Comparison of Two Mathematical Models for Describing Heat-Induced Cell Killing

Author(s): Joseph L. Roti Roti and Kurt J. Henle

Source: Radiation Research, Vol. 81, No. 3 (Mar., 1980), pp. 374-383
Published by: Radiation Research Society
Stable URL: http://www.jstor.org/stable/3575196
Accessed: 15-12-2015 04:43 UTC

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RADIATIONRESEARCH81, 374-383 (1980)

Comparisonof Two MathematicalModels forDescribing

Heat-InducedCell Killing
JOSEPH L. ROTI ROTI AND KURT J. HENLE

Departmentof Radiology, Universityof Utah Medical Center,Salt Lake City, Utah 84132

ROTI ROTI, J. L., AND HENLE, K. J. Comparisonof Two MathematicalModels forDe-

Heat-Induced
scribing Cell Killing.Radiat.Res. 81, 374-383(1980).
A computer-based minimization algorithm is utilizedto obtaintheoptimum "fits"oftwo
modelsto hyperthermic cell killingdata.The modelschosenare themultitarget, single-hit
equation,whichis ingeneraluse,andthelinear-quadratic equation,whichhasbeenapplied
tocellkilling
byionizing irradiationbutnottoheat-induced cellkilling.
Thelinear-quadratic
equationfitshyperthermic cellkillingdataas wellas themultitarget, equation.Both
single-hit
parameters ofthelinear-quadratic equationobeytheArrhenius law,whereasonlyoneofthe
twoparameters of themultitarget, single-hitequationobeystheArrhenius law. Thusthe
function
linear-quadratic can completely definecell killing as a functionofbothtimeand
temperature. Inaddition,thelinear-quadratic modelwillprovidea simplified approachtothe
studyofthesynergism betweenheatandX irradiation.

INTRODUCTION

The effectsof hyperthermia(42-460C) on mammalian cells can be readily

assayed in termsof cell killing.The inactivationof mammaliancells by heat is
generallydescribedby the multitarget, single-hitequation,S/So = 1 - (1 -
e-tto?)n,
where S/So is the fractionof survivingcells, t is the heatingtime at a specific
temperature, and tois theheatingtimewhichwouldkill63% ofthecell populationif
theextrapolationnumber,n, were equal to one. Apparently,thisequationis chosen
largelybecause of the similaritybetween heat survivalcurves and those obtained
withionizingirradiation.The use ofthisequation has two advantages.It allows for
theeasy comparisonofcellularheatresponseat varioustemperatures, T, byusingto
(1-I0) and an Arrhenius analysis of The
1/to. latter feature has allowed the
calculationoftheactivationenthalpyforcell killing(2, 4, 6-10). The importanceof
thiscalculationis thatitprovidesa parameterforcomparingthethermodynamics of
cell killingwiththose of various molecularprocesses. One intriguing aspect of this
work,however,is thedifference in activationenthalpyabove and below 43?C (2, 6,
9, 10) which will later be discussed in greaterdetail.
Up to the presenttime,the analysis of heat-inducedcell killinghas been based
primarilyon the exponentialportionof the heat survivalcurve, therebyutilizing
onlythe value ofto(1-10). The interpretation of the extrapolationnumber,n, has
been complicatedby thepresenceofa temperature transient,thetimeduringwhich
cells pass from370C to thefinaltemperature.Onlya smallnumberofstudiescontain
detailed measurementsof thistemperaturetransient(2, 9, 10) whichis dependent

0033-7587/80/030374-10$02.00/0 374
Copyright? 1980 by Academic Press, Inc.
All rightsof reproductionin any formreserved.

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 MODELS FOR CELL KILLING BY HEAT 375

