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ICTR 2000 Biology

LOW-DOSE HYPERSENSITIVITY: CURRENT STATUS ANDPOSSIBLE MECHANISMS

MICHAEL C. JOINER, PH.D.,* BRIAN MARPLES, PH.D.,* PHILIPPE LAMBIN , M.D., PH.D.,†

SUSAN C. SHORT, M.B., PH.D.,* AND INGELA TURESSON, M.D., PH.D.‡

*Gray Laboratory Cancer Research Trust, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom;†Department of RadiationOncology, University of Maastricht, Maastricht, The Netherlands;‡Department of Oncology, Academic Hospital, Uppsala, Sweden

Purpose: To retain cell viability, mammalian cells can increase damage repair in response to excessive radiation-induced injury. The adaptive response to small radiation doses is an example of this induced resistance and hasbeen studied for many years, particularly in human lymphocytes. This review focuses on another manifestationof actively increased resistance that is of potential interest for developing improved radiotherapy, specifically thephenomenon in which cells die from excessive sensitivity to small single doses of ionizing radiation but remainmore resistant (per unit dose) to larger single doses. In this paper, we propose possible mechanisms to explainthis phenomenon based on our data accumulated over the last decade and a review of the literature.Conclusion: Typically, most cell lines exhibit hyper-radiosensitivity (HRS) to very low radiation doses (<10 cGy)that is not predicted by back-extrapolating the cell survival response from higher doses. As the dose is increasedabove about 30 cGy, there is increased radioresistance (IRR) until at doses beyond about 1 Gy, radioresistanceis maximal, and the cell survival follows the usual downward-bending curve with increasing dose. The preciseoperational and activational mechanism of the process is still unclear, but we propose two hypotheses. Thegreater amount of injury produced by larger doses either (1) is above a putative damage-sensing threshold fortriggering faster or more efficient DNA repair or ( 2) causes changes in DNA structure or organization thatfacilitates constitutive repair. In both scenarios, this enhanced repair ability is decreased again on a similar timescale to the rate of removal of DNA damage. © 2001 Elsevier Science Inc.

Radiation, Hypersensitivity, Induced radioresistance.

INTRODUCTION

Over the last decade, work performed principally at theGray Laboratory has identified a region of high sensitivityin the radiation survival response of mammalian cells atdoses below;0.5 Gy. This phenomenon, which has beentermed hyperradiosensitivity (HRS), precedes the occur-rence of a relative resistance (per unit of dose) to cell killingby radiation over the dose range;0.5–1 Gy. The latterphenomenon has been named increased radioresistance(IRR). A number of laboratories have now reported HRS/IRR-type responses after exposure to ionizing radiation,UV, and chemotherapeutic agents in a number of cell sys-tems. Here, we review the current state of knowledge onHRS/IRR, with the aim of proposing a mechanistic frame-work for the interrelated phenomena.

HRS/IRR-type responses in nonmammalian systemsEarly HRS/IRR-type experiments on irradiated maize plants

described hypersensitivity to doses less than 50 cGy compared

with higher doses, for both mutation induction and lethality inpollen grains after acute low-doseg-ray exposures (1, 2).Calkins (3) raised the idea of a threshold dose above whichcells acquired radioresistance to explain the shapes of thesedose–survival relationships and, in the protozoanTetrahymenapyriformis, a real increase in cell survival as the radiation dosewas raised above this hypothetical level. Survival curves ofbudding yeast (4) and algae (5) also demonstrate a similarHRS/IRR-type pattern in the low-dose region. “Adaptive re-sponses” have also been detected in lower cell systems, forexample in Chlamydomonas (6) and in sporelings of the fernOsmunda, where Hendry (7) showed that prior irradiationincreased radioresistance by a factor of 3–4 to subsequentexposures given 5 h later. This resistance decayed by 24 h.Yeasts demonstrate similar adaptive responses to ionizing ra-diation, as summarized by Boreham and Mitchel (8), as well asthe HRS/IRR cell survival pattern (4).

The variation of radiosensitivity of cells during progres-sion in the cell cycle was initially proposed to explain the

Address reprint requests to: Michael Joiner, Ph.D., Experimen-tal Oncology Group, Gray Laboratory Cancer Research Trust, P.O.Box 100, Mount Vernon Hospital, Northwood, Middlesex HA62JR, U.K. Tel: 144 192 382 8611; Fax:144 192 383 5210;E-mail: [email protected]

Presented at ICTR 2000, Lugano, Switzerland, March 5–8, 2000.Accepted for publication 31 August 2000.

