[CANCER RESEARCH 33, 415-421, February 1973]

Studies on the Quantitative Biology of Hyperthermic Killingof HeLa Cells1

Robert J. Palzer and Charles Heidelberger2

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

SUMMARY

The quantitative biology of hyperthermic killing of HeLacells was studied. Plots of cell survival versus doses ofhyperthermia did not show first-order kinetics. The rate ofHeLa cell killing shows a striking temperature-dependentrelationship in the 41.0-43.0° temperature range. There is a

delay of approximately 1 day in the division of cells heated to42.0°, after which time some cells resume normal growth,

whereas others divide at least once before death. Cells selectedfor their capacity to survive prolonged periods ofhyperthermia are killed at approximately the same rate duringsubsequent heat treatments as cells that had not been heatedpreviously. Hyperthermic cell killing is reduced in cells that areheated in the presence of certain compounds that areinhibitors of DNA and protein synthesis. Fractionated doseexperiments indicate that cells recover from sublethalhyperthermic damage. Furthermore, hyperthermic killing is atleast a two-step process, and cells are capable of recovery frompotentially lethal damage, particularly in the presence ofcycloheximide and high levels of thymidine.

INTRODUCTION

Cavaliere et al. (2) have provided an extensive literaturereview of clinical and experimental evidence indicating thattumor cells may be selectively killed by hyperthermia. Theyalso reported (2) on the use of localized hyperthermicperfusions to obtain complete remissions in 4 out of 7 patientswith melanomas of the limbs. These remissions have persistedto date, which is now more than 5 to 7 years since the initialtreatment (R. Cavaliere, personal communication). Morerecently, Stehlin (22) has reported that localized hyperthermicperfusions of the limbs with phenylalanine mustard producedtumor regressions more effectively than perfusions with thesame alkylating agent alone.

Harris (7, 8) was the first to quantitate hyperthermic killingof mammalian cells, using a cloning assay originally developedby Puck and Marcus (18) to study cell killing by X-irradiation.More recently, Westra and Dewey (24) have also used cloningassays to quantitate hyperthermic killing of synchronizedcultures of Chinese hamster ovary cells. Giovanella et al. (5)used a sensitive in vivo bioassay to quantitate the influence of

various drugs on the hyperthermic killing of LI 210 leukemiacells.

The present study was undertaken to provide a biologicalbackground for future biochemical investigations ofhyperthermic cell killing, the mechanism of which is currentlypoorly understood. We are attempting to answer the followingquestions. Are there a limited number of temperature-sensitiveevents that cause hyperthermic cell killing? Does hyperthermiccell killing occur immediately, or can cell division precededeath? Do cells repair hyperthermic damage, and if so, bywhat means? Are some cells resistant to hyperthermia, and ifso, how can heat resistance be induced?

We chose HeLa cells for this study for several reasons. Theywere originally derived from a human cervical carcinoma andtherefore are of malignant origin. Since HeLa cells have beenroutinely passaged in many laboratories since 1952, there is aconsiderable literature on their biochemistry under a variety ofconditions. HeLa cells attach to a surface with a relatively highplating efficiency; consequently, cloning assays can be used toquantitate cell killing. However, since there are no suitablenormal cell controls for HeLa cells, we do not deal with thequestion of whether tumor cells are selectively killed byhyperthermia.

MATERIALS AND METHODS

Cell Culture. HeLa 83 cells were kindly provided by Dr.Roland Rueckert of the University of Wisconsin and weremaintained in log phase by regular passage in suspensionculture in Spinner MEM3 supplemented with 10%(heat-inactivated) calf serum, 0.04% Pluronic, 0.005%streptomycin, and penicillin, 50 units/ml, in an atmosphere of5% COa in air, hereafter referred to as complete medium.Monolayers were used for the heating experiments with MEMsubstituted for Spinner MEM in the complete medium. Thecells were negative for My coplasma contamination on periodictesting for their characteristic growth on pleuropneumonia-likeorganism agar (10). Cells growing on monolayers weredetached with 0.1% trypsin for 5 min at room temperature. Insome experiments cloned sublines of HeLa were used afterhaving been obtained by a ring isolation technique (19).

