int. j. radiat. biol 2001, vol. 77, no. 6, 655 ± 664

Low-dose hypersensitivity after fractionated low-dose irradiationin vitro

S. C. SHORT, J. KELLY, C. R. MAYES, M. WOODCOCK and M. C. JOINER*

(Received 8 September 2000; accepted 30 January 2001)

Abstract. fully enable repair and therefore exhibit maximalPurpose : It was demonstrated previously that some radioresistant radioresistance. If this is the case, the radioresistancetumour cell lines respond to decreasing single, low radiation seen after doses > 1 Gy may mainly re� ect this extradoses by becoming increasingly radiosensitive. This paper reports repair. This is termed ‘increased radioresistance’the response of four radioresistant human glioma cell lines to

(IRR). A modi� cation of the linear-quadratic (LQ)multiple low-dose radiation exposures given at various intervals.Three of the cell lines (T98G, U87, A7) were proven already to equation has been used to model the response ofshow low-dose hyper-radiosensitivity (HRS) after single low cell lines that behave in this way. In this ‘induced-doses; the fourth, U373, does not show HRS after acute doses. repair’ (IR) model, the sensitivity parameter of theMaterials and methods : Clonogenic cell-survival measurements were cell population, a, is not constant but is typicallymade in vitro using the Dynamic Microscopic Image Processing

> 1 Gy Õ1 at very low doses and decreases as the doseScanner (DMIPS) or Cell Sorter (CS) following exposure to

240 kVp X-rays one or more times. increases. Statistical � tting of this model to the dataResults: A consistent, time-dependent hypersensitive response to from cell lines showing HRS has con� rmed thata second, or subsequent, dose was observed in the cell lines that this concept of dose-dependent radioresistance is ademonstrated HRS. This time-dependent change in radiosensit-

tenable explanation of low-dose radiation e Ú ectsivity did not occur in the radioresistant cell line that did not(Lambin et al. 1993, Short et al. 1999b). In the IRshow HRS (U373). In one cell line that demonstrated strong

HRS, T98G, a similar time-dependent hypersensitive response model, low-dose hypersensitivity is explained as thewas also seen when the cells were irradiated whilst held in the constitutive response of cells without the enabling ofG1-phase of the cell cycle. In this same cell line, signi� cantly repair mechanisms triggered by higher doses.increased cell kill was demonstrated when three very low doses

The single-dose IRR phenomenon has similarities(0.4 Gy) were given per day, 4 h apart, for 5 days, comparedto the adaptive response seen in some cell lines inwith the same total dose given as once-daily 1.2 Gy fractions.

Conclusions : These data demonstrate the possibility that a multiple- which a small initial ‘priming’ radiation dose (usuallydose per day, low-dose per fraction regimen, termed ‘ultra- < 0.5 Gy) can induce resistance to a second, higherfractionation’, could produce increased tumour cell kill in ‘challenge’ dose given some time later (Wol Ú et al.radioresistant tumours compared with the same total dose given

1988, Fan et al. 1990, Shadley 1994, Seong et al.as conventional-sized 2 Gy fractions.1995) . This adaptive e Ú ect has been most thoroughlystudied in human lymphocyte populations and, in

1. Introduction these cell lines, is time-dependent; an interval of atleast 4 h is necessary between the ‘priming’ dose andThere is now overwhelming evidence that somethe ‘challenge’ dose for the maximum adaptive radio-radioresistant cell lines respond to very low radiationresistance to be seen. Like IRR, adaption is assumeddoses (< 1 Gy) with an increasing cell kill per gray.to be related to an enabling or induction of repairThis phenomenon is termed ‘low-dose hyper-mechanisms, in this situation, as a response to theradiosensitivity’ or HRS (Lambin et al. 1993, 1994,� rst dose. However, Wouters and Skarsgard (1997)1996, Marples and Joiner 1993, Joiner 1994, Singhargued that IRR and the adaptive response must beet al. 1994, Skarsgard et al. 1996, Wouters et al. 1996,separate phenomena. They investigated the e Ú ect ofMarples et al. 1997, Joiner et al. 1999, Short et al.a small priming dose on the survival of HT29 cells,1999a, b). HRS has now been demonstrated in morewhich demonstrate HRS, and found that a primingthan 26 human cell lines in vitro. It has been suggesteddose abolished the hypersensitive response to dosesthat it re� ects a di Ú erential triggering or induction< 1 Gy but did not increase radioresistance to higherof cellular repair mechanisms, so that only after thedoses given after the same priming dose. This indi-levels of damage that occur at high doses do cellscates that IRR at low doses can be triggered withoutinducing a classical adaptive response at high doses*Author for correspondence; e-mail: joiner@graylab.ac.ukand that the two mechanisms of increased radio-Gray Laboratory Cancer Research Trust, PO Box 100, Mount