Int. J. Radiation Oncology Biol. Phys., Vol. 49, No. 2, pp. 379–389, 2001Copyright © 2001 Elsevier Science Inc.Printed in the USA. All rights reserved

0360-3016/01/$–see front matter

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induced-resistance phenomena. For HRS/IRR, the argumentwas that low doses of radiation would eliminate cells pre-dominantly in sensitive phases of the cycle and that higherdoses would then be needed to kill cells in resistant phases.(This “two-population” explanation is similar to the situa-tion of oxic and hypoxic cell populations in tumors, whichrespond with differing sensitivity.) Joineret al. (9) havediscounted this two-population hypothesis. For the adaptiveresponse, the argument was that a first dose of radiationwould produce partial synchrony by eliminating cells pre-dominantly in sensitive phases of the cycle. The survivingcohort(s) of cells would then re-present in resistant phasesof the cycle some time later for the second, larger radiationdose. This hypothesis has also been challenged in nonmam-malian cell systems. For example, Howard and Cowie (10)showed that in Closterium, initial doses (which were only10% of those needed to produce any measurable response“off the shoulder”) induced an almost doubling in bothincremental (increasedD0) and absolute radioresistance to asubsequent larger dose. This increased radioresistance wassignificant within 1 h following the small conditioning dosebut required 6 h to reach maximum. This could not beexplained by the known variation in radiosensitivity of thesecells within the cell cycle; subsequently the same authors(11) showed that cells kept in darkness (hence in cyclearrest) demonstrated the same adaptive response and thatthis response could be inhibited by the presence of cyclo-heximide during the period between the conditioning doseand the subsequent challenge dose. Other studies reachedsimilar conclusions; for example, Horsley and Laszlo (12,13) examined synchronous cultures of Oedogonium andfound that a first dose of radiation induced resistance to asubsequent dose that was vastly in excess of any change insensitivity that could be explained on the basis of cell cycleprogression between the two doses. Bryant (14) also testedChlamydomonas, and Santieret al. (15) tested Chlorella,and the same picture emerged.

Koval (16) demonstrated HRS/IRR in the lepidopterancell line TN-368 irradiated in either air or nitrogen. Asimilar oxygen enhancement ratio was found for both thelow-dose (sensitive) component and the high-dose (moreresistant) components of survival. This survival curve sub-structure did result from genuine induction of radioresis-tance with increasing dose, but the mechanism was notdetermined (17, 18). According to Beamet al. (4), thesubstructure in these cell survival curves could only beexplainable as the sum of the individual responses of two ormore cell populations with differing radiosensitivity if thesensitivity of one of the populations was negative in thelow-dose range; in other words, survival in response toradiation would be greater than 100% for that cell popula-tion alone, which is clearly nonsensical. Based on the evi-dence for adaptive responses in photosynthesizing cells, it ismore likely that these survival curve shapes result fromdose-dependent radiosensitivity, i.e., increased or inducedradioresistance with increasing dose.

HRS/IRR in mammalian systemsImprovements in the methodology of clonogenic assays

within the last decade (19–25) have also made it possible toexamine mammalian cells with sufficient accuracy to re-solve changes in radiosensitivity at doses much less than100 cGy where cell survival approaches 100%. Conven-tional colony assays cannot reliably measure radiation-pro-duced mammalian cell death in this low-dose region. Beforethese developments, similar studies were generally possibleonly in the nonmammalian systems that respond at higherdoses, as reviewed above. These methods for improving theaccuracy of cell survival measurement determine exactlythe number of cells “at risk” in a colony-forming assay. Thisis achieved using either a fluorescence-activated cell sorter(FACS) to plate an exact number of cells (25) or micro-scopic scanning to identify an exact number of cellsafterplating (22). Using the latter technique, Marples and Joiner(26) and Marpleset al. (27) were first to define HRS andIRR in the dose range less than 100 cGy in mammalian cells(V79 hamster fibroblasts). These data demonstrated HRSbelow 10 cGy, but also HRS/IRR features following X-ir-radiation but not following single-dose irradiation withhigh-LET (linear energy transfer) neutrons. As with thenonmammalian systems cited above, the low-dose X-raysubstructure was not explained by differential sensitivity ofcells in different phases of the cell cycle (26). Therefore,there is significant homology in the HRS/IRR phenomenonbetween mammalian and nonmammalian systems, leadingone to suspect that it results from a conserved stress-re-sponse mechanism.

