int. j. radiat. biol 1997, vol. 72, no. 1, 101 ± 109

Modi® cation of potentially lethal damage in irradiated Chinesehamster V79 cells after incorporation of halogenated pyrim idines

N.A.P. FRANKEN ² *, C. VAN BREE² ³ , J.B.A . KIPP² and G.W. BARENDSEN²

(R eceived 2 D ecemb er 1 9 96 ; accept ed 1 1 M arch 1 9 97 )

Abstract. Radiosensitization of exponential ly grow ing and BrdUrd, IdUrd) into DNA is known to sensitize cellsplateau phase Chinese hamster V79 cells by incorporation of to ionizing radiation (Lett et al. 1964, Szybalski 1974,halogen ated pyrimidines (HP) was investigated for di å erent Iliakis et al. 1987a, 1989, M iller et al. 1992a,b). Theculture conditions that in¯ uenced repair. For this purpose cells

induced radiosensitization increases w ith the degreewere grown for 72 h with 0, 1, 2 and 4 mm of chloro-(CldUrd),of thymidine-rep lacement (Erikson and Szybalskibromo- (BrdUrd) or iodo-deoxyuridine (IdUrd) and were sub-

sequently irradiated with gam ma-rays from a 137Cs source, either 1963, Phillips et al. 1989). The mechanism of radio-in exponential grow th or in plateau -phase. Cell survival after sensitization by the HP has been suggested to beirradiation was determ ined by clonogen ic assay. In exponential ly either an increase in the amount of DNA damagegrow ing cultures thymidine-replacem ent in the DNA of the cells

induced by radiation, (Kinsella et al. 1984, Iliakisafter incubation with 4 mm of CldUrd, BrdUrd and IdUrd waset al. 1989, 1991, Ling and Ward 1990, Webb et al.22´3, 32´7 and 12´7%, respectively. In plateau -phase cultures the

percentage thymidine replacement in the DNA of the cells after 1993, Jones et al. 1995), an in¯ uence on repair ofincubation during grow th with 4 mm CldUrd, BrdUrd and IdUrd sublethal damage (SLD), and/or an enhancedwas 27´5, 33´8 and 10´7%, respectively. Linear-quadratic analy ses expression of potentially lethal damage (PLD) (Iliakisof the radiation survival curves were performed. In exponential ly

et al. 1987a,b, Wang and Iliakis 1992, Wang et al.grow ing cells a marked increase by a factor 2 ± 3 of the value of1994). Since di å erent processes are involved in thesea was obtained . The b term signi® cantly increased only in cells

which were grown in the presence of BrdUrd and which were phenomena several mechanisms might contribute totrypsinized and replated immediately after irradiation. In plat- radiosensitization.eau-phase cells which were trypsinized and plated immediately Additional complexity of the mechanisms causingafter irradiation both a and b increased up to a factor 2 ± 3 with

HP-induced radiosensitization is suggested by resultsincreasing incorporation of halogen ated pyrimidines. In plateauof Iliakis and co-workers, who showed that the degreephase cells which were allowed to repair potentially lethal

damage (PLD) for 6 h and subsequently trypsinized and plated , of sensitization can be lower in plateau -phase cellsa increased by a factor 3 ± 4. In these latter conditions changes as compared to proliferating cells (Iliakis et al. 1987a,in b were smaller. In exponential ly grow ing cells in which repair Iliakis et al. 1992, Wang and Iliakis 1992, Wang et al.was allowed after irradiation by plating prior to the treatm ent,

1994). These authors hypothesized that alterationsthe a values decreased for all the HP drugs tested as com paredin chromatin conformation as cells enter a restingto the a of cells plated immediately after irradiation. In contrast,

delay of plating for plateau phase cells yielded increased a values G 0 like phase, stabilizes chromatin and enablesnot only when com pared with the a of plateau phase cells plated repair of lesions that would have been ® xed inimmediately after treatm ent but also when com pared with the a proliferating cells .value of radiosensitized exponential ly grow ing cells. The increase

HP have been suggested to provide an advantageof a might be interpreted as an enhancement in the expressionin radiotherapy as radiosensitizers of cells in rapidlyof PLD. The larger contribution of ® xation of PLD might be

due to initial DNA damage and/or to inhibition of PLD repair growing tumours, in particular in clinical conditionsresulting from incorporation of HP. The increase of b might be in which critical normal tissues show lim ited prolif-attributed to enhanced interaction or to ® xation of sublethal eration and, as a consequence, take up less HP.damage (SLD). In view of clinical applications of HP it is of

Labelling depends on the growth fraction, cell loss,interest that sensitization is not abolished in plateau -phase cells.cell cycle time and potential doubling time (Steel1977, Rodriguez et al. 1994). Of special importance1. Introductionfor sensitization is the rate at which non-cycling cells

Incorporation of halogenated pyrim idines (HP), are recru ited into the proliferative compartmentchloro-, bromo- and iodo-deoxyuridine (CldUrd, during exposure to HP and a course of radiotherapy.