Vernon Hospital, Northwood, Middlesex HA6 2JR, UK. resistance must therefore be di Ú erent. The precise

International Journal of Radiation Biology ISSN 0955-3002 print/ISSN 1362-3095 online © 2001 Taylor & Francis Ltdhttp://www.tandf.co.uk/journals

DOI: 10.1080/0955300011004132 6

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656 S. C. Short et al.

mechanisms involved in these responses are currently Hospital, from his own cell collection in December1996. U373 is a human-grade III astrocytoma cellunknown, but there is evidence that protein synthesis

and nucleic acid turnover are involved in both line supplied by Dr G. Perlman in 1996 from a cellline originally established by J. Ponten and held atadaption and IRR (Youngblom et al. 1989, Marples

and Joiner 1995) . Any of the many examples of gene the American Type Culture Collection (ATCC). Allcells were maintained in exponential growth in mono-regulation that have been demonstrated following

radiation exposure to higher doses could also be layer culture in vitro in Eagle’s minimum essentialmedium (EMEM) supplemented with 10% foetal calfinvolved in the induction of radioresistance after low

doses. Some groups have demonstrated induction of serum and 0.01% sodium pyruvate. The cells weremaintained in a 90% N2 , 5% CO2 , 5% O2 atmo-early response genes, stress genes and increased ‘heat

shock protein-like’ molecules following very low- sphere at 37 ß C. They were routinely passaged twicea week using a calcium-free salt solution with 0.05%dose radiation (Yamaoka et al. 1994, Prasad et al.

1995, Sadekova et al. 1997, Amundson et al. 1999) . trypsin and 0.02% EDTA. Single-dose cell survivalcurves for these cell lines have been published (ShortHowever, whilst DNA repair mechanisms seem likely

to be involved in both responses ( Joiner et al. 1999), et al. 1999a, b), and it has been shown that T98Gcells that have been treated with serum deprivationthis remains to be de� nitively proven.

There is speculation about the possible utilization to produce a reversible G1 arrest also demonstrateHRS after single low doses (Short et al. 1999a).of the HRS response clinically. If HRS is prevalent

in radioresistant cell lines, then it is possible thatradiotherapy using multiple very low doses per frac-tion might be more e Ú ective in radioresistant tumours 2.1. Experiments using asynchronously growing cells andthan conventional radiotherapy regimens using 2 Gy two consecutive low dosesper fraction. This prediction will only hold true ifthere is a hypersensitive response to each dose in a The Dynamic Microscopic Image Processing

Scanner (DMIPS) and the Cell Sorter (CS) werefractionated regimen. This might not necessarily bethe case if, for example, the e Ú ect of a subsequent used to measure clonogenic cell survival after expo-

sure to very low doses of 240 kVp X-rays. Thedose is in� uenced by an adaptive response to thepreceding one, as discussed above, thus producing a DMIPS is an automatic scanning microscope that

locates and counts cells on the surface of a cultureresistant response rather than a sensitive one. Smithet al. (1999) investigated the response of two cell lines, � ask and logs their positions in two dimensions so

that each cell position can be revisited after anC3H 10T1/2 and V79, to consecutive low doses totest the above prediction. They concluded that when appropriate interval to establish whether colony

formation has occurred. This allows accurate estima-multiple 0.3 Gy doses were administered at 3-h inter-vals in vitro, the resulting cell survival did not provide tion of the surviving fraction (SF) even when the

values are close to unity (Marples and Joiner 1993) .� rm evidence to support the presence of HRS.Although their results were consistent with the pres- To measure clonogenic survival after consecutive low

doses, T98G tumour cells were harvested from theence of some hypersensitivity to low doses, overallthey were adequately modelled using the LQ formula. growth surface using EDTA/trypsin whilst in expo-

nential growth, washed and diluted to 3 Ö 103The data presented here represent a further, moredetailed study, in which the authors set out to cells ml Õ 1 then plated to Nunclon high optical clarity

� asks at 3 Ö 103 cells per � ask with 5 ml medium.investigate the e Ú ect of consecutive and multiple verylow doses on a series of human cell lines already These cells were allowed to attach to the growth

surface (30 min) then the medium was decanted o Úknown to demonstrate low-dose hypersensitivity. Thisstudy provided a test of the principle of ‘ultrafrac- and the � asks re� lled completely with fresh medium

and sealed. Flasks were then irradiated with consecut-tionation’, i.e. the use of very low doses per fractionto overcome radioresistance. It also investigated ive doses of 0.4 or 0.5 Gy at 37 ß C using a Pantak

