heat resistance in mammalian cells: lessons and challenges

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Heat Resistance in Mammalian Cells:Lessons and Challengesa

ANDREI LASZLOb AND ANIKO VENETIANERc

bSection of Cancer Biology, Radiation Oncology Center, MallinckrodtInstitute of Radiology, Washington University School of Medicine,St. Louis, Missouri 63108 USAcInstitute of Genetics, Biological Research Center of the HungarianAcademy of Sciences, H-6701 Szeged, Hungary

The purpose of this brief review is to collect information concerning the resis-tance of mammalian cells to exposure to elevated temperatures. Such resistancecan be transient, such as that observed in induced thermotolerance, or perma-nent, such as that observed in spontaneously occurring heat-resistant variants,those selected after mutagenesis and those in which the expression of a particu-lar heat-shock protein has been elevated through molecular genetic techniques.The phenomenon of induced thermotolerance will be reviewed briefly followedby a more detailed review of the properties and physiology of various heat-resis-tant cell lines that have been established and investigated over the last fifteenyears. For a more complete review of thermotolerance, the reader is referred toseveral recent reviews.1–5

TRANSIENT THERMOTOLERANCE

Exposure of mammalian cells in culture to temperatures above normal growthtemperatures, usually higher than ~ 40°C, leads to reproductive cell death. Thisprocess has been quantitated by so-called survival curves, in which the fraction ofsurviving cells, as monitored by the clonogenic survival assay, is plotted semiloga-rithmically against the total time of exposure to a particular temperature. Theobserved survival is dependent on both the particular temperature and the dura-tion of the exposure. The survival curves obtained in this manner are reminiscentof those that are associated with the exposure of cells to ionizing radiation: thecurves exhibit an initial shoulder, followed by a region of exponential killing.Thermodynamic and rate theory analysis of the process of heat-induced cell killinghave indicated that the activation energy associated with heat-induced cell killingis usually between 110 to 150 kcal/mole, a value that has been interpreted as indi-cating that protein denaturation may be a rate-limiting step in the process of heat-induced cell death. However, because the effects of heat on cellular physiology arepleiotropic, affecting a constellation of parameters, the exact mechanisms involvedin heat-induced cell killing are still to be elucidated. There has been a debate con-cerning the primary target in heat-induced cell killing, in which the plasma mem-brane or the nucleus have been proposed as being the ultimate target. This review

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aThe work in the laboratory of Andrei Laszlo has been generously supported by theNational Institutes of Health (CA-R01-49018). The work in the laboratory of AnikoVenetianer has been generously supported by the National Scientific Research Fund (OTKAT016060, F013102) and the Research Fund of the Health Research Council (ETT 129/1966).

will focus on clonogenic cell death as an endpoint, which by definition mustinvolve the loss of reproductive integrity of the nucleus. Thus, although the pri-mary target may be elsewhere, ultimately it is the cell nucleus that suffers a lesionthat leads to reproductive death. Although hyperthermia has been shown toinduce apoptosis in cells derived from the hematopoietic lineage, in the cell linesof epithelial or mesenchymal origin that have been used extensively in hyperther-mia biology, apoptosis has not been found to be a major mode of cell death. Timelapse cinematographic studies have indicated that there is some interphase celldeath after hyperthermia, but most cells die after undergoing at least one moreround of cell division. For a more detailed discussion of the physiology of hyper-thermic exposure and of mechanisms involved in heat-induced cell killing, severaldetailed reviews may be consulted.6–8 In the consideration of the topics to be dis-cussed in this review, it will be useful to keep in mind that the mechanismsinvolved in heat-induced clonogenic cell killing are still not clear and that no bio-chemical determinants of this process have been established unequivocally.