However, even in rapidly growing tumours, cells*Author for correspondence. may, after proliferative cycles, move into a non-² Department of Radiotherapy, Laboratory for Radiobiology,

proliferative stage. This might compromise the³ Department of Internal Medicine, Academic Medical Centre,degree of radiosensitization if resting cells are lessUniversity of Amsterdam, PO Box 22700, 1100 DE Amsterdam,

The Netherlan ds. a å ected by HP, or are better able to cope with

0955 ± 3002/97 $12.00 Ñ 1997 Taylor & Francis Ltd

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.

102 N . A . P . F ranken et al.

additional damage by repair of PLD. Further In the present communication radiosensitization isreported of exponentially growing and plateau -phaseinformation and insight in the mechanisms involved is

therefore of interest for the selection and optimization Chinese hamster V79 cells , studied in conditions w ithand without repair of PLD, respectively .of treatment schedules in clinical applications.

Sensitization of cells by HP has been commonlydescribed in term s of various parameters of survival

2. M aterials and m ethodscurves, either using the single-hit multitarget modelw ith the extrapolation number N and the ® nal slope 2.1. C e ll cul turegiven by D0 as parameters , or the linear-quadratic

V79 Chinese hamster cells ( Joshi et al. 1977) weremodel (LQ) describing lethal events as a function ofgrown as monolayers in Costar tissue culture ¯ asksthe dose D with two parameters : F (D )=aD + bD

2.(75 cm 2 ) in minimal essential medium (GIBCO-BRL)This model is based on well accepted biophysicalsupplemented with 10% foetal bovine serum, glutam-concepts, involv ing the assumption that lethaline and penicillin (MEM -s) at 37 ß C in an atmospheredamage can be induced by single-particle tracks andof 2% CO2 in air. About 5 Ö 103 cells were plated inby accumulation of multiple-particle induced4 ml M EM-s in a 30 mm culture dish when exponen-damage. It has been found to describe the low-dosetially growing cells were needed, or 7 Ö 104 cells wereregion of the survival curves better than the single-plated in 2 ml M EM-s when plateau -phase cells werehit multitarget model. Furthermore the LQ-modelneeded at the time of irradiation. A fter plating thishas been shown to describe, adequately, fractionationnumber of cells , in exponentially growing cultures,e å ects for normal tissue tolerance and for experi-cells went through 5 ± 6 cell divisions and in plateaumental tumours. The LQ-model has also the advant-phase cultures, cells went through 4 ± 5 cell cyclesage that it requires only two parameters to describebefore irradiation treatment. The cells were incub-survival curves. It allows the separate analysis ofated for 72 h in M EM-s in the presence of 0, 1, 2 orchanges in e å ectiveness in the low dose range, mainly4 mm of CldUrd, BrdUrd or IdUrd, while 2´5 mmdeterm ined by the linear term and in the high dosethymidine was added to mimic the average level ofrange determ ined mainly by the quadratic termthymidine in rodent plasma (Rodriguez et al. 1994)(Barendsen 1982, Joiner 1993). An additional advant-and to minimise HP-incorporation ¯ uctuations dueage of the LQ model is that its parameters can beto variations of thymidine concentrations of thediscussed in term s of speci® c mechanisms of cellserum (Limoli and Ward 1993). The distribution ofinactivation by radiation (Barendsen 1990, 1994).cells in the various phases of the cell cycle wasLinear-quadratic analyses of HP-induced radiosensit-monitored by ¯ ow cytometry . In exponentially grow-ization have been reported for exponentially growinging cultures at the time of irradiation treatment, cellshuman tumour cells and for four di å erent experi-were 45, 25 and 30% in G 0+G 1, S, and G 2+Mmental rodent cell lines (Miller et al. 1992a,b, vanphase respectively , and in plateau -phase cultures atBree et al. in press). An increase of the a-componenttime of treatment over 95% of the cells were inwas observed , corresponding to an enhanced lethalG 0+G 1 phase.damage at low doses. The b-component, which is

assumed to depend on the interaction of sublethallesions (SLD), was hardly a å ected by the observed