240 kVp X-ray set (HVL 1.3 mm Cu) with varyingwhether an adaptive response could be elicited aftervery low doses and how this relates to the induction intervals between doses (0–12 h). After irradiation,

the � asks were scanned using the DMIPS to recordof resistance at high doses.the positions of 200–300 cells. The � asks were thenreturned to the incubator at 37 ß C. After 7 days, the2. Materials and methods � asks were returned to the DMIPS and rescannedusing a revisiting programme to check manually forT98G and U87 glioblastoma cells were obtained

from ECACC in December 1996. A7 cells were a colony formation at each previously logged cell posi-tion. SF was calculated for each irradiated � ask bygift from Professor H. Suit, Massachusetts General

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657Low-dose hypersensitivity after fractionated low-dose irradiation

Table 1. Fraction of the T98G cell population in each phasedividing its plating e Ý ciency by the mean platingof the cell cycle following serum deprivation.e Ý ciency of three unirradiated control � asks.

U87, A7 and U373 cells were unsuitable for ana- Cell-cycle phase Fraction (%)*lysis using the DMIPS because of their non-discrete

G1 84.4 Ô 0.83colony morphologies. The DMIPS protocol was alsoS 8.55 Ô 0.63di Ý cult to use when more than two consecutive dosesG2 7.62 Ô 1.29were given, because of the time required to scan

each � ask, which had to be delayed until after the * Values are fraction Ô SD.� nal irradiation. Therefore alternative protocols,using the CS (U87 and U373) and conventional population, con� rming that > 80% of cells had G1survival assays (A7), were used in experiments with DNA content at the time of irradiation. These condi-these cells and in which more than two consecutive tions were used to reduce the potential confoundingdoses were given. The survival of A7 cells was e Ú ect of cell-cycle progression during experiments inassessed using conventional assays because these cells which cells were irradiated with consecutive very lowhad a low plating e Ý ciency following cell sorting. doses. T98G cells were plated and irradiated asIn the cell sort protocol, cells were harvested as above but serum-de� cient medium (0.5% FCS) wasdescribed above and plated using a modi� cation of used in place of standard EMEM (10% FCS). Whenthe technique � rst described by Durand (1986) . In the irradiations were completed, 3.5 ml mediumthis protocol, a FACSVantage equipped with a sort was removed from each sealed 40-ml � ask and rep-enhancement module (Becton Dickinson) was used laced with 3.5 ml FCS, thereby restoring the serumto dispense droplets containing single cells directly to concentration to 10%.25 cm2 culture � asks containing 5 ml medium. Cellswere identi� ed by their forward and side scatter 2.3. Fractionated very low doseswithout the use of � uorescent stains. Four hundredto 1000 cells were counted into each dish, left to In a further set of experiments using T98G cells,attach for 1 h in an incubator at 37 ß C in a 90% N2 , the SF after 15 fractions of 0.4 Gy, given three times5% CO2 , 5% O2 atmosphere, then irradiated using a day, was compared with the same total dose givena Pantak X-ray set, as described above, to give 0.4 Gy as once-daily 1.2 Gy fractions. Six � asks, each withor 0.5 Gy Ö 3 with intervals of 0–6 h. The � asks were 3 Ö 103 cells were prepared as described above andreturned to the incubator for 12–14 days, then irradiated with three fractions per day at 4-h intervalswashed and stained with crystal violet and colonies (09:00, 13:00 and 17:00 hours each day) each day> 50–100 cells were manually scored as survivors. for 5 days. A parallel set of � asks received 1.2 Gy asSF was calculated relative to the plating e Ý ciencies a single dose once per day for 5 days. On day 6, thefor three non-irradiated controls, as in the DMIPS cells in each � ask were removed from the growthexperiments. These two methods have been com- surface using trypsin/EDTA and the total cellpared by Short et al. (1999a) . In conventional assays, number in each � ask was measured using a haemo-cells were harvested as described then the cell sus- cytometer to count cell numbers in a known dilution.pension was diluted and pipetted to 25 cm2 � asks Five hundred cells from each � ask were then replatedcontaining 3 Ö 103 cells per � ask. These � asks were to 6 cm Petri dishes and incubated at 37 ß C in a 90%then irradiated as described above, and SF was calcu- N2 , 5% CO2 , 5% O2 atmosphere for 14 days tolated in the same way as in DMIPS and cell-sort allow them to grow to 50–100 cell colonies. Theexperiments. medium was then removed from the dishes and they

were stained with crystal violet. Colonies with > 50cells were manually scored as survivors and SF was2.2. Consecutive doses in growth-arrested cells calculated as described above. Relative clonogensper � ask was calculated asT98G cells enter a reversible G1 arrest in condi-

tions of serum deprivation or high cell density (con-� uence arrest) (Stein 1979) . This has been con� rmed

Final cells per flask (treated)