HRS/IRR in human cellsThere are now data on the very low-dose responses of

more than 26 different human cell lines, obtained using boththe FACS and the microscopic cell location assays (28–35).The HT29 line has additionally been independently con-firmed HRS positive by two different laboratories (25, 32,35), and the T98G glioma cell line has been found HRSpositive by both the FACS and microscopic cell locationassays. Therefore, low-dose hypersensitivity in human cellshas now been well documented by different laboratoriesusing different assay techniques and different conditions ofcell growth, handling, and irradiation. Figure 1 illustratesHRS/IRR in T98G human glioma cells (33). The phenom-enon is generally more pronounced in human cell lines thanin V79 cells, and the much steeper reduction in cell survivalat low doses compared with high doses is clearly visible.This comparison is indicated in Fig. 1 by the slopesas andar, which are sensitivity parameters in the induced repair(IR) mathematical equation (31) used to model these typesof data, as shown by the solid line. The linear-quadraticmodel substantially underestimates the effect of low radia-tion doses. Figure 2 summarizes the data on HRS/IRR inmammalian cell lines tested to date. These consist of colo-rectal carcinoma, bladder carcinoma, melanoma, prostatecarcinoma, cervical squamous carcinoma, lung adenocarci-noma, neuroblastoma, glioma, one nonmalignant lung epi-

380 I. J. Radiation Oncology● Biology ● Physics Volume 49, Number 2, 2001

thelial line, and one primary human fibroblast line. HRS istherefore widespread. Generally, it is those cell lines mostradioresistant to 2-Gy doses that demonstrate the mostmarked low-dose HRS, but this trend is no longer significantdespite earlier evaluations on more limited data sets (31).The radiosensitivity in the HRS region of the survival curve(as) is similar for all the cell lines regardless of the extent oftheir high-dose radiation response and is generally greaterthan 1 Gy21, which is very large. Two of the cell lineslacking an HRS response are very radiosensitive at 2 Gy,but in U373 glioma (surviving fraction at 2 Gy: SF25 0.63)and SiHa cervix (SF25 0.64) lines, HRS is also undetect-able. These lines should prove good candidates for studieson mechanisms, because they can be compared with othercell lines of similar high-dose radioresistance but whichstrongly express HRS/IRR (e.g., T98G, A7, Be11, HGL21,RT112).

HRS/IRRin vivoThere is evidence that hypersensitivityin vitro translates

into additional effectiveness of fractionated radiotherapy

given in very small doses per fraction. Thus when the doseper fraction is reduced below 1 Gy, the total dose needed toproduce damage decreases in mouse skin (36), kidney (37),and lung (38). This “reverse fractionation” effect is pre-cisely that expected from the HRS/IRR pattern of cellsurvival following low doses in cell lines, but, importantly,also implies arapid decayof adaptive resistance in thesemammalian systems over the period between fractions. Inthese studies, this interval was 7–8 h. Recent work on threehuman glioma cell linesin vitro has confirmed the rapidrecovery of HRS between successive doses as shown in Fig.3. This means that HRS could be exploitable in radiotherapyby using very many dose fractions optimally around 0.5 Gy,an approach we have termed “ultrafractionation” (39). Ob-taining therapeutic gain with ultrafractionation requires thatmore excess sensitivity occur in the tumor than in criticalnormal tissues. This amount of increased sensitivity at lowerdoses depends on the parameteras/ar shown in Fig. 2, butalso on therate at which the transition fromar to as occurswith decreasing dose. The “worst case” normal tissue testedso far is the kidney of the mouse (37), and if this iscompared with many of the glioma lines shown in Fig. 2,the predictions are promising. For example, if T98G werethe target tumor with kidney the critical normal tissue, 141fractions of 0.5 Gy per fraction to a total of 70.5 Gy wouldbe equivalent to 117 Gy in 2-Gy fractions to the tumor and60 Gy in equivalent 2-Gy fractions to the normal tissue.This is an overall therapeutic gain of almost 200%.

Whether low-dose hypersensitivity occurs in human nor-mal tissues, and to what extent, is clearly an important issue.On the one hand, demonstration of HRS would provideproof of the principle that the phenomenon so far observedin vitro and in animal models translates to the clinic; on theother hand, if the magnitude of HRS is too high in humannormal tissues, this would argue against a therapeutic gainfrom ultrafractionation. However, despite the impressivedocumentation of the HRS/IRR phenomenonin vitro, in-cluding human cell lines, andin vivo for various normaltissues in mice, there has been no clear-cut clinical evidencepublished. Stimulated by the various therapeutic possibili-ties of hyperradiosensitivity to very low doses, a clinicalstudy on this issue is going on in collaboration between thedepartments of oncology in Gothenburg and Uppsala inSweden (I. Turessonet al., personal communication). Theresults emerging from this work provide convincing evi-dence of the existence of a reverse fractionation effect fordoses per fraction below 1 Gy in human skin.