2.2. D eterm ination of percentage thym idine- replacementradiosensitization. In both studies the most radiores-istant cell lines were more sensitized than the radio- Exponentially growing and plateau -phase cells

were plated at the densities mentioned earlier andsensitive lines.As mentioned earlier , in the literature data have treated with CldUrd, BrdUrd and IdUrd as described

above. Percentage thymidine-rep lacement wasbeen presented concerning di å erences in radiosensit-ization between exponentially growing and plateau - measured by the technique described by Belanger

et al. (1987) and modi® ed by M iller et al. (1992a). Inphase cells and in the repair of additional lesionsproduced by HP uptake (Iliakis et al. 1987a, Wang short, the cells were trypsinized , pelleted and lysed

in water. A fter isolation from the lysate, DNA wasand Iliakis 1992). Analyses of these data were notbased on the LQ model, but nevertheless a large digested in order to liberate nucleosides. The digest

was analysed by high-pressure liquid chromato-contribution of repair of PLD was shown. A furtherstudy of sensitization of cells in plateau phase and of graphy. Percentage thymidine-rep lacement was cal-

culated as the concentration of incorporatedPLD repair in HP sensitized cells using the LQformula is evidently of interest in order to assess HP Ö 100 divided by the sum of the concentrations

of thymidine and incorporated HP. The ratio of thequantitatively the in¯ uence of HP in the range oflow doses of 0 ± 3 Gy, important in radiotherapy. concentration guanidine and the sum of the concen-

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.

103M odi® cation of P LD in irradiated V 7 9 ce lls after H P incorporation

trations of thymidine and incorporated HP was calcu-lated for each sample. This ratio was used to verifythat the recoveries of HP and thymidine were equiva-lent regardless of the percentage of rep lacementinto DNA.

2.3. R adiation survival curves

After incubation with CldUrd, BrdUrd or IdUrd,exponentially growing cells were trypsinized andrep lated in appropriate dilutions in 6-well macro-plates (Greiner) 4 h prior to irradiation. For anappropriate comparison with procedures applied tocells in plateau -phase, a separate set of cultures w ithcells in exponential growth were irradiated ® rst andimmediately after irradiation trypsinized and plated

Figure 1. Percentage thymidine-replacem ent in the DNA ofin appropriate dilutions in macroplates. Plateau- exponential ly grow ing (ex) and plateau -phase (pl) V79phase cells were irradiated, and 0 or 6 h after cells grown for 72 h in the presence of 0, 1, 2 and 4 mmirradiation trypsinized and rep lated in appropriate of CldUrd, BrdUrd and IdUrd. (Means of three

determ inations Ô s.e.m.)dilutions in 6-well macroplates. Irradiations wereperformed with gamma-ray s from a 137Cs source,yield ing a dose rate of about 1 Gy/min. Seven days 72 h growth in the presence of HP, are still exponenti-

ally growing was not very di å erent from the percent-after inoculation in the 6-well macroplates the colon-ies were ® xed and stained in 6% glutaraldehyde with age of thymidine-rep lacement in the DNA of cells

which had reached plateau phase after 72 h. In0´05% crystalviolet. Colonies of 50 cells or morewere scored as originating from a single clonogenic exponentially growing cultures thymidine-rep lace-

ment in the DNA of the cells after incubation withcell.Surviving fractions (S(D)/S(0)) after dose D , cor- 4 mm of CldUrd, BrdUrd and IdUrd was 22´3, 32´7

and 12´7% respectively . In plateau -phase cultures therected for toxicity of CldUrd, BrdUrd and IdUrdalone, (S(0)), were calculated and survival curves percentage thymidine-rep lacement in the DNA of

the cells after incubation during growth with 4 mmwere analysed using BM DP (Los Angeles, USA)statistical software by means of a ® t of the data by CldUrd, BrdUrd and IdUrd was 27´5, 33´8 and

10´7% respectively .multiple regression, accord ing to the linear-quadraticformula:

S ( D )/S (O )=exp Õ (aD +bD2 ) (1) 3.2. R adiation dose survival curves of ce lls in exponential

grow th plated b efore or after irradiation

A few examples of the survival curves obtainedare shown in Figure 2, illu strating radiation dose-3. Resultssurvival curves of exponentially growing V79 cells

3.1. P ercentage of thym idine- replacement and e å ec ts onirradiated before or after plating and of plateau -

proliferation after cul turing ce lls in the presence of H Pphase V79 cells w ith or w ithout plating delay afterirradiation. The four curves in Figure 2A di å er bothThe 72-h exposure to the CldUrd, BrdUrd and