on the tissue cultureflasks,the volume of the mediumoverlyingthe cells, and the

specificdetailsof cell heating.The timeto reach 63% ofthe finaltemperature(i.e.,
1/eor that time which is equal to the reciprocalof the rate constant) should be
independentof the final temperatureprovided the heating conditions remain
constant and the thermal conductivityof the culture flask is temperature
independent in the range of 37-480C (2, 11). At least one study reported
measurementscontraryto this(10). As a resultof the transientproblem,analysis
has been restrictedto the "linear" portionofthesurvivaldata. Thus onlya fraction
of the available survival data is employed to determineto (2, 7-10). Further-
more, a decision regardingwhich data points (i.e., those off the shoulder) to
include in the to calculation is generallyarbitrary.To obtain an analysis which
employsall thedata, a computer-basedminimization proceduremustbe used. This
procedure can be used in fittingcomplete sets of data provided a reasonable
temperaturetransientcorrectionis made.
Studies of cell survivalfollowinghyperthermia have universallyemployedthe
multitarget,single-hitmodel. However, itis possible thatalternativemodels might
describe the cell survivaldata as adequately as thismodel. One alternativeis the
linear-quadratric model, S/So = e-w+(t)t, firstproposed by Kellerer and Rossi
(12). Later Chadwick and Leenhouts (13) derived this equation based on the
molecular nature of the events which lead to cell death. Thus, if the
linear-quadratic model could adequately describe the hyperthermic cell-killing
data, itmayprovidenew insightsintoeventsleadingto heat-inducedcell death. For
example, it would be of interestto determineifthe constantsa and /3obeyed the
Arrheniuslaw. This resultwouldallow a completeparameterization ofcell killingas
a functionofbothtimeand temperature.In addition,thelinear-quadraticmodel,if
applicable to hyperthermia,should facilitate the description of heat-ionizing
radiationsynergismbecause all the parametersare in the exponentialpartof the
expression.

MATERIALS AND METHODS

Computeralgorithm.All computeroperations(survivalcurve fitting and linear

regressionanalysis) are conducted using a Tektronix4051 minicomputer.The
Marquardtleast-squaresmethoddescribedby Bevington(14) is used to obtainthe
best-fitsurvival curves. In our program,the X2 statisticin Tables I and II is
calculated usingthe logarithmof the survivalvalues as follows:

X2 = (log S - log S)2,

whereS is the calculated survivingfraction,S; is the observed survivingfraction,

and n is the numberof data points. Accordingto thisprocedure,the "best-fit"is
obtained when the value of X2is a minimum(see Discussion).
Experimentaldata. All experimentaldata are takenfrompublishedor in press
reports.We consider only those studies whichinclude survivalcurves at fouror
more temperatures,with a minimumof four or more data points at each
temperature.For ease of presentation,the data sets are noted in an abbreviated

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376 ROTI ROTI AND HENLE

formatas follows:CHO: B&H (9), W&D (2), and S,ea (7); HeLa: RR, H&W (8),
and G,ea (6); pig kidney:H (1). Each of these data sets is correctedfora
temperaturetransient 2.5 minfromreported
bysubtracting heatingtimes(2, 15). In
thecase ofa ratgliosarcoma:RR&L (10), a 3.5-mincorrection is madebecause
theseauthorsdescribea longertemperature transient
fortheirconditions.

RESULTS

To determine ifthelinear-quadraticequationwilldescribehyperthermic
cell
killingas adequatelyas does themultitarget, model,bothequationsare
single-hit
optimally "fit"tothesurvival dataateachtemperature.The modelwhichgivesthe
lowestX2valueis themoreconsistent withthedata. FortheCHO data(Table I),
two data sets (B&H and S&D) generallyshow a lowerX2value forthe linear
quadraticfit.In thesecases, onlyone survivalcurveoutofseven(B&H) andtwo
outofnine(S,ea) havea lowerX2valueforthemultitarget, model.The
single-hit
otherdataset(W&D) showsa slighttrendwiththelinear-quadratic modelhaving
thelowerX2at twotemperatures and themultitarget, modelhavingthe
single-hit

TABLE I

Survival Curve ParametersforCHO Cells

Data Temper-
refer- ature
ence (C) X2a n to X2a

B&H 41.5 9.94 x 10-3 -2.70 x 10-5 6.25 x 10-5 + 0.44 459.31 1.54 x 10-
42.0 1.58 x 102 -2.74 x 10-5 5.90 x 10-3 0.51 132.90 2.90 x 10-3 +
43.0 3.47 x 10-2 5.56 x 10-5 4.25 x 10" + 2.00 20.80 3.60 x 10-3 +
44.0 6.50 x 10-2 7.31 x 10-4 1.35 x 10-2 + 4.14 7.60 2.17 x 10-2
45.0 1.14 x 10-1 2.08 x 10-3 1.04 x 102 + 1.83 5.16 2.15 x 10-2
46.5 4.66 x 10-1 4.54 x 10-3 4.70 x 10-3 + 1.24 1.88 5.50 x 10-3
48.0 1.42 4.03 x 10-2 9.40 x 10-3 + 1.17 0.62 1.22 x 10-2