In the study, skin biopsies of 3-mm diameter are takenbefore and regularly during the course of radiotherapy ofprostate cancer patients. Prescribed tumor dose is 35 frac-tions of 2 Gy in 7 weeks using 11 or 15 MV photons.Biopsies taken from opposed lateral fields, with 5-mm bolusapplied to the left field, and at 1.5 and 3 cm outside thelateral fields, allow assessments after skin doses of 0.07,0.20, 0.45, and 1.10 Gy per fraction. The end point is thebasal cell density (BCD) in the epidermis. The Ki-67 index

Fig. 1. Survival of asynchronous T98G human glioma cells irra-diated with 240 kVp X-rays, measured using the cell-sort protocol.Each data point represents 10–12 measurements. The solid lineand dashed lines show the fits of the induced repair (IR) model andlinear-quadratic (LQ) models, respectively. At doses below 1 Gy,the LQ model, using an initial slopear, substantially underesti-mates the effect of irradiation, and this domain is better describedby the IR model using a much steeper initial slopeas.

381Low-dose radiation hypersensitivity● M. C. JOINER et al.

is also measured to serve as an indicator of the proliferationrate in the basal cell layer.

The mean dose responses for BCD after 0.45 and 1.10 Gyper fraction in 40 patients are compared in Fig. 4. Thesedata show a highly significant reverse fractionation effect,meaning a higher effectiveness per unit of dose of 0.45 Gycompared with 1.1 Gy in the depletion of basal cells in theepidermis. The ratio between the slopes of these dose–response relationships (dose modifying factor [DMF]) is1.8.

A confounding effect of cellular repopulation on thesedose–response relationships might lead to misinterpretationof a reverse fractionation effect. However, during the first 3weeks of radiotherapy, the Ki-67 index is depressed belowthe value in unirradiated skin, followed by a significantincrease during the next 4 weeks, in a similar pattern forboth 0.45 and 1.1 Gy per fraction. The dose–response slopesfor BCD established separately for these 2 periods giveDMFs of 2.1 and 1.7, respectively. This means that cellularrepopulation to some extent actually obscures the effect ofthe HRS/IRR phenomenon.

In a subgroup of 14 patients, BCD has also been assessedafter very low doses per fraction. Figure 5 shows the re-sponse in the dose per fraction range of 0.07 to 1.10 Gy,evaluated on biopsies taken after 20 fractions in 4 weeks.The dose–response relationship reveals a region of steepdecrease in BCD and hyperradiosensitivity up to 0.20 Gyper fraction, which precedes a plateau in BCD and increasedradioresistance. If the linear-quadratic description applied,the response for doses per fraction below 1.10 Gy should lieabove the dashed line in Fig. 5. On the contrary, the BCD at0.07, 0.20, and 0.45 Gy per fraction is found to be signifi-cantly below that line. The DMF is 3.8 and 3.4 for 0.07 and

0.20 Gy per fraction, respectively. These clinical findingsare confirmation that the experimental determinations ofHRS/IRRin vitro andin vivoactually translate to the humansituation.

Clinical evidence of hyperradiosensitivity to low radia-tion doses has also been seen in a study evaluating post-therapeutic salivary gland function in relation to radiationdose, using a functional assay (P. Lambinet al., personalcommunication). Twenty-one patients with a histologicallyproven carcinoma of head and neck of any stage treatedexclusively by radiotherapy were included in the study.They had both a CT and a scintigraphy with free 99mTc-pertechnetate in the treatment position with mask, beforeand one month after RT. The scintigraphy has a dynamicpart for evaluation of the function of each salivary gland bycalculating the cumulative gland uptake and salivary excre-tion fraction (SEF). This last parameter is a quantitativemeasure of the excretion response to carbachol, adminis-tered s.c. at the moment that the uptake reaches a plateau.Registration of dosimetric CT and SPECT images wasneeded to build up a dose–response curve for each individ-ual patient. Six patients had a large gradient of doses in theirparotid with a low minimal dose,20 Gy in 35 fractions,therefore 0.57 Gy per fraction or less. This allowed theconstruction of dose–response curves, as every 5 mm theabsorbed radiation dose could be calculated, based on thedosimetric CT and the decrease of parotid function based onthe scintigraphy before and after radiation. Dose–responsecurves were found with some evidence of hypersensitivityat low doses per fraction; Fig. 6 shows one example. Scin-tigraphy is now being performed at 6 months and 1 year tosee if this apparent hyperradiosensitivity at low doses is stillpresent.