'This work was supported in part by Grants CA-07175 andCRTY-5002 from the National Cancer Institute, NIH.

2American Cancer Society Professor of Oncology.

Received September 13, 1972;accepted November 8, 1972.3The abbreviations used

medium; TdR, thymidine.

are: MEM, Eagle's minimal essential

FEBRUARY 1973 415

Robert J. Falzer and Charles Heidelberger

Qoning Experiments. A 5-ml suspension containing 50single cells/ml of complete medium was pipetted into a 30-mlplastic Falcon No. 3012 tissue culture flask, gassed with 5%COj in air, stoppered with an 00 rubber stopper, and culturedat 37°. Five hr after plating, the cells were heated by

immersion in a water bath. Immediately prior tohyperthermia, the multiplicity was approximately 1.2cells/microcolony, and cell attachment was more than 90%complete, as indicated by a failure to lose cells during a changeof medium.

Immediately after heating, the cells were returned to a 37°

incubator where they remained undisturbed for 9 to 11 days,at which time they were fixed with methanol and stained withGiemsa. Only colonies consisting of at least 50 cells werecounted by direct visual examination according to the methodof Puck and Marcus (18). The results were scored only whenthe unheated control flasks, which had remained in a 37°

incubator, liad a plating efficiency greater than 70%.Inhibitor Studies. In experiments with inhibitors, the

compound was dissolved in phosphate-buffered saline, and 200¡i\were pipetted into plastic tissue culture flasks or Petri dishescontaining attached cells. At the appropriate time the inhibitorwas removed by replacing the medium witli drug-free complete

medium. In each experiment, unheated controls were exposedto inhibitors for equivalent times during culture at 37°.The

inhibitors were used at concentrations that produced less than25% kill at 37°.

Chemicals. Tissue culture media and heat-inactivated calfserum were obtained from Grand Island Biological Co. (GrandIsland, N. Y.). Streptomycin and penicillin were obtained fromChase. Pfizer & Co., Inc. (New York, N. Y.), cycloheximide(Actidione) and TdR (A grade) were purchased fromCalbiochem (Los Angeles, Calif.), and Giemsa-Lösung stainwas obtained from Roboz Surgical Instrument Co.(Washington, D. C.).

Equipment and Supplies. Petri dishes and tissue cultureflasks were obtained from Falcon Plastics Co. (Los Angeles,Calif.). Shaker cultures were maintained at 37°in a Gyrotory

Model G-25 air incubator (New Brunswick Equipment Co.,New Brunswick, N. }.). For the heating experiments,monolayer culture flasks were heated to within 0.05°of the

desired temperature by immersion of the flasks into waterbaths (our design) that were heated by a Bronwill ModelCTC20 temperature circulator (Bronwill Scientific, Inc.,Rochester, N. Y.). Cells were counted with a Coulter Model Bcounter (Coulter Electronics, Hialeah, Fl.).

RESULTS

Cell Survival Curves. We found that exposure of ourcomplete media to 50°for 5 hr did not influence cell survival

when added to unheated cells. Selawry et al. (21) alsoobserved no effect with a different heated medium.

Chart I shows the survival of HeLa cells that were heated at41.0—43.0°for various periods of time. Survival was notdecreased appreciably when cells were heated to 41.0°for up

to 5 hr. However, a critical temperature range for cell survivalwas found between 41.5 and 42.5°(Chart 1), at which 30 to

90% of the cells were killed following a 2-hr treatment.

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Chart 1. Composite cell survival curves. This is a semilog plot of cellsurvival versus the time of hyperthermic exposure of cells at varioustemperatures. Cell survival is given as a percentage of the 37°control.

Numbers in parentheses, number of experiments used to obtain thecurve. The standard deviation is given for each point.

Exposure of cells to 43.0° produced considerably more cell

killing.We chose 42.0° for the remainder of our studies because

this temperature produced more than 50% cell killing and yetis within acceptable limits for potential clinical treatment ofcancer (2).