IdUrd did not result in reduced cell numbers, neither in the linear term determ ining the initial slope (aparameter) and in the quadratic term mainly deter-in exponentially growing cultures nor in plateau

phase cultures at the end of the incubation periods mining the continuously curving high dose region (b-parameter). As can be deduced from the a and b(data not shown). Plating eæ ciency decreased to 85,

77 and 65% after incubation with 1, 2 and 4 mm of values, exponentially growing cells w ithout IdUrdare only slightly more sensitive if plated immediatelyClUrd respectively , but declined less for BrdUrd and

IdUrd (data not shown). after irradiation as compared to cells plated beforeirradiation. The extent of radiosensitization of theseDeterm ination of the percentage thymidine-

rep lacement after drug exposure showed that the cells grown in the presence of 4 mm IdUrd, a factorof about 2 in a and a factor 1´3 in b, is notincorporation of halogenated pyrim idines is concen-

tration-dependent (Figure 1). The percentage of thy- signi® cantly di å erent for the cells plated before orafter irradiation.midine-rep lacement in the DNA of cells which, after

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.

104 N . A . P . F ranken et al.

3.3. R adiation dose survival curves of plateau- phase ce lls

plated imm ediately and 6 h after irradiation and

compared w ith proliferating ce lls

In Figure 2B a few survival curves for plateau -phase cells are illu strated . As comparison of the datafor 0% thymidine rep lacement w ith data fromFigure 2A shows, a values for plateau -phase cellsplated immediately after irradiation are smaller by afactor 2 and b values are larger by a factor of about1´5 than for exponentially growing cells . The valuesof both a and b of plateau -phase cells w ithout HPplated 6 h after irradiation are smaller than thecorresponding values of plateau -phase cells platedimmediately after irradiation, showing a relativ elysmall in¯ uence of repair of PLD. It can further bededuced from Figure 2B, that radiosensitization ofplateau -phase cells by 4 mm IdUrd causes an increaseby a factor 2 in a and b if trypsinization and platingis performed immediately, while for delayed platinga increases more strongly.

3.4. Linear- quadratic analy ses of radiosenstisation w ith

di å erent H P in exponentiall y grow ing and plateau-

phase ce lls : e å ec t on the l inear parameter a

Radiosensitization was obtained in exponentiallygrowing and in plateau -phase cells for all threehalogenated pyrim idines. The values of a and b

derived by linear-quadratic analyses of survivalcurves of exponentially growing cells and plateau -phase cells are presented as a function of the percent-age thymidine rep lacement in Figures 3 and 4respectively . The increase of a for exponentiallygrowing cells after incubation with 4 mm of HP wasequal to factors between 2 and 3 (Figure 3A and B).The plating conditions, i.e. plating before or after

Figure 2. Radiation dose-survival curves of exponential ly grow -ing (Panel A) cells without HP (open symbols) and afterincubation with 4 mm IdUrd (closed symbols), and plateau -phase (Panel B ) cells without HP (open symbols) and afterincubation with 4 mm IdUrd (closed symbols). Lines rep-resent polynomial ® ts to the data. Each point representsthe mean value of nine di å erent experiments Ô s.e.m.Linear-quadratic parameters of these cells are:Exponential ly grow ing cells plated immediately after irra-diation: Without HP: a=0´18 Ô 0´02 b=0´017 Ô 0´003%. 4 mm IdUrd: a=0´38 Ô 0´04 b=0´023 Ô 0´007 &.

Exponential ly grow ing cells plated prior to irradiation:Without HP: a=0´15 Ô 0´02 b=0´013 Ô 0´003 #. 4 mmIdUrd: a=0´29 Ô 0´03 b=0´016 Ô 0´004 $.

Plateau -phase cells plated immediately after irradiation:Without HP: a=0´09 Ô 0´03 b=0´026 Ô 0´004 %. 4 mmIdUrd: a=0´17 Ô 0´02 b=0´062 Ô 0´005 &.

Plateau -phase cells plated 6 h after irradiation: WithoutHP: a=0´07 Ô 0´02 b=0´020 Ô 0´002 #. 4 mm IdUrd:a=0´30 Ô 0´03 b=0´024 Ô 0´004 $.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.