Fig. 2. The ratio of the survival curve slope measured at very low doses (as) to the slope extrapolated from the high-doseresponse (ar) is plotted against high-dose radioresistance as indicated by the surviving fraction measured at 2 Gy. Thereis a trend for cell lines that are more high-dose radioresistant to demonstrate the greatest gain in radiosensitivity as thedose is reduced to less than 10 cGy, but this is not significant. Dark symbols, human cell data from Gray Laboratorystudies; shaded symbols, human cell data from Ref. 35; open symbols, V79 cells under ambient, oxic, and hypoxicconditions from Refs. 26, 109.


Are HRS/IRR and the adaptive response homologousphenomena?

If the adaptive response and HRS/IRR were conse-quences of the same underlying mechanisms, after a smallconditioning (or “priming”) dose there should be no HRS inresponse to a second challenge dose. In V79 hamster cells,small priming doses of X-rays induced resistance to a “chal-lenge” dose of radiation given a few hours later (40). Thisadaption was dose dependent, with priming doses of 20 cGybeing more effective in abolishing challenge-dose HRS thanhigher priming doses. The induction of increased radiore-sistance after single doses of X-rays in V79 cells wasinhibited by cycloheximide, which demonstrates the needfor protein synthesis in the development of IRR (40). Cy-cloheximide also inhibits the adaptive response in the samecell line, illustrating further similarities between the twoinduced-resistance phenomena.

In these experiments with V79 cells, 4–6 h were neededfor full induction of resistance, and a return to the hyper-sensitive state occurs later. However in the HT29, RT112,T98G, U87, and A7 human cell lines, this time course ismuch faster, and a full return to a hypersensitive state occursby 3–6 h following an initial small dose (41). This rapiddecay of induced radioresistance explains the effect of re-peated exposure to very low doses, as in the normal tissuestudies described above, in which each successive radiation

Fig. 3. Survival of asynchronous T98G human glioma cells irra-diated with 240 kVp X-rays, measured using the cell-sort protocol.Three doses of 0.4 Gy were given, separated by the intervalsshown. Data points are mean6 standard error. The broken lineindicates the predicted surviving fraction assuming HRS wouldoccur at every dose, calculated using the response shown in Fig. 1.The solid line indicates the cell survival measured after a singledose of 1.2 Gy, i.e., interval5 0. These data show that HRSrecovers, provided a more than 3-h interval is left between suc-cessive doses; the apparent increase in cell survival beyond 4 h isan artifact due to cell proliferation (41).

Fig. 4. Dose response for basal cell density in skin, following dailyfractions of 0.45 and 1.10 Gy. Mean values and 95% confidenceintervals, 40 patients. The difference in radiosensitivity (DMF51.8) is highly significant and demonstrates HRS/IRR in the humansituation (Data and figure courtesy of I. Turesson, M. Flogegård,K-A. Johansson, P. Svensson, S. Tucker, T. Wahlgren, and J.Nyman; personal communication).

Fig. 5. Dose response for basal cell density in skin, assessedfollowing daily fractions of 0.07, 0.20, 0.45, and 1.10 Gy to 20fractions in 4 weeks. The linear-quadratic (LQ) model wouldpredict a response above the dashed line; however, the data indi-cate human HRS/IRR (Data and figure courtesy of I. Turesson, M.Flogegård, K-A. Johansson, P. Svensson, S. Tucker, T. Wahlgren,and J. Nyman; personal communication).


treatment must produce the hypersensitive response to de-rive an increased effect of the overall schedule. The fasterreturn to HRS in the human cell lines tested so far isevidence that HRS/IRR maynot be related to the adaptiveresponse where these time courses are much longer. ForHT29 cells, Wouters and Skarsgard (42) have concludedthat IRR is indeed distinct from the adaptive response.

The fast return to HRS after an initial dose also impliesthat continuous, very protracted exposure at dose rates lessthan about 10 cGy per hour would result in a hypersensitivelethal response. Our preliminary tests in the T98G cell linehave produced evidence to support this hypothesis. To sum-marize, while HRS/IRR has some similarities to the adap-tive response, there are also contradictions. The issue ofwhether the two phenomena have a common mechanismremains to be resolved.