In the mid 1970s, two research groups discovered independently that split doseexposure to hyperthermia, modeled after similar studies of ionizing radiation, ledto less cell killing than a single dose of equivalent duration. Thus, the exposure ofcells to elevated temperatures for a brief period of time, a treatment that led to atmost 20 to 50% of cell killing, followed by recovery for several hours at 37°C, ledto the development of the ability of cells to survive another more acute heat shock,which would have been otherwise severely lethal (cell survival of less that 10– 4 to10–5). This transient resistance, which had a definite kinetics of development anddecay, was called thermotolerance.9,10 The search for possible mechanismsinvolved in thermotolerance led to the proposal by three different groups in 1982,supported by convincing circumstantial evidence, that the heat-shock proteins(Hsps) were involved.11–13 An important feature of the proposed mechanism wasthe demonstration that several agents that induced elevated synthesis of the Hspswere also capable of inducing thermotolerance. This proposal was timely in thesense that at that particular time, although the molecular mechanisms involved inthe heat shock response were being delineated, there was no clear biochemicalfunction associated with the Hsps; the concept of molecular chaperoning was stillfive years away. This proposal that the Hsps play a role in clonogenic thermotol-erance led to a flurry of research activity that examined the questions of whetheror not the elevated expression of the Hsps was necessary and/or sufficient for thedevelopment of clonogenic thermotolerance. This was motivated by the demon-stration that it was possible to induce thermotolerance under conditions in whichthe synthesis of the Hsps was inhibited. It is clear now that there are at least twostates of thermotolerance, which have been called protein synthesis dependentand protein synthesis independent thermotolerance, respectively.14 The latter hasbeen shown to be induced mainly after exposure to heat, but not to other agents,such as sodium arsenite.15,16 However, it is interesting to note that evidence hasbeen presented that the Hsps present in the cell at the time of exposure to heatprobably play an important role in the protein synthesis independent state of ther-motolerance.15,16

The physiology of the thermotolerant state has been studied extensively(reviewed in Refs. 2, 3, and 7). At least two mechanisms can be involved in thethermotolerant cells’ ability to survive otherwise lethal heat treatments. The cellscan either be protected against heat-induced damage to the thermotolerant cell, orthe process of repair of such damage can be more efficient in such cells. Becauseevidence for both processes has been presented in a variety of cell lines, both

170 ANNALS NEW YORK ACADEMY OF SCIENCES

mechanisms are probably involved. Protection from initial damage has beenfound for the effects of heat on the plasma membrane, cytoskeleton, and somenuclear parameters. On the other hand, the absence of protection from initial dam-age in several cytoplasmic and nuclear processes has also been reported, followedby more rapid recovery. The kinetics of development and decay of both protectionfrom initial damage and the more efficient repair of similar levels of initial dam-age have been shown to parallel closely the kinetics of development and decay ofclonogenic thermotolerance.17,18 Although these observations fit nicely with thecurrent paradigm that the Hsps are molecular chaperones involved in intracellu-lar protein economy, including metabolism, localization, and function, the exactdetails of how protection from heat-induced cell killing is carried out by the Hspshave remained elusive. This is due, in major part, to our lack of understanding ofthe mechanisms of heat-induced cell killing, as discussed above. Nevertheless,thermotolerance remains an important and challenging phenomenon, one thathas been demonstrated from bacteria to cells in culture to whole experimental ani-mals. The detailed elucidation of the mechanisms involved should lead to impor-tant insights into basic cell biology and physiology.

PERMANENTLY HEAT-RESISTANT CELL LINES

The intrinsic heat resistance of animal cells in culture varies significantly.19

Relative heat resistance has been associated with the body temperature of theorganisms from which the cells were derived. Thus, cells from chickens and pigs,which have a body temperature between 41° to 42°C, are quite resistant to expo-sure at 45°C, a temperature that is quite lethal to cells derived from rodents,which have a body temperature of 36° to 37°C. Interestingly, human cells aremore resistant than rodent cells above 43°C, but many human cell lines are moresensitive than rodent cells to temperatures below 42°C.20,21 One approach to gain-ing insight concerning these fascinating differences has been to isolate heat-resistant cell lines. Pioneering studies were performed with heat-resistant pigkidney and Chinese hamster cells.22,23 However, the only physiological differ-ences found in these early studies was the ability of the heat-resistant pig kidneycells to resist the heat-induced leakiness of small metabolites from cells, espe-cially nucleic acid precursors.24

The suggestion that the Hsps played a role in transient clonogenic thermotol-erance11–13 stimulated further investigations of heat-resistant variants, followingthe logic that if the Hsps did play a role in thermotolerance, then permanent heatresistance may be associated with genetic alterations of constitutive levels ofmacromolecules transiently induced in thermotolerant cells. Several differentheat-resistant cell lines were established and studied, each with its individualcharacteristics, involving several different mechanisms. These cell lines will nowbe discussed in detail.