Chart 2 represents a typical individual survival curve of acloned population of cells. This characteristic S-shaped curvewas frequently observed following exposure of any of 10different clones of HeLa cells to 42.0°.Since the location of

the point of inflection varied either among determinations ofthe cell survival of different clones made at the same time oramong determination of the survival of the same clone made atdifferent times, the composite cell survival curve at 42.0°was

nearly linear.Attempts to Produce Heat-resistant Cells. Preliminary

experiments revealed that clones resulting from cells that wereheated for 42.0°for 18 hr did not possess any detectable heat

resistance compared with cells that had not been heatedpreviously.

Since whatever properties that had conferred heat resistanceon the surviving fraction of cells had been lost during theperiod required to grow enough cells for a 2nd comparativeexperiment, we adopted the following procedure. Five-literbottles were plated with 2 X IO7 cells, cultured for 5 hr at37°,and heated to 42.0°for 44 hr. Following hyperthermia,

the bottles were shaken vigorously to dislodge dead, looselyattached cells from the monolayer surface. The remainingviable cells were trypsinized, transferred to plastic tissueculture flasks, cultured at 37°for 5 hr, and heated to 42.0°for

various times. Flasks containing cells that had not beenpreviously exposed to hyperthermia were simultaneously

416 CANCER RESEARCH VOL. 33

Hyperthermic Killing of HeLa Cells

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Chart 2. A typical cell survival curve based on 1 experiment.Replicate flasks containing a cloned subline of HeLa were exposed to42.0° for various times. Cell survival versus time is expressed as a

percentage of the unheated control.

heated in the same water bath.A significant number of cells survived prolonged exposure

to 42.0° under conditions estimated to produce more than a

5-log kill if cell killing continued at the initial exponential rateduring this period. However, cells obtained by this 1-stepthermal selection procedure did not possess any heritable heatresistance during subsequent heat treatments. Furthermore,cells obtained by thermal selection were either more sensitiveor more resistant than the original population, depending onthe timing of subsequent heat treatments (Chart 3). The mostlikely explanation for these results is that our 1-step thermalselection procedure selected primarily for a phenotypicexpression of genetically equivalent cells. The interpretationhas been supported by additional studies with synchronizedcells, which are reported in the following paper (17).

Delayed Hyperthermic Cell Killing. Colonies that developedfrom heated cells were consistently smaller and of morevariable size than the unheated control colonies. Therefore,the following experiment was carried out to determinewhether hyperthermia kills cells immediately or whether celldivision precedes death. Replicate flasks were plated with 300cells, cultured for 5 hr at 37°,heated to 42.0°for 2.5 hr, andcultured again at 37°.At daily intervals duplicate flasks were

fixed and stained, and the number of cells in the microcolonieswas counted.

Chart 4a shows that 77% of the cells heated to 42.0°for 2.5

hr remained as single cells 1 day after hyperthermia, while theunheated control cells doubled as expected. On the 2nd dayafter hyperthermia 63% of the cells had divided at least once(Chart 46). By the 8th day (Chart 4c), cells that had beenheated for 2.5 hr at 42.0°existed as 2 distinct populations; 1

group consisted of small colonies, which were observedmicroscopically to consist of dying cells and the 2nd group of

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Chart 3. Hyperthermic killing of cells after thermal selection.Replicate flasks containing cells that had been heated for 44 hr at 42.0°

were heated a 2nd time (B) 5 hr, (C) 1 day and (D) 2 days later. Thesecells were heated in the same water bath with cells (A) that had notbeen exposed to hyperthermia previously. The cell survival after variousdurations of hyperthermia is plotted as a percentage of the 37°control.

relatively large colonies consisted of cells that appeared to begrowing normally. However, the size of the colonies in theheated flasks indicated that an average of 1 to 2 fewer celldivisions had occurred. These results show that cell divisionwas delayed in most cells that were heated to 42.0°for 25 hr,

after which time some cells resumed normal growth, whereasother cells divided at least once more before death.