105M odi® cation of P LD in irradiated V 7 9 ce lls after H P incorporation

Figure 3. Linear parameter a plotted against the percentage of thymidine replacement in exponential ly grow ing cells platedimmediately after irradiation (A), plated prior to irradiation (B) and in plateau -phase cells plated immediately after irradiation(C), and plated 6 h after irradiation (D). Each point represents the mean value of three di å erent experiments Ô s.e.m.

irradiation, had no in¯ uence on the factor of increase and BrdUrd with cells plated immediately after irra-diation might indicate that a maximum of aboutof the value of a . For all three HP in the exponentially

growing cells which were plated before irradiation 0´5 Gy Õ1 is approached already after incubation

with 1 mm.the a increases w ith thymidine rep lacement. Thedata are not suæ cient to conclude whether a max- For plateau -phase cells which were trypsinized and

plated immediately after irradiation (Panel 3C), animum is attained, although the results for Cldurd

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.

106 N . A . P . F ranken et al.

Figure 4. Quadratic parameter b plotted against the percentage of thymidine replacement in exponential ly grow ing cells platedimmediately after irradiation (A), plated prior to irradiation (B) and in plateau -phase cells plated immediately after irradiation(C), and plated 6 h after irradiation (D). Each point represents the mean value of three di å erent experiments Ô s.e.m.

increase of a by a factor of about 2 is obtained for 3.5. Linear- quadratic analy ses of radiosensitiz ation w ith

H P in exponentiall y grow ing and plateau- phase ce lls :IdUrd, but for CldUrd the increases are smaller. Bye å ec t on the quadratic parameter bcontrast, for plateau -phase cells plated after a delay

of 6 h (Panel 3D) a larger increase of a by a factor The results obtained for the parameter b of the3 to 4 is derived after incubation with 4 mm Cld- quadratic term , which dominates responses at largerUrd, BrdUrd and IdUrd. IdUrd is the most potent doses, are subject to rather larger uncertain ties than

the values of a . For exponentially growing cells aradiosensitizer at low thymidine-rep lacement.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.

107M odi® cation of P LD in irradiated V 7 9 ce lls after H P incorporation

signi® cant increase in b is obtained only for BrdUrd radiosensitization is mainly due to an increase in thelinear parameter a . The quadratic parameter onlyin cells that were plated immediately after irradiation

(panel 4A). For cells plated prior to irradiation b increased when BdUrd was used as the radiosensitizerand the cells were rep lated immediately after irradi-values were not signi® cantly in¯ uenced by either of

the HP (panel 4B). In plateau -phase cells plated ation. The data on the a obtained for radiosensitizedexponentially growing cells are in agreem ent w ithwithout delay a signi® cant increase in b of a factor

2 to 3 is obtained for all three HP (panel 4C), but results reported by M iller et al. (1992a,b) whoobserved a clear increase in the a of two tumour cellfor delayed plating the increase is less pronounced

for all HP (panel 4D). lines growing in vitro and were sensitized with BrdUrdor IdUrd. However they observed a small increasein the b in only one of the cell lines after sensitization

4. D iscussionwith BrdUrd (Miller 1992b). The e å ect might be cellline dependent. On the other hand the value of b isThe major aim of the reported experiments is to

evaluate di å erences in sensitization by the three especially, in cases of high values of a , subject tolarge uncertain ties as shown by the relativ ely largehalogenated pyrim idines CldUrd, BrdUrd and IdUrd

in V79 cells which have incorporated these com- error bars in Figure 4.In Figure 3A the relation between a of exponenti-pounds in DNA and subsequently either remain in

the proliferating phase or pass into a resting phase. ally growing cells trypsinized and rep lated immedi-ately after irradiation, and the increasing % ofIn particular repair of potentially lethal damage was

assessed because of its relevance to radiation thymidine rep lacement is presented as linear.However, after incubation with 1 mm of CldUrd orresponses of tumours as well as normal tissues.BrdUrd or 2 mm of IdUrd, almost maximal increasesof the a were observed suggesting that a saturation

4.1. I ncorporation of H Pin radiosensitization was reached, associated with thetrypsinization and rep lating procedure immediatelyHP incorporation does reduce the plating eæ ci-

ency compared to controls, mainly w ith CldUrd and after irradiation. When the cells were plated beforeirradiation a clear linear relation between a andless w ith BrdUrd, but in the assessment of parameters

of survival curves this is taken into account. Uptake thymidine rep lacement could be obtained. In thesecells PLD repair could occur, because the cells areof HP di å ered between the compounds tested . Data

on incorporation of HP presented in Figure 1 show not disturbed after irradiation. A fter the lower % ofthymidine rep lacement, this repair could probablythe expected increase w ith the concentration in the

medium for exponentially growing cells , w ith less occur more easily than after a high % of thymidinerep lacement by HP.incorporation of IdUrd by a factor of about 2 ± 3 as

compared to CldUrd and BrdUrd. The lower incorp-oration of IdUrd may be explained by the larger 4.2.2. LQ analy ses of radiosensitiz ed plateau- phase ce lls and

comparison w ith exponentiall y grow ing ce lls . In plateau -radius of the iodine atom (Elkind and Whitmore1967). phase cells which were trypsinized and rep lated

immediately after irradiation both the linear and thequadratic parameters , a and b, changed . The