PIG KIDNEY AND HAMSTER CELLS22,23

These interesting cell lines were generated in the laboratory of Morgan Harris,one of the pioneers of somatic cell genetics. However, besides the physiological

LASZLO & VENETIANER: HEAT RESISTANCE 171

characterization mentioned above,24 these cell lines were never studied withrespect to the Hsp content or induction after exposure to hyperthermia. Thus,these cell lines have not contributed to the debate concerning the heat protectivefunctions of the Hsps.

B16 MOUSE MELANOMA CELLS

Permanently heat-resistant cell lines were isolated after repeated exposures ofB16 melanoma cells and surface-reacting, agglutinin-resistant variants derivedfrom them, to 43°C, followed by the growing up of the survivors.25,26 Subsequently,heat-resistant cell lines were selected from the same cell types, following repeatedexposures to 45°C; these cell lines were found to be more heat-resistant than thecells selected after exposure to 43°C.27 The expression of the major Hsps wasexamined by one-dimensional gel electrophoretic analysis of three of the later celllines, one derived from wild-type B16 cells, one from a concanavalin A–resistant variant and one from a wheat germ agglutinin (WGA)-resistant variant.28

Analysis of the extracts of cells labeled with radioactive amino acids for 48 hours,followed by quantitation of autoradiograms by densitometry and counting of gelslices, did not indicate a detectable difference in the expression of the major heat-shock proteins, Hsp70, 90, and 110. Other, more sensitive techniques, such as two-dimensional gel electrophoresis and immunoblotting techniques, were not used,making the conclusions that heat resistance in these cell lines was not associatedwith an increased expression of the major Hsps somewhat weak. Furthermore, thestatus of Hsp27, which has also been implicated in heat resistance (see below), wasnot examined. The lipid composition of the plasma membrane of these cells werealso studied in detail.29 In the heat-resistant variant derived from the WGA-resis-tant subclone, there was a decrease in cholesterol content, accompanied byincreased membrane fluidity. The fatty acid composition and the unsaturationindex of the plasma membrane were similar in the parent and heat resistant vari-ant cells. However, in the other two heat-resistant cell lines that were also exam-ined, derived from wild-type B16 and a Con-A-resistant subline, the cholesterollevel was higher, but this was compensated for by an increase in phospholipidcontent, so that the molar ratio of cholesterol to phospholipid was similar to thatfound in the parental cell lines. Thus no consistent alterations in the characteris-tics of the plasma membrane were found to be associated with the permanentlyheat-resistant state. The studies performed with these cell lines did demonstratethat permanent heat resistance can be achieved in the absence of drastic increasesin the expression of the major Hsps.

HEAT-RESISTANT VARIANT DERIVED FROM HA-1 CHINESE HAMSTER CELLS30

The HA-1 subline of Chinese hamster ovary fibroblasts has been used in sev-eral studies of thermotolerance (reviewed in Ref. 3) and in one of the studies link-ing the increased expression of the Hsps and thermotolerance.11 Heat-resistantvariants were selected from nonmutagenized cells using the following selection


scheme. Exponentially growing cells were treated at 43°C for 6 hours (survival <10– 4). Survivors were grown as a mass population and then exposed to 45°C for 45min. Individual surviving clones were picked and grown. Three clones resultingfrom this procedure, 3012, 3011, and 2214, were characterized further, and foundto be heat resistant.30 The heat resistance was associated with the increased expres-sion of Hsc70, the cognate form of Hsp70, under normal growing conditions. Thisdifference was detected by two-dimensional gel electrophoresis and was shown tobe due to increased levels of the Hsc70 mRNA, as indicated by in vitro translation.Subsequently, the Hsc70 gene from hamster cells was cloned and used for genomicSouthern and in situ hybridization, which revealed that the increased expressionof Hsc70 in the heat-resistant cells was due to amplification of the Hsc70 gene.31