S,ea 41.5 6.54 x 10-4 1.43 x 10- 2.93 x 10-5 + 2.45 142.66 8.63 x 10-2
42.0 4.80 x 10-3 4.90 x 10-5 6.29 x 10-5 + 2.06 62.14 3.77 x 10-4
42.5 2.02 x 10-2 1.87 x 10-5 4.20 x 10-3 1.90 36.08 5.87 x 10- +
43.0 2.54 x 10-3 4.75 x 10-4 1.90 x 10-3 + 12.14 13.75 8.30 x 10-3
43.5 3.17 x 1.01 x 10-3 3.20 x 10-3 + 61.66 7.37 1.98 x 10-2
10-
44.0 3.98 x 10" 2.29 x 10-3 6.10 x 10-3 78.28 4.87 5.60 x 10-3 +
44.5 3.15 x 102 3.34 x 10-3 5.92 x 10-4 + 18.97 4.22 1.19 x 102
45.0 -3.46 x 106 1.42 x 10-2 2.25 x 102 + 28.58 2.18 7.57 x 10-2
46.5 -2.36 x 101 8.70 x 10-2 2.73 x 10-2 + 708.20 0.80 3.84 x 10-2
W&D 43.5 2.52 x 10-2 8.39 x 10- 9.58 x 10-2 90.51 6.53 1.91 x 10-2 +
44.0 5.88 x 10-2 1.32 x 10-3 6.95 x 102 22.73 5.17 5.68 x 102 +
44.5 3.86 x 10-2 4.20 x 10-3 1.59 x 10-1 + 41.34 3.33 2.00 x 10-1
45.0 1.12 x 101 7.37 x 10-3 1.10 x 10-2 + 7.47 2.67 4.51 x 102
45.5 2.61 x 101 2.94 x 10-3 1.11 x 10-2 1.70 2.91 5.80 x 10-3 +
46.0 5.29 x 10- 6.88 x 10-3 1.71 x 101 4.53 1.34 1.26 x 10-1 +
46.5 5.28 x 10- 4.11 x 10-2 1.02 x 101 9.90 0.85 3.54 x 10-2 +
a
The value ofX2is givenper degree of freedom.The + indicatesthe model withthe lowerX2value.

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 MODELS FOR CELL KILLING BY HEAT 377

TABLE II

Survival Curve Parameters

Temper-
Data ature
reference (oC a X2a n to X2a

HeLa 42 4.19 x 10-3 2.09 x 10-7 1.90 x 10-3 0.88 263.38 1.70 x 10-3 +
G,ea 43 1.62 x 102 -1.56 x 10-6 5.60 x + 0.99 63.24 5.92 x 10-4
10-
44 3.43 x 102 2.60 x 10-5 2.92 x 10-2 + 1.28 24.78 3.21 x 10-2
45 8.14 x 102 5.66 x 10-5 3.40 x 10-3 + 1.22 11.21 4.90 x 10-3

RR,H&W 44 2.86 x 10-2 2.86 x 10-5 1.10 x 10-3 +/- 1.09 31.60 1.10 x 10-3 +/-
45 8.86 x 10-2 -2.03 x 10- 2.80 x 10-3 + 0.78 14.23 4.20 x 10-3
46 1.17 x 10-' 7.34 x 2.20 x 10-3 + 1.61 6.34 3.00 x 10-3
10-
48 4.28 x 10-' 1.51 x 10-2 2.04 x 10-2 2.53 1.47 1.81 x 102 +
Pig kidney 44 1.15 x 102 3.78 x 10-5 1.70 x 10-3 1.41 53.88 1.50 x 10-3 +
H. 45 2.67 x 10-2 1.55 x 10-4 4.20 x 10-3 2.46 19.27 1.69 x 10-4 +
46 8.97 x 102 8.59 x 2.07 x 10-2 1.39 10.60 1.51 x 10-2 +
10-
47 1.22 x 10-' 4.37 x 10-4 2.67 x 10-2 + 1.63 6.40 3.46 x 10-2