Effects of TdR and Cycloheximide on Hyperthermic CellKilling. Various investigators have reported that the synthesesof DNA, RNA, and protein are profoundly inhibited byhyperthermic exposure of cells (1, 11, 12, 14, 15, 23).However, the relationship of this inhibition to hyperthermickilling is poorly understood; therefore, the followingexperiment was conducted. Cells were heated to 42.0°in the

presence of 2 mM TdR or 1 /¿gof cycloheximide per ml, whichblock DNA (25) and protein (17) synthesis, respectively. Chart5 shows that cell viability was higher when cells were heated to42.0° in the presence of either of these compounds as

compared to cells heated without inhibitors. The effects ofhyperthermia on cells exposed to inhibitors of RNA synthesisare reported in the following paper (17).

Cellular Repair of Hyperthermic Damage. We used thefollowing radiobiological techniques to determine whethercells recover from sublethal or potentially lethal hyperthermicdamage. (For a review of these techniques, see Refs. 4 and 16.)

One convenient method of measuring the capacity of a cellto recover from sublethal damage is to compare a fractionateddose with the same dose of a lethal agent given at 1 time. In an

FEBRUARY 1973 417

Robert J. Falzer and Charles Heidelberger

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Chart 4. Delayed cell killing following hyperthermia. Cells wereplated into replicate flasks and heated to 42.0° for 2.5 hr. Afterheating, the flasks were incubated at 37°.Duplicate flasks were fixed

and stained at: a, 1 day; ft, 2 days; c, 8 days after hyperthermia. Theplot is a percentage of the total observed microscopically to consist of(A) single cells, (B) 2 cells, (C) 3 to 4 cells, (D) 5 to 8 cells, (£")7 to 16

cells, (F) 17 to 32 cells, (G) 33 to 64 cells, (//) 65 to 128 cells, and (/)more than 128 cells.

experiment in which cells are exposed to either 1 dose of alethal agent or an equivalent dose that has been divided into 2increments, there are 3 possible outcomes: (a) cell viability ishigher in cells exposed to a fractionated dose treatment,indicating that cells have recovered from sublethal damage inthe interval between treatments; (b) cell viability is lower incells that have been exposed to a fractionated dose, indicatingthat sublethal damage lias been converted into lethal damagein the interval between treatments; (c) there is comparablesurvival in cells that have been exposed to either a fractionatedor an unfractionated dose of the lethal agent, indicating eitherthat no sublethal damage was produced by the 1st incrementin the fractionated dose treatment or that cells are incapableof recovering from such damage in the interval between doses.

A 2nd type of recovery, in addition to recovery fromsublethal damage, is recovery from potentially lethal damage.This is most commonly measured by exposing cells to variousmetabolic inhibitors after the initial treatment with potentiallylethal agents (such as hyperthermia). If cell viability is eitherincreased or decreased by exposure to these compounds after

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hyperthermia. This shows the survival of cells heated to 42.0° for

various times in the presence of 2 mM TdR (a) (27) or cycloheximide,1 jug/ml (X), compared with cells heated to 42.0°in the absence of the

inhibitors (o).

hyperthermia, cells are considered to be capable of recoveryfrom potentially lethal damage.

Table 1 indicates that cell killing varied with the length oftime for which hyperthermia was applied after the cells wereplated. This may be related to a partial synchronization of thecells produced by medium changes and other manipulationsassociated with cell plating, as has been shown by others (26).Table 1 also shows that there was up to a 33% increase in eel]viability when hyperthermia was fractionated into three 1-hrtreatments at 42.0°, as compared with one 3-hr exposure at42.0°, indicating that cells can recover from sublethal

hyperthermic damage.However, in Table 2 we have shown that when the time

interval between 2 fractionated doses of hyperthermia isincreased beyond 6 hr, cell survival fails either to increase overthat of an equivalent unfractionated dose or to plateau at ahigh level as might have been expected from an extension ofthe results in Table 1. Furthermore, we have shown in Table 2that, when fractionated heat treatments are separated by atime interval of between 8 and 10 hr, cell survival falls to aminimum that approaches that produced by an equivalentunfractionated heat treatment. In addition, when the timeinterval between heat treatments exceeds 10 hr, cell survivalonce again increases. These results suggest that the net cellsurvival presented in Table 2 represents a summation of atleast 2 conflicting phenomena, recovery from heat-inducedsublethal damage and an increased sensitivity that results froma heat-induced cell synchrony, as will be discussed.