The gene amplification event was associated with a complex chromosomalrearrangement characteristic of the early stages of the amplification of the struc-tural gene of dihydrofolate reductase in response to methotrexate treatment.32 Thiswas the first reported instance of gene amplification induced by heat and theamplification of a heat-shock gene product. Transient thermotolerance could beinduced in the HR variants, but only to the level that was found in the parentalcell line.30 There was no change in chromosomal number, cell size, cell cycle tran-sit time, cell cycle distribution, or in cell morphology.30 This is in contrast to the HRhamster line characterized by M. Harris, which displayed significantly differentmorphology.23 The induction of the heat-shock response was altered in the HRcells. Higher amounts of Hsc70 and Hsp110 and lower amounts of Hsp70 andHsp89 were induced by all treatments tested, as measured at the level of therespective proteins.33 The effects on the expression of Hsp70, Hsc70, and Hsp89have been shown to be transcriptional, as demonstrated by Northern blots andtranscription assays.34 The HR cells were not resistant to ionizing radiation, butdid display significant resistance to adriamycin.35 However, they were not resis-tant to BSO or protoporphyrin used in photodynamic therapy.36,37 The heat-resis-tant phenotype was stable after 200 passages.

HEAT-RESISTANT VARIANT ISOLATED FROM CHO CELLS38

CHO cells were first exposed to the mutagen ethane methane sulfonate (EMS),with 45% survival and then subjected to two rounds of selection by an 18- to 20-min exposure to 46°C. Single colonies from the second selection were grown anda clone demonstrating heat resistance, called HR-01, was studied further.Transient thermotolerance could be induced in the HR-01 cells to the same finalheat resistance, as was the case with the HR HA-1 cells described above. A moreelongated cell morphology was found in the HR-01 cells. The doubling time wassimilar to that of the wild-type cells. Two-dimensional gel electrophoretic analysisindicated the increased expression of Hsp90, but no changes in the expression ofHsc70 or Hsp70 in the HR-01 cells. The HR phenotype was maintained for up to450 passages. These cells have not been characterized further.

HEAT-RESISTANT VARIANTS ISOLATED FROM 0-23 CHINESE HAMSTER CELLS39

0-23 Chinese hamster lung cells were treated with EMS to 50% survival andthen exposed to a single-step selection procedure, involving a heat treatment of 4hours at 44°C. Sixty individual HR strains were isolated, and four were studied


further. In two of these strains, the HR phenotype was stable for at least 4 and 10months. Two-dimensional gel electrophoretic analysis revealed that the HR phe-notype was associated with the increased expression of Hsp27, due to increasedlevels of the mRNA encoding this protein in three out of the four characterized celllines. No increases in the levels of the other major Hsps were detected in these cellsat the level of the proteins (Hsp70, Hsc70, Hsp89, Hsp110) or at the level of themRNA (Hsp70 and Hsc70).39 These mutants were not characterized further, as thisresearch group generated hamster and mouse cells transfected with the humanHsp27 in their further studies of the role of this protein in heat resistance.40–42

HEAT-RESISTANT VARIANTS ISOLATED FROM RIF-1 CELLS43

In order to study HR in vivo, HR variants were selected from mouse radiation-induced fibrosarcoma cells (RIF), an in vivo/in vitro transplantable tumor modelsystem. In vitro–grown cells in the confluent state were exposed to 45°C for 60 min(survival of 10–5), and the surviving colonies were grown and treated again. Aftereach cycle of selection, the thermal response was monitored; increased HR wasobserved after three cycles of selection. No further increase in HR was observedafter seven cycles of selection. Twenty individual clones were isolated after 10cycles of selection, and four were characterized further. No changes in morphol-ogy were associated with the HR phenotype. A similar degree of thermotolerancedeveloped in wild-type (WT), and HR cells were exposed to equisurvival trigger-ing treatments. The response to ionizing radiation was not modified in the HRcells; exposure to equisurvival doses led to greater heat-induced radiosensitiza-tion (HIR) in the HR cells, whereas equidose exposure led to a similar degree ofHIR.40 The HR cells were transplanted into mice, allowed to form tumors, andthen heated. As measured by in vitro clonogenicity, the HR phenotype was main-tained in vivo, and it was not found to be associated with increased antigenicity.