Rat gliosar- 42 2.68 x 10- 6.85 x 10-7 2.90 x 10-3 1.32 281.81 2.47 x 10-3 +
coma (9L) 42.5 2.78 x 10-3 2.94 x 10-5 3.24 x 10-3 + 6.71 54.91 5.69 x 10-3
RR&L 43 -3.00 x 10- 2.20 x 10-4 6.39 x 10-3 31.60 18.34 4.27 x 10-3 +
44 -9.59 x 1.00 x 10-3 3.72 x 10-3 + 68.90 8.03 9.84 x 10-3
10-
45 3.13 x 10-2 2.87 x 10-3 6.91 x 10-3 7.42 5.34 1.42 x 10-2 +

a The value of
f is given per degree of freedom.The + indicatesthe model withthe lower X2value.

lowerX2value forthe remainder.The remainingdata sets (Table II) are small(less

than five survivalcurves) and no specifictrendscan be observed. Both models
show a lowerX2value in eightsurvivalcurves. Overall,itis impossibleto establish,
on a statisticalbasis, thatone of these models produces bettersurvivalcurve fits,
but these results do show that the linear-quadratic equation can describe
hyperthermic cell killingas adequately as does the multitarget, single-hitmodel.
Several investigators 4,
(2, 6-10) have used the Arrhenius law and thereciprocal
of toin the multitarget, model
single-hit to calculate the activation energyof cell
the
killing.Considering complex natureof cellular the
systems, interpretation ofthe
activationenthalpyobtained fromthiscalculationis open to debate. However, it
can be a usefulparameterforcharacterizingand comparingresults(2, 4, 6-10).
Thus we have examined the behavior of the linear-quadratic model under the
Arrheniuslaw. In thissense, thea termcould representa rateconstantfora killing
process in whichthe probabilityof cell death is a linearfunctionof time,whereas
the p3term could representa rate constant for a killingprocess in which the
probabilityof cell death is a functionof time squared. By analogy to ionizing
radiation(13), we willreferto these,respectively,as one-hitand two-hitprocesses.
From the foregoing,one would expect that a plot of In a and In /3versus the
reciprocalof the absolute temperaturewould give straightlines. As can be seen
(Figs. I and 2), theseplotsgivereasonablygood straightlinesforall sets ofdata. An
interesting aspect of these plots is thatthe break in the Arrheniusplot of (lI/to)at
43?C is not evident in the a plots. Also, it appears to be absent in the /3plot;
however,onlythreedata sets have positivevalues of /3below 43?C. Thus thereis a

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378 ROTI ROTI AND HENLE

?C
40 42 44 46 48
ion I I 1
_

CHO

100

a/
A/

10
,
'
/
0 O
,

/,/,
10~~0
//
2?x /)

0
:
1-5_
I.

319.5
319.5 315.5
315.5 31.5
311.5

FIG. 1. Arrheniusplotofthea and /3parametersfromCHO data. The values ofthea parameter(open

symbols) or the values of the /3parameter(solid symbols) are plottedagainst the reciprocolof the
absolute temperature.The correspondingtemperaturein 0C is plottedat the top for reference.The
variouslyshaped symbolsreferto differentsets of data as follows:B&H, (9) O and O; W&D (2) A and
A; and S,ea (7), O and U.

constantactivationenthalpyforthe one-hitprocess throughoutthe temperature

rangestudied,and the same is probablytruefortheactivationenthalpyofthetwo-
hitprocess. These energiesare giveninTable III. Ifthedata sets are pooled (except
S,ea.), overallactivationentropiescan be obtained.These activationentropiesare
161.6 + 15.9 and 283.9 55.6 kcal/mole?K for the one-hit and the two-hit
processes, respectively.In_ both cases, the linear correlationcoefficients
suggest
linearityto the ?95% confidencelimit(16). Also, it should be mentionedthat
survivalcurveswitha negativecawerenotincludedinFigs. 1and 2 (see Discussion).

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 MODELS FOR CELL KILLING BY HEAT 379

0C
40 42 44 46 48
l0

HeLa
PK
RGS

oro
0 8

10-2

a
10-
iO-

105

106'

I0"7 i
I

319.5 315.5 311.5

(x 105)
FIG. 2. Arrhenius plotofthea and/3parametersfrom HeLa, pigkidney, andratgliosarcoma cells.The
values of the a parameter(open symbols)and the values of the/3parameter(solid symbols)are plotted
Thecorresponding
oftheabsolutetemperature.
againstthereciprocol temperaturein?Cis plotted
atthe
topforreference. shapedsymbolsreferto different
The variously setsofdataas follows:HeLa, RR,
H&W (8) O and@; HeLa, G,ea,(16) O andf pigkidney,H, (1) A andA; andratgliosarcoma,RR&L,
(10) V and V.