4.2. R adiosensitiz ation w ith H P increase of the a was less than a factor 2 while theincrease of b was about a factor 2 ± 3. In delayedBoth increased ® xation of PLD and increased

repair of PLD (reviewed by Iliakis et al. 1992), have plated plateau -phase cells , the a further increasedwhile the b returned to values as found in cellsbeen suggested to depend on trypsinization and

culture conditions. In addition these conditions might w ithout HP. The factor of increase of the a wasmuch greater than in plateau -phase cells w ithoutalso in¯ uence the modi® cation of cellu lar sensitiv ity

by HP. Because of this possibility the sensitization plating delay and also much greater than in radio-sensitized exponentially growing cells . These resultsby HP has been assessed for exponentially growing

and plateau -phase cells plated immediately after on plateau -phase cells seem to be di å erent fromresults of Wang and Iliakis (1992) and Iliakis et al.irradiation or after allow ing the cells to repair PLD.(1987a). They reported a decreased radiosensitiv ityin IdUrd or BrdUrd containing plateau -phase CHO4.2.1. LQ analy ses of radiosensitiz ed exponentiall y grow ing

ce lls . In Figures 3 and 4, results are presented of the and C3H10T1/2 cells plated after a delay as com-pared with cells that were immediately plated afteranalyses by the linear-quadratic formula plotted

as a function of the % of thymidine rep lacement. irradiation. This decreased radiosensitization wasobserved at radiation doses of 4 Gy and higher. OurIn exponentially growing cells HP-induced-

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.

108 N . A . P . F ranken et al.

LQ analyses of survival curves of radiosensitized V79 quadratic parameters , derived from analyses of sur-vival curves between proliferating as compared withcells demonstrated decreased radiosensitization after

doses of 4 Gy and higher, resulting in lower b values. resting cells . From the results in this study it can beconcluded that radiosensitization of mammalian cellsHowever, increased radiosensitization was observed

after doses of 1 ± 3 Gy resulting in higher values of a by HP is due to a complex of mechanisms involvingrepair and ® xation of damage, acting di å erently infor delayed plated plateau -phase cells as compared

with immediately plated cells . dependence on the proliferative state of the cells andon cell handling after irradiation. The direct compar-ison between immediate and delayed plating of

4.3. M echanism s of H P - induced radiosensitiz ationplateau -phase cells and between plateau phase andexponentially growing cells shows signi® cantDi å erent mechanisms might be involved in the

radiosensitization induced by halogenated pyrim id- quantitative di å erences. The data on the linear andquadratic parameters described in this paper provideines in exponentially growing compared with plateau -

phase cells . Wang et al. (1994) already suggested various new insights in the interpretation of radio-sensitization of delay plated plateau -phase cells . Inthat in exponentially growing cells increased DNA

damage production was the major component of particular it is shown that the sensitization by HP ofnon-cycling cells is not abolished by enhanced repairradiosensitization while in plateau -phase cells

radiosensitization occurred through inhibited repair of PLD. The possibility that HP increase the a inproliferating as well as in resting cells suggest possibleand/or enhanced ® xation of potentially lethal

damage. The increase of the a values for exponenti- advantages for clinical applications especially at lowradiation doses, used in radiotherapy. Further studiesally growing cells as found in this study, indicates an

increase in the number of directly lethal events due with other cell lines are required to elucidate whether,quantitatively, contributions to the increased lethalityto the HP. This is in agreem ent w ith observations of

Webb et al. (1993) and Jones et al. (1995) which are generally applicable. Insight into the modi® cationof these contributions is required if suggestions aboutsuggest that an important mechanism of radiosensit-

ization involves an increase of e å ective DNA double therapeutic applications are to be derived.strand breaks (dsb). M iller et al. (1992a,b) havesuggested that radiation-induced damage in cells Acknowledgem entswhich have HP incorporated into the DNA after

This work is supported by the Interuniversitylow-LET irradiation resembles the damage producedResearch Institute of Radiopathology and Radiationby high-LET radiation.Protection (IRS project #: 7.1.7.). The M aurits andIn plateau -phase cells plated immediately afterAnna de Kock Foundation is acknowledged for spon-irradiation the increase of a might be due to thesoring laboratory equipment.same mechanism as involved in exponentially grow-

ing cells . In these cells also an increase of b wasobserved indicating that accumulation of sublethal Referenceslesions contributed signi® cantly (Barendsen 1990).