Mechanisms underlying induced radioresistanceEvidence for DNA repair.At low single doses (,10

cGy), where HRS dominates, cells can be in excess of 20times more sensitive than at doses greater than 1 Gy (whichtrigger IRR), where measurements of radiation-induced cellkilling are usually made. For the cell lines tested so far, theamount of this change in radiosensitivity over the first grayis broadly correlated with intrinsic radioresistance to higherdoses (see Fig. 2). Moreover, hypersensitivity to low singledoses (compared with doses.1 Gy) is not seen with somevery “sensitive” cell lines, for example SW48 and HX142.If this change reflects induced repair mechanisms as pro-posed above, thenintrinsic radiosensitivity (to doses of 1

Gy and above) could be linked to the induced repair abilityof cells. There is accumulating evidence that induced radio-resistance, inferred from Figs. 1 and 2, and adaptive re-sponses to small conditioning doses of radiation, may bothbe due to increased repair capacity or repair fidelity but oververy different time scales as noted in the previous section.For example, the adaptive response in lymphocytes isblocked by 3-aminobenzamide (43), suggesting that repairpathways involving poly(ADP-ribose) polymerase (PARP)may be involved. 3-aminobenzamide also blocks the devel-opment of IRR in single-dose survival studies in V79 cells(44), so that radiation response continues to follow thelow-dose hypersensitive pattern out to higher doses. Directevidence that repair can be induced by DNA damage comesfrom studies measuring the reactivation of radiation-dam-aged virus (e.g., adenovirus 5) by the DNA repair mecha-nisms of a subsequently infected host mammalian cell. Thehost cells are usually not irradiated, so the end point is afunctional test only of host cell repair and repair fidelity(45). However, if the host cells are pretreated with smalldoses of UV (46) or gamma rays (47), increased ability toreactivate damaged virus can be measured compared withuntreated host cells. Recently, Francis and Rainbow (48)reported that UV treatment results in an induced repair ofUV-damaged DNA in the transcribed strand of active genesthrough the enhancement of transcription-coupled repair.

In further support for the involvement of repair, the rad52mutant ofSaccharomyces cerevisiae, which is deficient inrecombinational repair, does not show an adaptive response(49). In the context of HRS/IRR, Skovet al. (50) measuredthe low-dose response of 3 hamster cell lines defective inDNA repair, compared with their parental lines. The V79-derived double-strand break repair–deficient line (XR-V15B) showed a purely exponential survival response withno increased radioresistance (IRR) in the zero to half-grayrange compared with the V79B wild type. XR-V15B cellline is defective in the Ku80 subunit of the DNA-PK com-plex that repairs DNA dsbs (double-strand breaks) (51). TheUV-20 line, which is defective in nucleotide excision repair,also appeared to respond exponentially with no evidence ofinduced radioresistance. However, the base excision repair–deficient line EM9 did show some low-dose hypersensitiv-ity and induced radioresistance, suggesting that HRS/IRRmay be linked to double-strand break and nucleotide exci-sion repair mechanisms. Support for adirect link betweeninduced radiation resistance and DNA dsb repair comesfrom Wojewodzkaet al. (52) in studies with human lym-phocytes and Ikushimaet al. (53) in studies with V79 cells.Both have indicated that small conditioning doses of X-raysor hydrogen peroxide lead to faster and more completerejoining of DNA double-strand breaks following exposureto a higher challenge dose of radiation several hours later.Recently, Robsonet al. (54) described a novel gene,DIR1,that is transiently repressed after exposure to low radiationdoses. They postulate that theDIR1 gene has a role ininduced radioresistance through a mechanism that increasesthe rate of DNA repair. Further compelling evidence linking

Fig. 6. Dose response of one parotid of one patient. They axisshows the loss of function of the parotid after irradiation, com-puted as the difference between the salivary excretion fractionbefore and one month after irradiation (DSEF). Thex axis showsthe radiation dose; each slice of 5 mm of the parotid has beenirradiated at various total doses in a fixed number of 25 fractions;therefore 10 Gy corresponds to a dose per fraction of 0.4 Gy. Thefit to the data is linear, except at the 2 lowest doses, whichdemonstrate increased radiosensitivity. Data points are mean6 SD(Data and figure courtesy of P. Lambin, A. Maes, F. Van Acker, P.Flamen, C. Weltens, P. Dupont, L. Mortelmans, and W. Van DenBogaert; personal communication).


DNA dsb repair to HRS/IRR comes recently from theLaboratory of Bourhis at the Insitut Gustave Roussy,France. In a functional assay measuring the activity of theDNA-PK complex, a marked decrease in DNA-PK activitywas found in six cell lines exhibiting HRS (55). In contrast,an increase in DNA-PK activity was observed for the fourcell lines that did not exhibit HRS.