DISCUSSION

The survivalcurve fitting presentedin thispaper suggeststhatthelinear-quad-

raticformulation can describehyperthermic cell survivaldata as adequatelyas does
themultitarget, single-hitformulation. However, forseveralofthesurvivalcurves,
the value of eithera or /3is negative.This resultis nonintuitive and requiressome
additionaldiscussion. The firstquestion to consideris whetherthese resultsare

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380 ROTI ROTI AND HENLE

TABLE III

ActivationEnthalpies Kcal/moleoK

Cell line One-hitprocess LCCa Two-hitprocess LCCa

Cell line One-hitprocess LCC" Two-hitprocess LCC"

CHO
B&H 150.6 + 4.2 0.988 237.8 ? 35.2 0.969
S,ea 167.8 101.0 0.641 x 397.4 62.4 0.943
W&D 221.4 _ 28.8 0.960 209.8 ?_ 50.2 0.882
_
HeLa
G,ea 192.1 ? 18.0 0.991 387.1 79.5 0.980 x
RR,H&W 129.3 ? 17.4 0.982 316.1 +_ 5.1 1.000

Pig kidney
H. 167.2 ? 24.7 0.979 89.4 ? 17.8 0.335 x
Rat gliosarcoma L9
RR&L 172.7 + 25.1 0.990 x 493.0 ? 157.5 0.953 x

a
Linear correlationcoefficient,x indicates those data sets which are not statisticallylinear (P
> 0.95, 16).

unique to the linear-quadratic model or if thereare correspondingresults(e.g.,

n < 1.0 or n verylarge) in multitarget,single-hitmodels. Whenever/3is negative,
n > 20. These observationssuggestthatthe negativevalues of a or /3are due to
certainaspects of the data in those cases and not to unique problemswitheither
model of the minimizationalgorithm.Survival studiesat 43?C or less suggestthe
existenceofa thermotolerant subpopulation(9, 17), and negativevalues of/3appear
to be due to thisthermotolerant subpopulationat thetransitionzone betweenthe
original survival curve and the thermotolerant phase. We do not have a clear
explanationforthe negativea and the large values of n. It is possible thatthese
resultscould be due in part to an incompletetemperaturetransientcorrection.
The computer algorithmwe used provides for three optional methods of
weightingthe X2statisticin the form:

X2 = C Wi(logS, -
i=i
log S)2 (14).

S is thecalculated survivingfraction,Sl is theobserved survivingfraction,n is the

numberof data points,and Wiis optionalweightingfunctionwhichcan have three
forms:(a) W?= 1 or all pointsgiven equal weighing;(b) W, = 1/log SI , whichin
linearformatwillfavorthelowersurvivalvalues butinlogformatslightly favorsthe
highersurvivalvalues; or (c) Wi = 1/o~whichweightsthe data pointsaccordingto
the uncertaintyof each point. The analyses presentedin thispaper are repeated,
insofaras the available informationallows, withall threemethods.In the case of
1/o weighting, is determinedbythreemethods:(a) usingthestandarddeviations
o-
ofrepeatexperiments;(b) determining theuncertainty based on thenumberofcells
plated assuminga binomialdistribution; and (c) determiningtheuncertainty based
on thenumberofcolonies countedassuminga Poisson distribution. Althoughthese

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 MODELS FOR CELL KILLING BY HEAT 381