Final cells per flask (control)Ö surviving fraction

using propidium iodide staining and FACS analysisof T98G cells under di Ú erent growth conditions inthis laboratory (Short et al. 1999a). Growth in EMEM 3. Resultswith only 0.5% foetal calf serum allowed cell survival 3.1. Consecutive low doseswithout reducing plating e Ý ciency but produceda reversible G1 arrest. Table 1 shows the results Figure 1 shows the results of irradiating asynchron-

ous T98G cells with two consecutive 0.4 Gy doses atof FACS analysis of the G1-arrested T98G cell

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658 S. C. Short et al.

analysis of variance on these data (table 3) con� rmsa signi� cantly reduced SF at these times. Figures 3and 4 show the data from experiments with and A7and U87 cells using conventional plating and count-ing and the CS protocol respectively to measuresurvival after three 0.4 or 0.5 Gy doses at intervalsbetween 0 and 12 h. In A7 cells, the lowest survivalsoccurred with 4 and 6 h intervals ( p< 0.0003; table 3).In U87 cells, survival was reduced at intervalsbetween 1 and 4 h although this just failed to reachsigni� cance (table 3). In all three cell lines, the SFremained high when low doses were spaced by up to2 h and at intervals > 6 h the SF increased again.

These data are consistent with a repeated hyper-sensitive response after each low dose when thedoses are spaced by certain intervals. However, in� gures 1–4, these experiments were carried out withasynchronously growing cells, so movement throughthe cell cycle and/or cell division during the timetaken to irradiate the cells could have confoundedthe results. For example, cell proliferation occurringduring the time taken to irradiate the cells wouldspuriously increase the measured SF by increasing

Figure 1. Survival of asynchronously growing T98G cells after the numbers of cells in the irradiated populations.irradiation with two doses of 0.4 Gy separated by theTo explore this possibility, experiments were repeatedintervals shown, measured using the DMIPS. Data pointsusing T98G cells that were held in G1 arrest for theare mean SF, error bars are SEM. The solid line and

shading shows the measured SF after a single 0.8 Gy duration of the irradiations. It has been shown thatdose. The broken line and stippled shading (layered G1 arrest does not abolish HRS in these cells (Shortunder) are the predicted SF assuming HRS occurs after et al. 1999a). These time-course data are shown ineach dose (table 2).

� gure 5, which indicates that G1-arrested T98G cellsrespond in a similar time-dependent manner to asecond low dose; there appears to be a radioresistantintervals between 1 and 12 h. SF was measured using

the DMIPS. The response to a single 0.8 Gy dose response to a second dose when given at 0–2 hintervals, then a sensitive response when the second(‘zero interval’) is emphasized by the solid horizontal

line with accompanying solid shading indicating the dose is given between 3 and 8 h. At longer intervals,the SF appears to increase, but this is less markedrange of standard error. The dashed line and stippled

area ( Ô SEM) show the survival predicted after two than in asynchronous cells.consecutive doses, based on the single 0.8 Gyresponse and the T98G cell survival curve published 3.2. Consecutive doses in a cell line that does not showpreviously (table 2). The split-dose data demonstrate HRSa time-dependent response to the second dose: SFbelow that of the single 0.8 Gy dose occurred, and The e Ú ect of consecutive low-dose fractions was

also measured in U373, a radioresistant humanin line with the prediction, when the second irradi-ation was between 2 and 6 h after the � rst, but at glioma cell line which does not show single-dose

HRS (Short et al. 1999b). The results of irradiatingintervals < 2 h and > 8 h the SF was larger. Analysisof variance of all the data in � gure 1 con� rms this U373 cells with three consecutive 0.4 Gy doses are

shown in � gure 6. These data show no enhanced celltrend (table 3) but in this two-dose study the time-related variation in response does not reach signi� c- killing after consecutive low doses compared with the

same total dose given as a single fraction. At noance. Therefore, to magnify the possible di Ú erencebetween single and split doses, T98G cells were interval tested did the e Ú ect of three consecutive low

doses produce a SF lower than that of the singleexposed to three consecutive 0.4 Gy doses at variousintervals. Figure 2 shows the cell survival measured 1.2 Gy dose. This corresponds well with the predic-

tion for this cell line based on the evidence forusing the CS. In these experiments the lowest SF, inline with prediction (table 2), occurred when three complete absence of HRS published previously

(table 2).doses of 0.4 Gy were spaced by 2, 3 and 4 h and

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659Low-dose hypersensitivity after fractionated low-dose irradiation

Table 2. Comparison between cell survival predicted following multidose exposure and survival measured following a single dose.