Evidence against apoptosis and cell cycle delay.We havetested two other hypotheses to explain HRS/IRR. First, HRSmight reflect apoptosis (hence high sensitivity) at low dosesas a means of removing genomically unstable cells from thepopulation; as the dose increased, apoptosis would bedownregulated to allow cell population survival as a prior-ity. In a range of cell lines demonstrating HRS/IRR, wehave found no evidence to support this hypothesis (56).Moreover, in 10 cell lines of differing HRS/IRR response,Vaganay-Jue´ry et al. (55) have shown that the level ofDNA-PK is unchanged in cells irradiated with either 0.2 or0.5 Gy. These data therefore also argue against apoptosis asthe primary underlying mechanism of HRS/IRR, as thecleavage of the DNA-PK complex is known to be a markerof apoptosis (57). Second, cell cycle delay increases withdose in many cell lines; this would imply a longer availabletime for repair with larger doses and hence more sensitivityto lower doses. However, in a range of cell lines, we havefound no consistent correlation between HRS/IRR and a cellcycle delay vs. dose relationship. This parallels similarconclusions from the work with nonmammalian systemsreviewed above.

Different DNA lesions as triggering events.Priming orconditioning treatment with small doses of 60Cog raysprotects human fibroblasts against subsequent doses of X-rays (58). We observed similar radioprotection after prim-ing treatment with X-rays in V79 cells (40). However,mammalian cell survival studies withsingledoses (32, 59)indicate that high-LET radiation is less able to induce ra-dioresistance than X-rays at similar levels of cell killing. Inyeast, small neutron conditioning doses are less efficientthan X-rays at producing adaptive protection against subse-quent large X-ray exposures (8). Yet, Marples and Skov(60) reported that 20-cGy doses of high-LET (d (4)-Be)neutronscouldproduce an adaptive resistance (dual dosing)to subsequent 1-Gy challenge doses of X-rays in V79 cellsand that this adaption was at least as large as that induced by20-cGy doses of X-rays. This apparent contradiction withthe lack of HRS/IRR noted in previous work with singleneutron doses in this cell line can be resolved by consider-ing that the overall response of these cells to neutrons isextremely steep, insignificantly different from a linear rela-tionship between log survival and dose. In single-dose ex-posures, therefore, cells are killed by complex lesions onwhich repair is largely ineffective, and hence increasedresistance cannot be detected after high-LET irradiation byan assay that scores a clonogenic survival end point. How-ever, in those cells that survived a small dose of neutrons,the mechanism is activated, and those cells are effectively

adapted to subsequent doses of lower-LET radiation, whererepair is important in determining response.

Small doses of hydrogen peroxide, as well as X-rays, caninduce protection against subsequent doses of ionizing ra-diation, supporting the possibility of oxidative species orsingle-strand breaks (ssb) as inducers (40, 61). In addition,priming treatment with cisplatin can induce protectionagainst subsequent irradiation, despite the inability of cis-platin to directly cause DNA strand breaks. However, therepair of cisplatin-induced DNA lesions can result in theproduction of DNA strand breaks. In contrast, 0.25 cGypriming treatments ofg-rays, but not 5 cGy,could induce aprotective effect to subsequent low-dose cisplatin in a hu-man squamous carcinoma cell line (62). This cell line ex-hibits low-dose hypersensitivity and increased resistance tohigher concentrations ofsingledoses of cisplatin, a patternof survival that corresponds to HRS/IRR (62). In summary,after single exposures, damage by free radicals and low-LET radiation might be particularly good at inducing radio-protection compared with the “clustered” damage producedby high-LET radiation (60), but in dual-dosing experimentsboth are equally effective.

Evidence for the involvement of specific cellular repairpathways.The systems that monitor and signal DNA dam-age are highly regulated to conserve the integrity of DNAduring replication. They ensure that the DNA strands are notbroken and that the necessary enzymes are present forpassage from one cell cycle phase to another (63). Thesesystems are tightly interrelated with those that detect un-scheduled DNA breaks (64). Single-stranded DNA damageis normally rapidly and efficiently repaired by base excisionrepair (65) and has therefore been considered noncritical inradiation-induced cell inactivation. A second excision repairpathway, nucleotide excision repair, has also been thoughtnot to have a major role in the repair of radiation-inducedDNA damage (66). In contrast, a radiation-induced break onboth DNA strands at a single site (DNA dsb) is regarded asdifficult to repair and is therefore considered the primelesion in radiation-induced inactivation. Failure to repair (ormisrepair) radiation-induced DNA dsbs is a potential causeof cell death. The established significance of dsbs in radia-tion-induced cell inactivation gives credence to the hypoth-esis presented earlier that the repair of double-strand breaksis important in the phenomenon of induced resistance, andhence absence of such repair would be characteristic ofHRS.