variousweighting schemesgiveratherdifferent valuesto theX2statistic, thefinal

valuesofa and p/3 or n andtoare ingeneralonlyslightly affected. In addition,the
particular modelwhichgivesa lowerX2is rarelyaffected (5 cases outof40). The
linearityoftheArrhenius plotswasnotaffected although theactivation energiesare
slightlylowerifthe1/logS weighing is used.Sincethemeasureoftheuncertainty
is oftenunavailableinpublishedsurvivalcurvesandourpurposeis toconsistently
comparetwomodels,we havechosentheunitweighting andrefrained frommaking
a statistical interpretation of theX2values. In addition,we have linearizedthe
linear-quadratic modelanddetermined a and/3bylinearregression analyses.This
procedurealso givessimilarresults.
The necessityforan accuratetemperature transient correction represents a
limitation forbothmodelssincea goodtemperature transientcorrection is required
foran accurateestimation of botha and /3in thelinear-quadratic modeland is
affectedsignificantly by the temperature transient in the multitarget, single-hit
model(15). Thustemperature transientcorrections arerequired bybothmodelsfor
a completemathematical description.
TheArrhenius lawhasbeenappliedfrequently toheat-induced cellkillingwithin
theframework ofthemultitarget, single-hitmodel(2, 4, 6-10). For thispurpose,
thereciprocalofthetois employedas a rateconstantforcellkilling.One curious
aspectofthesestudiesis thatthereappearsto be an activationenthalpy forcell
killingabove 430Cwhichis approximately threetimeslowerthanthatbelow430C
(2, 6-10). Previouslyit has been postulatedthatthe difference in activation
enthalpy aboveandbelow430Cwasduetotheexistenceoftwodifferent cell-killing
mechanisms (6, 7,9). However,thesecondparameter, n,ofthemultitarget, single-
hitmodeldoes notappearto obeytheArrhenius law. Thusthismodeldoes not
represent a completely determined survivalfunction. WhentheArrhenius law is
appliedtothea and/3parameters, straightlinesresult(Figs. 1and2) formostofthe
datasets.Inthecases wherethelinearcorrelation coefficientis low(TableIII), the
datado notappearto fita nonlinear function (Figs. 1 and 2); ratherthedata seem
scattered abouta line.Fromtheseresults,itis attractive to speculatethatthereis
one mechanismforthe first-order process(a) and anothermechanismforthe
second-order process(/3).However,sincetheoverallactivation entropy forthe
two-step processis within experimentalerroroftwicethatfortheone-step process,
it is possiblethatthe rate-limiting stepsof bothprocessesare the same. Then
combinations ofthesetwoprocessesgiverisetothedifferent activation enthalpies
aboveandbelow430Cseenintheli/to Arrhenius the
plot.Also, finding thata andP
each have a singleactivationenthalpy is ofinterest in thatit maybe possibleto
mathematically describe thecomplete survival responsebasedon thermodynamic
parameters, i.e., the activation and
entropy enthalpy usingtheEyringformofthe
Arrhenius law (18).
Another advantageofthelinearquadraticmodelis itspotential use to studythe
interaction ofheatand X irradiation. A previousstudyofthisproblemusingthe
multitarget, single-hitmodel resultedin an equation with seven arbitrary
parameters inspiteofthefactthatthetimebetweenheatandX irradiation washeld
constant (19,20). Use ofthelinear-quadratic equationcouldsimplify thisstudyas
follows:Thekilling ofcellsbyheatalonecouldbe describedby(S/So)H= e-a+t

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382 ROTI ROTI AND HENLE

and thekillingof cells by X irradiationalone by (S/So)x = e -a'+?f'3D , wherea' and

/3'are the parametersforX irradiationand D is the X-raydose. The cell killingby
heat and X irradiationwould then be given by (S/So)x+H = -[(a+#t)t+(a'a'D)D+@]
where 4 is an interactiontermwhichis a functionof the timebetweenheat and X
irradiation,the temperature,the X-rayand heat dose, and several otherpossible
parameters.The importantpoint of this developmentis that those parameters
whichaffectthe interactionof heat and X rays can be isolated fromthe effectsof
heat alone and X rayalone. We can combinetheabove equationsand eliminatethe
termsdue to heat alone and X ray alone obtaining:
(S/So)x+H

[(S/So)H][(S/So)x]
Of course,the undefined
function5 could be verycomplicated;however,we
believe thatthisresultis themoststraightforward
approachto a quantifiedstudyof
the heat and X-irradiationsynergism.

ACKNOWLEDGMENTS

This workwas supportedin partby GrantCA 16465.J. L. Roti Roti was supportedby RCDA Grant
CA 00254. Both grantswere awardedby theNational Cancer Institute,DHEW. The authorswould like
to thankDr. W. B. Stanishforderivingthe expressionforo2 based on the numberof cells plated,Drs.
L. A. Dethlefsen,S. P. Tomasovic, and R. L. Wartersforcriticalreviewofthemanuscript,R. Wakifor
computerprograming,and S. Stewartfortyping.

RECEIVED: December 4, 1978; REVISED:May 9, 1979

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