Single dose SF ratio PredictedDoses Figure SF multi/single dose multidose SF Reference

2 Ö 0.4 1 0.941 0.840 0.791 Short et al. (1999a)(0.821–1.062) (0.690–0.892)

3 Ö 0.4 2 0.851 0.793 0.674 Short et al. (1999b)(0.799–0.903) (0.633–0.716)

3 Ö 0.4 3 0.921 0.531 0.489 Short et al. (1999b)(0.882–0.960) (0.468–0.509)

3 Ö 0.5 4 0.661 0.907 0.599 Short et al. (1999b)(0.610–0.711) (0.553–0.645)

2 Ö 0.4 5 0.915 0.920 0.842 Short et al. (1999a)(0.794–1.036) (0.731–0.953)

3 Ö 0.4 6 0.909 1.129 1.026 Short et al. (1999b)(0.759–1.059) (0.857–1.195)

Column 5 shows the predicted cell SF (with SE range), shown in � gures 1–6 as the stipple-shaded area, following multidose exposureand assuming that each dose elicits HRS. Obtained by multiplying the measured surviving fraction (column 3) following a single dose(equal to the sum of the 0.4 or 0.5 Gy doses indicated in column 1) and presented in � gures 1–6 as the zero-time data point and solid-shaded area, by the expected ratio of survival after multidose exposure compared with exposure to the single dose (equal to the sumof the 0.4 and 0.5 Gy doses indicated in column 1) given in column 4 and obtained from the full dose–response curves cited in column 6.

Table 3. F values and signi� cance ( p) in the analysis of varianceof the cell-survival data presented in � gures 1–6.

Figure Interval (h) F p

1 all data 1.34 0.26µ 0 to # 6 0.682 0.67µ 2 to < 6 0.866 0.50µ 6 to < 12 21.2 0.0036

2 all data 4.25 0.0044µ 0 to # 3 6.26 0.0057µ 3 to # 6 5.94 0.0071

3 all data 5.49 0.0001µ 0 to # 4 7.41 0.0003µ 0 to # 6 7.81 0.0001µ 4 to # 12 8.98 0.0006µ 6 to # 12 5.07 0.021

4 all data 2.66 0.027µ 0 to # 1 4.04 0.075µ 1 to # 16 3.07 0.019

5 all data 1.10 0.41µ 0 to # 4 1.18 0.38µ 4 to # 12 1.60 0.26

6 all data 0.519 0.79

p< 0.05 indicates a signi� cant change in cell survival over thetime interval indicated.

Figure 2. Survival of asynchronously growing T98G cells afterirradiation with three doses of 0.4 Gy separated by the3.3. Consecutive fractions of 0.4 Gy at 4-h intervalsintervals shown, measured using the CS. Data points aremean SF, error bars are SEM. The solid line and shadingThe SF after three doses of 0.4 Gy given at 4-hare the measured SF after a single 1.2 Gy dose. Theintervals was compared directly with that after 1.2broken line and stippled shading are the predicted SFand 2 Gy single doses. These data are shown in assuming HRS occurs after each dose (table 2).

� gure 7 and show that survival after three consecutive0.4 Gy fractions was signi� cantly lower than after thesame dose given as a single fraction ( p< 0.0002) . low doses on an asynchronously growing T98G cell

population is shown in � gure 8. These data demon-The e Ú ect of a 2 Gy single dose was not signi� cantlydi Ú erent to that of three consecutive 0.4 Gy fractions. strate a signi� cantly greater cell kill from multiple

small doses per day compared with the same nominalThe e Ú ect of a fractionated 5-day course of very

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660 S. C. Short et al.

Figure 3. Survival of asynchronously growing A7 cells after Figure 4. Survival of asynchronously growing U87 cells afterirradiation with three doses of 0.4 Gy separated by the irradiation with three doses of 0.5 Gy separated by theintervals shown, measured using conventional assays. The interval shown, measured using the CS. The data pointsdata points are mean SF, error bars are SEM. The solid are mean SF, error bars are SEM. The solid line andline and shading are the measured SF after a single shading are the measured SF after a single fraction offraction of 1.2 Gy. The broken line and stippled shading 1.5 Gy. The broken line and stippled shading (layeredare the predicted SF assuming HRS occurs after each under) are the predicted SF assuming HRS occurs afterdose (table 2). each dose (table 2).

doses. In V79 cells, which demonstrate HRS, adose given as single daily fractions. This was due toa reduction in cell number as well as a reduced ‘priming’ dose of X-rays or hydrogen peroxide given

6 h before ‘challenge’ doses down to 0.05 Gy inc-plating e Ý ciency. The plating e Ý ciency was reducedto a mean of 5.8% in the 0.4 Gy � asks and 8.2% in reased the SF in the low-dose region although it

did not completely abolish HRS (Marples and Joinerthe 1.2 Gy � asks compared with 15.9% in unirradi-ated controls. The total cell number on day 6 was 1995) . Wouters and Skarsgard (1997) studied the

e Ú ect of consecutive low doses on HT29 cells. Theirreduced to 1.16 Ö 104 in the 0.4 Gy � asks and to1.64 Ö 104 in the 1.2 Gy � asks relative to 2.62 Ö 104 data, however, also suggest that there is no priming

e Ú ect of these doses in the high-dose region, wherein control � asks. The � nal values of relative clonogensper � ask after irradiation in the two groups were an adaptive response would manifest itself.

