two cellular processes, t.otal protein and RNA synthesis, the faster recovery was observed after four different heat doses in transiently thermotolerant cells (Figs. 2 and 3) and two different heat doses in permanently heat-resistant cells (Fig. 4). It could be argued that the shortest heat challenge that we examined, namely 15 min at 45”C, already essentially saturated both protein and RNA synthesis with damage in control but not in thermoresistant cells. Therefore, the faster recovery observed in thermoresistant cells could have been an artifact stemming from such “hidden damage.” The observations that further inhibition of both RNA and protein synthesis was observed after longer heat challenges at 45°C in both normal and thermotolerant cells (Fig. 1, and Fig. 7 in [31]) and that no significant differences in initial effects on both RNA and protein synthesis were observed after heat challenges at 45°C that were less than 15 min (Laszlo and Davidson, unpublished observations) argue strongly against this possibility. It also could be argued that recovery could occur during the l-h labeling pulses that we used in our experiments; however, the fact that similar results were obtained after a 20 min-labeling pulse (Laszlo and Davidson, unpublished results) makes this possibility less likely. The initial heat-induced inhibition of protein and RNA synthesis after exposure to 45°C for various lengths of time in normal, thermotolerant, and buman hsp 70 transfected rat fibroblasts has also been reported to be similar [49]. Additional beat-induced perturbations in nuclear protein content and the organization of vimentin were observed in both normal and thermotolerant cells exposed to heat doses greater than those illustrated in Figs. 7 and 8 (Laszlo, unpublished observations). These results argue against the “‘hidden damage” interpretation of the data obtained with these two parameters as well, A difference between the recovery kinetics of total cellular protein and RNA synthesis in cells in which tbermotolerance was induced by exposure to sodium arsenite treatment or exposure to a mild heat shock was found after heat challenges including and above 30 min at 45°C (Figs. 2 and 3). Differences between sodium arsenite and heat-induced thermotolerance have been described in,work from this laboratory and that of others [12, 501. In the HA-1 cells used in this study, two features of the response of the hsp 70 gene family are clifferent in heated and sodium arsenite-treated cells [14]. First, the absolute increase in the mass of members of the hsp 70 family is smaller in the arsenite-treated cells; second, the heat-inducible form of hsp 70 is not induced

THERMORESHSTANT

STATE

529

by the arsenite treatment. Which of these differences in the response of the bsp 70 family is associated with the differences in the recovery of total cellular protein an RNA synthesis remains to be det~~rn~ne~. The finding rotein synthesis to that the response of total ccl hyperthermia was altered in cells in which the s Bsst is consistent ability to induce hsp 70 by h with the later possibility [48]. In order to determine the relatio~s~~ between the state of transient clonogenic ~hermo~o~era~ce and the more rapid recovery from heat-indexed perturbations in protein and RNA synthesis, we examine recovery from heat-induced perturbations in RNA and protein synthesis at various time points up to 96 h after the initial heat treatments, the time eriod during which event clonogenic thermotolerance cells ([4] and Fig. 12 in 1331). The decay of the ability to recover fast inhibition of protein and RNA synthesis paralleled the decay of clonogenic thermotolerance (Fig. 4) constitutes very strong circumstantial evidence that the two processes are related. The similar parallel decay of the elevated levels of the I-BP, especially bsp 70, and of clonogenie thermotolerance is one of the mai dence supporting the role of the phenomenon of transient ther The major fraction of tbe uridine into RNA that was observ this study is probably ribosomal been estimated as being from 65 to 70%, based upon the sensitivity of total RNA synthes 61 &ml of aetinomycin D (Laszlo, unpublished ervations), The nucleolus bas been known to be a very heat-sensitive structure and heat-induced i~~ibi~io~ of t.he synthesis and processing of ribosomal RNA has been reported in various mammalian cells, includin viewed in [ZO] and 1211). Since hs ciated with the nucleolus after beat to speculate that it could particip heat-induced damage in this structure. It is important to note in this context that the i~hib~t~~~ of the rapid recovery of RNA synthesis in tb~~rno~o~e~a~t cells bas been reported to abolish the expression of clonogenic thermotolerance in CHO cells 1321. If the hypothesis that a faster recovery from heat-induced alterations in RNA and protein synthesis is assostate is ciated with the transiently t exmot~le~ant correct, then it could be predicted as a corollary that the permanently heat-resistant state may also be associated with such faster recovery fro hea~-i~~~~ed alterations in these parameters. This notion was tested in permanently heat-resistant cells tbat express elevated leve e form of hsp 70, hse 72, which were deriv this study [149. we -1 cells that we found that in these permanen t-resistant cells the recovery from heat-inducedpe .tiomis of all parame-

530

ANDRE1

ters examined, including total RNA and protein synthesis (Fig. 5), cytoskeletal integrity (Fig. 7), and excess nuclear proteins (Fig. 8), was faster than that in their wild-type counterparts. These results indicate that the physiological state associated with the permanently heat-resistant phenotype is similar to that present in transiently thermotolerant cells. Since one common feature that is shared by cells displaying these two different types of heat resistance is the presence of elevated levels of the members of the hsp 70 family, a role of this heat shock protein in these two types of heat resistance is further supported. There has been a suggestion in the literature that the mechanism of heat-induced cell killing may be different at 43 and 45°C [52] due to the different kinetics of the accumulation of heat-induced damage. Thus, it is not inconceivable that when challenged at 43”C, there may be protection from heat-induced damage in thermotolerant cells for some of the cellular parameters that we have studied in this report. (It must be noted, however, that this is not the case with respect to the heat-induced ENL of hsp 70, as demonstrated in the accompanying report [33] .) Such observations would not contradict the conclusions reached from the work presented in this paper. The data presented in this report indicate that under conditions where similar levels of heat-induced initial damage can be demonstrated in control, transiently thermotolerant, and permanently heat-resistant cells, faster recovery from such damage is associated with the transiently thermotolerant and permanently heat-resistant state. A protection from initial damage in the same cellular parameters that we examined in this study which is distinct from the transient refractoriness to heat-induced perturbations that occurs in parallel with the development of the thermotolerant state may also be a feature of the heat-resistant state. This possibility is being currently investigated. In conclusion, the results obtained in this report indicate that the permanently heat-resistant state which is mediated by increased levels of hsc 72 and the transiently thermotolerant state share an important feature, namely the capacity for more rapid recovery from similar levels of initial heat-induced perturbations in cytoplasmic processes, including protein synthesis and cytoskeletal integrity, and nuclear processes, including RNA synthesis and the levels of DNA associated proteins. These observations are consistent with our current understanding of the function of the heat shock proteins [18, 19, 531. The exact molecular features of such repair mechanisms mediated by the elevated levels of the HSP remain to be delineated. I thank Dr. J. L. Roti Roti for important suggestions concerning the manuscript, Ms. T. Davidson and Ms. K. Bles for proofreading the manuscript. This research was supported by NIH-CA-49018.

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