Barendsen, G.W., 1982, Dose fractionation , dose rate and iso-Due to the immediate plating after irradiation this e å ect relationships for normal tissue responses. I nternationalsublethal damage might be ® xed. J ournal of R adiatio n O ncology , B io logy and P hy s ics, 8,

1981 ± 1997.The greates t increase in a was found in delayedBarendsen, G.W., 1990, Mechanisms of cell reproductive deathplated plateau -phase cells . This radiosensitization can

and shapes of radiation dose-survival curves of mamma-be interpreted as an enhanced ® xation of potentiallylian cells. I nternational J ournal of R adiatio n B io logy , 57,

lethal damage due to immediate DNA damage 885± 896.and/or to damaged DNA repair function in these Barendsen., G.W., 1994, The relationship between RBE and

LET for di å erent types of lethal damage in mammaliancells expressed during the interval before delayedcells: biophysical and molecu lar mechanisms. R adiatio nplating. The value of b in these cells returned toR esearch, 139, 257± 270.values as found in cells not containing HP. This

Belanger, K., Collins, J.M. and Klecker, R.W., 1987,demonstrates that sublethal damage has been Technique for detec tion of DNA nucleobases by reversed-repaired in HP-containing plateau -phase cells . phase high performance liquid chromatograp hy optimised

for quantitative determ ination of thymidine substitutionby iododeoxyuridine. J ournal of C hromatography , 417,

5. Concluding rem arks 57 ± 63.Bree, van C., Franken, N.A.P ., Bakker, P .J.M., Klomp-

In most earlier studies of radiosensitization by HP, Tukker, L.J., Barendsen, G.W. and Kipp, J.B.A., 1997,attention has not been focused on di å erences in Hypertherm ia and incorporation of halogen ated pyrimid-

ines: radiosensitisation in cultured rodent and humanrepair and associated changes of the linear and

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.

109M odi® cation of P LD in irradiated V 7 9 ce lls after H P incorporation

tumor cells. I nternational J ournal of R adiatio n O ncology , B io logy tusand future prospects: nonhypoxic cell sensitisers.I nternational J ournal of R adiatio n O ncology , B io logy and P hy s ics,and P hy s ics, (in press).10, 1399 ± 1406.Elkind, M.M. and Whitmore, G.F., 1967, The in¯ uence of

Lett, T., P arkins, G., Alexander, P . and Ormerod, M.G.,chem ical and physical factors on survival. In T he

1964, Mechanisms of sensitization to X-rays mammalianR adio b io logy of C ultured M ammalian C ell s, edited by M.M.cells by 5-bromodeoxyuridine. N ature , 203, 593± 596.Elkind and G.F. Whitmore (New York: Gordon and

Limoli, C.L. and Ward, J.F., 1993, A new method for introdu-Breach), pp. 145 ± 235.cing double-strand breaks into cellular DNA. R adiatio nErikson, E.L. and Szybalski, W., 1963, Molecu lar radiobiologyR esearch, 134, 160± 169.of human cell lines. V. Comparative radiosensitizing

Ling, L.L. and Ward, J.F., 1990, Radiosensitization of Chineseproperties of 5-halodeoxycytidines and 5-halo-hamster V79 cells by bromodeoxyuridine substitution ofdeoxyuridines. R adiatio n R esearch, 20, 252 ± 262.thymidine: enhancement of radiation-induced toxic ityIliakis, G., Kurtzman, S., P antelias, G. and Okayasu, R.,and DNA strand break production of mono® lar and1989, Mechanism of radiosensitization by halogen atedbi® lar substitution. R adiatio n R esearch, 121, 76 ± 83.pyrimidines: E å ect of BrdU on radiation induction of

Miller, E.M.,.Fowler, J.F. and Kinsella, T.J., 1992a, Linear-DNA and chromosom e damage and its correlation withquadratic analy sis of radiosensitization by halogen atedcell killing. R adiatio n R esearch, 119, 286± 304.pyrimidines, I. Radiosensitization of human colon cancerIliakis, G., P antelias, G. and Kurtzman, S., 1991, Mechanismcells by iododeoxyuridine. R adiatio n R esearch, 131, 81 ± 89.of radiosensitization by halogen ated pyrimidines: e å ect