In Smith et al. (1999), the response of two cell linessigni� cantly di Ú erent ( p 5 0.017) using a t-test.to multiple low doses was also measured. One ofthese lines, C3H 10T1/2, has not been studied in the4. Discussion low-dose region, so it is unknown whether HRSoccurs after acute single doses. Our own work hasAlthough the phenomenon of low-dose hypersens-

itivity is now well established, the e Ú ect of multiple shown that HRS is not a universal � nding, even inradioresistant cell lines, in which the e Ú ect is moreconsecutive low doses has not been studied in detail

before. The e Ú ect of the adaptive response on HRS common (Short et al. 1999b). Therefore, lack of HRSin C3H 10T1/2 cells after multiple low doses couldand the relationship between HRS/IRR and cellular

adaption after a small priming dose is uncertain. It be explained simply by the fact that HRS does notoccur at all in this cell line. The other cell line testedis possible that both IRR and the adaptive response

are di Ú erent manifestations of a single underlying in Smith et al. (1999), V79, has been studied in thelow-dose region, and is known to show HRS althoughprocess, and that, in some circumstances, a low

‘priming’ dose may abolish HRS to subsequent low the e Ú ect is much less than in the human tumour cell

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661Low-dose hypersensitivity after fractionated low-dose irradiation

Figure 6. Survival of asynchronously growing U373 cells afterFigure 5. Survival of serum-deprived G1-arrested T98G cellsirradiation with three doses of 0.4 Gy separated by theafter irradiation with two doses of 0.4 Gy separated byintervals shown, measured using the CS. The data pointsthe intervals shown, measured using the DMIPS. Theare mean SF, error bars are SEM. The solid line anddata points are mean SF, error bars are SEM. The solidshading are the measured SF after a single fraction ofline and shading are the measured SF after a single1.2 Gy. The broken line and stippled shading (layered0.8 Gy dose. The broken line and stippled shadingunder) are the predicted SF after three doses (table 2).( layered under) are the predicted SF assuming HRS

occurs after each dose (table 2).

consistent with repeated HRS, only occurs when thedoses are spaced by certain intervals. In T98G cells, alines reported here (Marples and Joiner 1993) . Smith

et al. used a 3-h interval between low-dose fractions second hypersensitive response was only demonstrablewhen the second dose was given at an interval ofin V79 cells and found no clear evidence of HRS

after 20 0.3-Gy fractions. The e Ú ect of consecutive between 2 and 6 h after the � rst. In U87 cells itoccurred when doses were spaced by between 1 andlow doses on V79 cells when they were spaced by

di Ú erent intervals, however, was not investigated. The 5 h and in A7 cells between 2 and 8 h. This time-course was broadly similar between the three diÚ erentdata presented here indicate that the low-dose

response is time-dependent, and Marples and Joiner cell lines studied and was also similar in a synchronizedpopulation of T98G cells that also showed signi� cantly(1995) have shown that pretreatment with X-rays can

produce a priming e Ú ect in V79 cells at certain reduced survival after two doses of 0.4 Gy spaced bybetween 3 and 8 h. A similar pattern was not seen,intervals and that 6–24 h were necessary between

fractions to allow for recovery of HRS. Therefore, however, in an HRS-negative cell line (U373). In allcell lines, the SF increased at longer interfractionlack of HRS in successive treatments with 3-h intervals

is readily explicable in the V79 rodent cell line. The intervals. These increasing SF observed after the long-est intervals, and therefore longest overall radiationabsence of HRS after consecutive doses based on

testing a single interfraction interval is clearly not times, might be explained by cell proliferation. Whenthe total time taken to irradiate these cell populationsadequate proof that multiple-dose HRS does not exist.

We have shown previously that HRS can be meas- is > 12 h, some cell division will have occurred so that,for the later irradiations, cells are irradiated as doublets.ured after single doses in glioma cell lines when

accurate methods such as the DMIPS and CS are This will lead to a spurious increase in the observedSF that obscures HRS. The exact contribution ofused to measure survival and that this also occurs in

G1-arrested T98G cells (Short et al. 1999a). The proliferation in these experiments can be calculated ifthe cell-cycle times of these cell lines are known, anddata in this study suggest that when consecutive low

doses are given, a lower than predicted cell survival, if the time at which signi� cant proliferation begins is

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662 S. C. Short et al.