We have also found that a 3- to 6-hr exposure of cells to

418 CANCER RESEARCH VOL. 33

Hypothermie Killing ofHcLa Cells

Table 1Comparison of cell killing following 1 or 3 fractionated Joses of hyperthermia

Replicate flasks were heated to 42.0" for 1 hr during the time intervals indicated by X. In allcases, the total hyperthermic treatment was 3 hr at 42.0°.Cell survival is given as a percentageof the 37.0°control. Five experiments are included.

% cellsurvival32

+422

±616±319±442±453±559±458

±9Time

after plating(hr)5678XXXXX

XXXXX9

1011X

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13 14 15 1617XXXXXXXXX

Table 2Comparison of cell killing following I or 2 fractionated doses of hyperthermia

Replicate flasks were heated to 42.0°for 1 hr during the time intervals indicated by X. In allcases, the total hyperthermic treatment was 2 hr at 42.0°.Cell survival is given as a percentageof the 37.0°control. Three experiments are included.

%cellsurvival66

í671+261

±464±466±854±154±350±752±351*4567XXXXXX

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after plating(hr)8

9 10 11 12 13 14 15 16 17 1819XXXXXX

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cycloheximide, 1 /Lig/ml, administered immediately afterhyperthermia, resulted in up to a 35% increase in cell survival(Table 3). There was also up to a 31% increase in cell viabilitywhen 2 mM TdR was present in the growth medium for 3 to 6hr following exposure of cells to 42.0°.Thus, HeLa cells are

capable of repairing potentially lethal hyperthermic damage.

DISCUSSION

We have shown within the narrow temperature range of41.0-43.0° that a 2-hr hyperthermic exposure of HeLa cells

produced killing from 10% to 2 logs, respectively. A similartemperature range was lethal to LI210 leukemia cells asreported by Giovanella et al. (5). A composite plot made fromseveral experiments of the logarithm of cell survival versustime at a given temperature approached linearity in the41.0-42.0° temperature range and had a shoulder at higher

temperatures. However, individual plots of cell survivalfollowing exposure of cells to 42.0° had a characteristic

shoulder, followed by a linear portion, indicating thathyperthermic cell killing did not follow Ist-order kinetics.Similar cell survival curves are obtained following X-irradiationof cells (4, 16). According to the single-hit, multitarget modelof Elkind and Whitmore (4), the initial shoulder in the cell

Table 3Influence of TdR and cycloheximide on cell killing when added

to cells following hyperthermiaReplicate flasks were heated to 42.0°for 3 hr and then either 2 mM

TdR or cycloheximide, 1 ¿ig/ml,was added, and the cells wereincubated at 37.0°.After a 3- to 6-hr interval following hyperthermia,the inhibitor was removed, fresh drug-free medium was added, and (hecells were again cultured at 37.0°for 9 to 11 days. Inhibitors were alsoadded and removed at the same time to flasks maintained at 37.0". The

percentage of cell survival in the presence of inhibitors were based onthese unheated controls, which were never less than 8()'i of thedrug-free controls. In this manner cell survival is corrected for thetoxicity of the inhibitor at 37°and any loss of cells during the change

of medium.

Treatment cell survival

Heat 3 hr, then add no inhibitor 22Heat 3 hr, then add 2 mM TdR for 3 hr after hyperthermia 43Heat 3 hr, then add 2 mM TdR for 6 hr after hyperthermia 53Heat 3 hr, then add cycloheximide. 1 Mg/ml, for 3 hr 40

after hyperthermiaHeat 3 hr, then add cycloheximide. I ¿ig/ml.for 6 hr 57

after hyperthermia

survival curve indicates that an accumulation of sublethalevents is required for cell killing. Our fractionated doseexperiments provided additional evidence that hyperthermia

FEBRUARY 1973 419

Robert J. Falzer and Charles Heidelberger

causes subie thai damage in HeLa cells and that cells canrecover from such damage.