Two-dimensional gel electrophoretic analysis of the HR strains yielded someinteresting results.44 The expression of several major Hsps, including Hsp28,Hsp60, Hsp68, and Hsp90, was elevated in the HR cells under normal growingconditions. In addition, a new protein was found to be present in the 70-kDaregion that cross-reacted with several anti-Hsp70 antibodies, but was only weaklyinduced by heat shock. Although the analysis of the membrane lipids showed nochange in cholesterol content, there was an increase in the proportion of the satu-rated fatty acids in the phospholipid fraction. The major changes observed werean increase in palmitic and oleic acid and a decline in the content of arachidonicacid; this resulted in a decrease in the unsaturation index from 4.5 in WT RIF cellsto 2.8 in HR cells, leading to a more “rigid” plasma membrane in the HR variants.

It has been proposed at this meeting (Maresca and Vigh45,46) that the degree ofunsaturation of the plasma membrane may serve as a set point for the cellularthermometer responsible for the induction of the heat-shock response. Making themembrane more rigid was shown to raise the threshold of the activation of theheat shock factor (HSF).47) However, the following observations with the HRstrains, which have a more rigid plasma membrane (see above), would indicatethat the mechanisms involved in temperature sensing may be more complex.48

Reverse-transcription PCR indicated that the HR cells express levels of Hsp70.1,Hsc70, and Hsp28 found in heat-shocked WT cells. Upon a heat shock, the HRcells demonstrated a more rapid onset of the induction of these same mRNAs,while the activated HSF and the induced Hsp70.1 mRNA decayed more slowly.


The HR cells also contained activated HSF at normal growth temperatures, indi-cating that they sense such temperatures such as to lock the cells permanently inthe “on” state of the heat-shock response.

HEAT-RESISTANT RAT HEPATOMA CELLS49–51

A rat hepatoma clone (clone 2) selected for dexamethasone resistance49 wasfound to be deficient in the induction of Hsp68 after exposure to heat and severalchemicals and also was unable to develop clonogenic thermotolerance after amild heat treatment or exposure to sodium arsenite.49,50 In order to examine therole of this protein in heat resistance, the HR variants were generated by meansof the following procedure. Cells were exposed to ten repeated cycles of 45°C for60, 70, or 80 min, with regrowth of the surviving cells. Individual subclones wereisolated from the survivors of the tenth cycle and characterized further. Cellsderived in this manner were found to be HR.51 No re-expression of the lost liver-specific differentiated functions associated with the glucocorticoid-resistant phe-notype of the cells subjected to heat selection was observed in the HR sublines.The differential induction of Hsp68 associated with clone 2 was observed aftermild, but not acute, heat shock in the HR cells.51 The kinetics of induction weremore rapid in the HR cells, probably because protein synthesis in these cells wasmore resilient to heat-induced perturbations in these cells. The data also indi-cated that the basal expression of several Hsps, including Hsp27, 70, 90, and 110was elevated in the HR cells.50,51 Thermotolerance could be induced in the HRcells, at least for the heat-induced inhibition of protein synthesis. Clone 2 cellsand the HR cells derived from them were transfected with a CMV-LUC plasmid(carrying the photinus pyralis luciferase under the control of the CMV promoter).A significant protection of luciferase activity against heat inactivation wasobserved in the HR cells.51 A most interesting feature of these HR cells is that theyalso overexpressed the mdr-1 gene, leading to resistance to several differentchemotherapeutic drugs.52

CONCLUSIONS

The numerous studies with HR variants have demonstrated two importantconcepts. First, whenever the expression of one of the major heat-shock proteins,Hsp27, 70, or 90, was elevated under normal growing conditions, resistance toheat-induced cytotoxicity, that is, cell killing, ensued at all elevated temperaturestested. Second, it is also clear that resistance to heat-induced cell killing can occurin the absence of elevated expression of the major heat-shock proteins. The eluci-dation of these mechanisms and how they may involve the endogenous levels ofthe major heat-shock proteins remains an exciting challenge for future research inthis area.


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heat resistance in mammalian cells: lessons and challenges

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