Figure 8. Results of irradiations over 5 days in asynchronousFigure 7. Survival of asynchronous T98G cells given either T98G cells given as either three 0.4 Gy doses at 4-h

three 0.4 Gy doses at 4-h intervals or 1.2 and 2 Gy single intervals each day or 1.2 Gy single doses once daily. Errordoses. Error bars are SEM. The dotted lines represent bars are SEM. Multiple low doses per day producedthe predicted SF after three doses of 0.4 Gy according to decreased clonogen survival compared with the samethe LQ equation (upper line) and the IR equation (lower total dose given as once daily fractions ( p< 0.017).line), from Short et al. (1999a) . The SF after three 0.4 Gydoses is signi� cantly lower than after the same dose as asingle fraction ( p< 0.0002) but not signi� cantly di Ú erent Table 4. Estimates of cell cycle times (TC ) of the cell lines usedfrom that after 2 Gy. in consecutive low-dose experiments.

Proliferation ProliferationCell line TC (h) factor T 5 6 h factor T 5 12 hknown also. We therefore measured both of these

parameters using BrdUrd labelling. Table 4 shows the T98G 24.2 1.19 1.41results of BrdUrd pulse-labelling experiments to meas- A7 33.8 1.13 1.28

U87 22.3 1.2 1.45ure cell-cycle times of T98G, U87, A7 and U373 cells.U373 27.9 1.16 1.35In addition, we established that cell-cycle progression

occurs within 2 h of plating these cells (data not shown). The proliferation factors are calculated as exp(T ln 2/TC )The in� uence of proliferation on overall SF as a and predict the factors by which SF increases due to proliferationfunction of dose (d ) can then be calculated using the at intervals (T ) after irradiation.formula

explained by cell proliferation. These comparisonsSF 5 AexpGT ln2

TC HB between calculated SF allowing for proliferation andthe experimental data for asynchronous T98G cellsare shown in table 5. The agreement between the

Ö expG Õ arA1 1 Aas

ar

Õ 1Be Õ d /d cBd Õ bd 2H calculated and experimental results is also consistentwith the G1-arrested T98G data. Of the cells used inthese experiments, only 80% were in the G1-phasewhere T is time from � rst irradiation and TC is the

cell cycle time, and also using the induced-repair of the cell-cycle as measured by propidium iodidelabelling, however they show a less marked inc-parameters as described by Short et al. (1999a).

The results of these calculations are also shown in rease in SF at longer intervals between low-dosefractions, which can be explained by a reduction intable 4. When compared with the experimental data,

they support the explanation that the increased SF proliferation in the G1-arrested cells.The fact that qualitatively similar changes in SFobserved after longer interfraction intervals can be

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663Low-dose hypersensitivity after fractionated low-dose irradiation

Table 5. Predicted surviving fractions of T98G cells after two or three equally spaced fractions of 0.4 Gy given within 6 or 12 h.

Prediction PredictionPrediction with proliferation Experimental Prediction with proliferation Experimental

T98G T 5 6 h T 5 6 h data T 5 12 h T 5 12 h data

2# 0.66 0.79 0.707 (0.047) 0.66 0.94 1.34 (0.13)3# 0.67 0.79 0.75 (0.047) 0.67 0.94 0.92 (0.061)

IR-predicted survival was calculated from single-dose data in DMIPS and CS experiments. The in� uence of proliferation wascalculated from the data shown in table 4. The experimental data shown are mean SF with SEM in parentheses.

with time between doses occurred in both asynchron- maximum limits to be placed on the amount ofpossible cell-cycle resensitization, i.e. assuming thatous and G1-arrested cells also suggests that cell-cycle

progression is not itself the underlying cause of the one of two subpopulations has SF 5 1 following irradi-ation. The following formulae give the lowest possibletime-dependent variation in survival following split

doses in these experiments, although the extent of SF (SFnet) following two or three doses as a functionof the single-dose SF (S)the HRS phenomenon may well be modulated

according to cell-cycle position. This is in agreementSFnet 5 Ó 2S Õ 1 (two doses)with other data which suggest that there is no

consistent e Ú ect of these low doses on cell-cycle SFnet 5 (2S Õ 1)1/3 (1 1 (2S Õ 1)1/3 )/2 (three doses)progression, and in particular no cell-cycle delaycausing cells to build up in radiosensitive phases such Applying these formulae to the values in column 3

of table 2, leads to the conclusion that even in theas G2 (Power 1998) . However, the movement ofcycling cells into more sensitive cell-cycle phases in worse-case scenario, cell-cycle resensitization could

not be responsible for the time-dependent changesthe interval between successive doses clearly has thepotential to lead to a net increase in response when in radiosensitivity described in � gures 1–5.