The 2nd shoulder in the cell survival curve at 42.0° in

individual experiments depicts decreased cell killing in aminority of the cells and was not observed following exposureof cells to X-irradiation (4, 16). This decreased rate of killingprobably represents a type of resistance. Our interpretationthat this resistance is not genetically determined came fromnumerous unsuccessful attempts to produce an enrichment ofcells resistant to hyperthermic killing in a mixed populationthat had survived prolonged exposure to 42.0°.Experiments

are now in progress to determine the origin of such phenotypicresistance to hyperthermia and its potential implication in theclinical treatment of cancer. Harris (8) and others (B.Giovanella, personal communication) have reported similarobservations that most cells surviving a single heat treatmentfailed to exhibit resistance during subsequent hyperthermictreatments. However, following repeated heat treatmentsHarris (9) has obtained heat-resistant cells..

We have observed several phenomena that are difficult toexplain. We cannot presently assess the significance of theapproximately 1-day delay of cell division following exposureof cells to 42.0°for 2.5 hr. A similar delay of cell division or a

marked decrease of mitotic cells following brief hyperthermiahas been observed with a variety of cells by others (3, 13,20,24). We are also unable to explain the delay in the lethalexpression of hyperthermic damage. Cells heated for 2.5 hr at42.0°underwent repeated division before death. Cells exposed

to minimal lethal doses of X-ray, but not of UV irradiation,can also undergo up to 4 to 5 cell divisions before death (4,16). This delay in the lethal expression of hyperthermia couldexplain why Harris (7) and Giovanella et al. (5) found that dyeexclusion was not a suitable indicator of viability followinghyperthermic exposure of cells.

The increased viability of cells treated with high levels ofTdR or cycloheximide after hyperthermia indicates that cellscan recover from potentially lethal hyperthermic damage.Therefore, hyperthermic cell killing is at least a 2-step process,involving a thermal alteration of 1 or more unidentifiedcellular components, followed by a conversion of thispotentially lethal damage into actual cell killing. Since cellularrecovery from potentially lethal damage occurred moreeffectively when DNA or protein synthesis was inhibited, thepossibility exists that the synthesis of these macromoleculeseither during or following hyperthermia may be required forthe conversion of potentially lethal damage into lethal damage.Furthermore, the protective effects of excess TdR andcycloheximide added to cells during hyperthermia providesadditional evidence but does not prove that the inhibition ofDNA and protein synthesis produced by hyperthermia (17)may protect cells against hyperthermic killing.

Numerous investigators have observed cell synchronyfollowing brief hyperthermic treatments (13, 20).Furthermore, we have shown (6, 17) that hyperthermic killingof HeLa cells is cell-cycle phase specific. Therefore, if weassume that heat induced partial cell synchrony, then at agiven time after an initial heat treatment there should be aperiod when a significant number of heated cells become moresensitive to a 2nd heat treatment, independent of any recoverythat then occurred following the 1st heat treatment. Our

results in Table 1 and 2 are consistent with this explanation, asare the many apparent inconsistencies in the literatureconcerning the effectiveness of fractionated heat treatments inproducing hyperthermic cell killing (3, 5, 21).

ACKNOWLEDGMENTS

The authors would like to thank Miss Sue Ann Lauer and Mr. GlennJohnson for providing skilled technical assistance.

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21. Selawry, O. S., Goldstein, M. N., and McCormick, T. Hyperthermiain Tissue-Cultured Cells of Malignant Origin. Cancer Res., 17:785-791, 1957.

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Hyperthcrmic Killing of HeLa Cells

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23. Warocquier, R., and Scherrer, K. RNA Metabolism in MammalianCells at Elevated Temperature. European J. Biochem., 10:362-370, 1969.

24. Westra, A., and Dewey, W. C. Variation in Sensitivity to HeatShock during the Cell-Cycle of Chinese Hamster Cells in vitro.Intern. J. Radiation Biol., 19: 467-477, 1971.

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