Miller, E.M., Fowler, J.F. and Kinsella, T.J., 1992b , Linear-of BrdU on cell killing and interphase chromosom equadratic analy sis of radiosensitization by halogen atedbreakage in radiation sensitive cells. R adiatio n R esearch,pyrimidines. II. Radiosensitization of human colon cancer125, 56 ± 64.cells by bromodeoxyuridine. R adiatio n R esearch, 131,Iliakis, G., Wang, Y., P antelias, G.E. and Metzger, L., 1992,90 ± 97.Mechanism of radiosensitization of halogen ated pyrimid-

P hillips, T.L., Bodell, W.J., Uhl, V., Ross, G.Y., Rasmussen,ines. E å ect of BrdU on repair of DNA breaks, interphaseJ. and Mitchell, J.B., 1989, Correlation between expo-chromatin breaks and potentially lethal damage in plat-sure time, concentration and incorporation of IdUrd ineau-phase CHO cells. R adiatio n R esearch, 129, 202± 211.V79 cells with radiation response. I nternational J ournal of

Iliakis, G., Wright, E. and Ngo, F.Q.H., 1987a, Possib leR adiatio n O ncology , B io logy and P hy s ics, 16, 1251 ± 1255.

importance of PLD repair in the modulation of BrdUrdRodriguez, R., Ritter, M.M., Fowler, J.F. and Kinsella,

and IdUrd-mediated radiosensitisation in plateau -phaseT.J., 1994, Kinetics of cell labeling and thymidine replace-

C3H10T1/2 mouse embryo cells. I nternational J ournal of ment after continuous infusion of halogen ated pyrimidinesR adiatio n B io logy , 51, 541 ± 548.

in vivo . I nternational J ournal of R adiatio n O ncology , B io logy andIliakis, G., Wright, E. and Ngo, F.Q.H., 1987b , Repair and

P hy s ics, 29, 105± 113.® xation of potentially lethal damage (PLD) as dem on- Steel, G.G., 1977, G row th K ine tics of T umours: C ell P opulationstrated by delayed plating or incubation with araA in

K ine tics in R elatio n to the G row th and T reatm ent of C ancer.contact inhibited refed plateau -phase C3H mouse embryo (Oxford, UK: Clarendon).10 T1/2 cells grown in the presence of BrdUrd. R adiatio n Szybalski, W., 1974, X ray sensitization by halopyrimidines.and E nvironmental B iophy s ics, 26, 47 ± 62. C ancer C hem othe rapy R eport, 58, 539± 557.

Joiner, M.C., 1993, Models of radiation cell killing. In B asic Wang Y. and Iliakis, G., 1992, E å ects of 5 ¾ iododeoxyuridineC l inical R adio b io logy fo r R adiatio n O ncologis ts , edited by G.G. on the repair of radiation induced potentially lethalSteel, (London, UK: Edward Arnold ), pp. 40 ± 54. damage interphase chromatin breaks and DNA

Jones, G.D.D., Ward, J.F., Limoli,C.L., Moyer, D.J. and doublestrand breaks in Chinese hamster ovary cells.Aguilera, J.A., 1995, Mechanisms of radiosensitization I nternational J ournal of R adiatio n O ncology , B io logy and P hy s ics,in iododeoxyuridine-substituted cells. I nternational J ournal 23, 353± 360.of R adiatio n B io logy , 76, 647± 653. Wang, Y., P antelias, G.E. and Iliakis, G., 1994, Mechanism

Joshi, D.S., Deys, B.F., Kipp, J.B.A., Barendsen, G.W. and of radiosensitization by halogen ated pyrimidines: theKralendonk, J., 1977, Comparison of three mammalian contribution of excess DNA and chromosom e damage incell-lines with respect to their sensitiv ities to hypertherm ia, BrdU radiosensitization may be m inimal in plateau cells.c-rays and U.V.-radiation. I nternational J ournal of R adiatio n International J ournal of R adiatio n B io logy , 66, 133± 142.B io logy , 31, 485± 492. Webb, C.F., Jones, G.D.D., Ward, J.F., Moyer, D.J., Aguilera,

Kinsella, T.J., Mitchell, J.R., Russo, A., Morstyn, G. and J.A. and Ling, L.L., 1993, Mechanisms of radiosensitiza-Glatstein, E., 1984, The use of halogen ated thymidine tion in bromodeoxyuridine-substituted cells. I nternational

J ournal of R adiatio n B io logy , 64, 695± 705.analogs as clinical radiosensitisers: rationa le, current sta-

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/30/

14Fo

r pe

rson

al u

se o

nly.