Likewise the radioresistance seen when doses area single dose is split into two or three fractions. Thisidea has been termed ‘cell-cycle resensitization’ and spaced by 1 and 2 h intervals cannot be explained

by proliferation, or cell-cycle e Ú ects. We suggest thathas been described in detail by Brenner et al. (1996).They illustrated the concept with a simple numerical this resistance is equivalent to a priming e Ú ect, which

occurs when enough initial damage occurs to triggerexample in which a hypothetical cell populationconsisted of half the cells in a very sensitive phase, increased repair. If this is the case, ‘adaption’ occurs

earlier in these cells and diminishes more quicklyand the other half in a very resistant phase. Byextrapolating ‘sensitive’ and ‘resistant’ to their than reported in human lymphocytes and other cell

lines, including rodent cell lines.extremes, a single dose would kill around half thepopulation but the same dose split into two with an These data suggest a cell-cycle-independent time

course that in� uences the response to consecutiveinterval allowing each subpopulation of cells to swapcompletely into the opposite phase, would kill almost low doses. However, it is likely that the response to

a second dose after a � rst low dose is time, cell line-every cell. Maximum resensitization is achieved witha two-subpopulation swap over and adding further and dose-dependent. An important implication from

these data is that if HRS is to be exploited clinically,subpopulations with intermediate response dilutesout the e Ú ect. Also, no extra resensitization is gained the minimum time between fractions will be critical.

We have demonstrated HRS after up to 15 consecut-by further splitting the total dose into smaller frac-tions; the maximum amount of resensitization is ive very low doses in vitro when these doses are given

at intervals of 4 h. The implication from the calcula-obtained when a single dose is split into two. Thismaximum resensitization is dependent on the single- tions discussed above, is that at longer intervals HRS

will also still occur. The fact that the response diddose response, since this must represent the averageof the response of the subpopulations. For example, not disappear when very low doses were repeated in

this way suggests that increasing total dose andif the SF after a single dose is 1.0, then two hypothet-ical subpopulations must also have responded each prolonging overall irradiation time had little e Ú ect

on expression of the phenomenon. The SF which wewith an SF 5 1.0 and there could be no cell-cycleresensitization. If the single-dose SF 5 0.5, then this obtained after this 5 day regimen were close to those

predicted by assuming a hypersensitive response towould allow one subpopulation to respond with SF 51.0 and one with SF 5 0, therefore allowing cell-cycle each dose; this suggests that HRS does also occur

when doses are spaced by longer intervals. Howeverresensitization to be extreme. This argument allows

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664 Low-dose hypersensitivity after fractionated low-dose irradiation

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low doses of ionizing radiation: a possible role in inducedsensitive response leads us to believe that the radiores-radioresistance. International Journal of Radiation Biology, 72,istance observed after short (1–2 h) intervals may be653–660.analogous to the adaptive response seen when a low Seong, J., Suh, C. O. and Kim, G. E., 1995, Adaptive response to

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Biology Physics, 33, 869–874.produces increased cell kill when applied to radiores-Shadley, J. D., 1994, Chromosomal adaptive response in humanistant cells in vitro if the doses are spaced appro-

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Fan, S., Vijayalaxmi, Mindek, G. and Burkart, W., 1990, Smith, L. G., Miller, R. C., Richards, M., Brenner, D. J. andAdaptive response to 2 low doses of X-rays in human blood Hall, E. J., 1999, Investigation of hypersensitivity to frac-lymphocytes. Mutation Research, 243, 53–56. tionated low-dose radiation exposure. International Journal of

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Joiner, M. C., Lambin, P . and Marples, B., 1999, Adaptive vitro. Journal of Cellular Physiology, 99, 43–54.response and induced resistance. Comptes Rendus de l’Academie Wolff, S., Afzal, V., Wiencke, J. K., Olivieri, G. and Michaeli,des Sciences Serie III, 322, 167–175. A., 1988, Human lymphocytes exposed to low doses of

Lambin, P ., Malaise, E. P . and Joiner, M. C., 1994, The eÚ ect ionizing radiations become refractory to high doses ofof very low radiation doses on the human bladder carcinoma radiation as well as to chemical mutagens that inducecell line RT112. Radiotherapy and Oncology, 32, 63–72. double-strand breaks in DNA. International Journal of Radiation

Lambin, P ., Malaise, E. P . and Joiner, M. C., 1996, Might Biology, 53, 39–47.intrinsic radioresistance of human tumour cells be induced Wouters, B. G. and Skarsgard , L. D., 1997, Low-dose radiationby radiation? International Journal of Radiation Biology, 69, sensitivity and induced radioresistance to cell killing in279–290. HT-29 cells is distinct from the ‘adaptive response’ and

Lambin, P ., Marples, B., Fertil, B., Malaise, E. P . and Joiner, cannot be explained by a subpopulation of sensitive cells.M. C., 1993, Hypersensitivity of a human tumour cell line Radiation Research, 148, 435–442.to very low radiation doses. International Journal of Radiation Wouters, B. G., Sy, A. M. and Skarsgard , L. D., 1996,Biology, 63, 639–650. Low-dose hypersensitivity and increased radioresistance

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