A number of mechanisms exist for repair of DNA dsbs. Inlower organisms homologous recombination dominates. Ineukaryotes, DNA dsbs can be repaired by at least threepathways: homologous recombination repair, DNA-PK de-pendent nonhomologous end joining (NHEJ) and direct-repeat end joining (reviewed in 67, 68). In mammalian cellsmost DNA dsbs are repaired by NHEJ. The major genesinvolved in NHEJ encode the three components of theDNA-dependent protein kinase (DNA-PK) complex and theXRCC4 protein. DNA-PK encompasses a heterodimeric


DNA-targeting component of Ku (70 and 80 kD subunits)and the catalytic component DNA-PKcs (69).

Our V79 cell data implicate DNA-PK in the developmentof induced resistance (70), as does the work of Vaganay-Juery et al. (55) discussed above. In addition, the extent ofHRS/IRR is also dependent on cell cycle phase (34) as isreported the regulation of DNA-PK activity, but unlike thelevels of Ku70 and Ku80 that remain unchanged through thecell cycle (71, 72). Furthermore, The XR-V15B cell line isdefective in the Ku80 subunit of the DNA-PK complex andfails to exhibit an IRR response (See earlier). DNA-PK hasbeen proposed as a damage sensor (initially via Ku bindingto DNA followed by DNA-PKcs recruitment) that initiatesa phosphorylation cascade as the “alarm” signal leading tothe binding of XRCC4 protein and ligase IV (73). Theimportance of the NHEJ process in the repair of radiation-induced DNA damage is demonstrated by the extreme ra-diosensitivity of cell lines deficient in this repair pathway.The mutant cell line xrs5 lacks Ku80 (74). Yet, the extremeradiosensitivity of xrs5 can be partially overcome by trans-fection with the human Ku80 gene (75). The radiosensitivehuman malignant glioma cell line MO59J fails to expressDNA-PKcs protein and is therefore defective in double-strand DNA break repair (76). MO59J cells are similarlyradiosensitive to xrs5 and;10-fold more sensitive than acorresponding DNA-PK competent cell line MO59K ob-tained from the same glioma biopsy specimen as MO59J(77). In contrast, the underlying basis for the extreme radi-osensitivity of AT cells is thought to involvemisrepairofDNA dsb rather than a reduced ability to rejoin dsb (74, 78).

The importance of the DNA-PK pathway in the repair ofradiation-induced damage is reported to be reduced withincreasing LET, leading to the suggestion that the DNA-PKpathway may be less critical in repairing the more complexlesions induced by high-LET particles (79). Interestingly,single-dose survival experiments with V79 cells irradiatedwith p-mesons from the TRIUMF facility in Vancouverindicated a correspondence between LET and IRR (60); thisis consistent with the hypothesis that DNA-PK is critical forIRR sincesingle doses of high-LET radiation are worseinducers of IRR than small doses of low-LET radiation (60,80). One hypothesis is that DNA dsb and the NHEJ repairpathway play a prime role in induced resistance, and thedisparate HRS/IRR responses reflect differences in NHEJ(or its constituent components) between cell lines.

Does existence of IRR reflect radiation-induced changesin DNA organization?

Chromatin structure and chromosomal organization arenow accepted as key controlling mechanisms in DNA rep-lication, transcription, and associated processes such as dif-ferentiation and cell cycle progression. It is also becomingclear that induction and processing of DNA damage issimilarly influenced by higher-order DNA structures. Ex-amples include the major radioprotective effect of chroma-tin-associated proteins such as histones (81–84), the effectof chromatin organization in the generation of clustered

DNA damage (85–87), the correlation between both cellcycle and anisotonic treatment–related changes in chroma-tin structure and changes in radiation sensitivity (83, 88–92), increased repair in transcribed vs. nontranscribed re-gions (93–96), and variations in chromosomal aberrationformation within euchromatic and heterochromatic chromo-somal regions (97–99). Radiation-sensitive cell lines andphenotypes often have associated alterations in chromatinstructure. Such changes are observed in both nuclear mor-phology and through biophysical and biochemical assays(100–104). It is possible, therefore, that the increasing num-ber of DNA strand breaks induced as the dose increasesover the range 0–1 Gy generates DNA conformationalchanges that trigger increased radioresistance. Such a pro-cess could lead to the induction of DNA repair processes perse or allow the greater access of repair enzymes to the sitesof damaged DNA, permitting repair. Whichever mechanismis correct, increased cell survival results.

A final note: Implications of HRS/IRR for cancer riskThe existence of HRS in the cell survival response im-

plies that cancer risk from small acute exposures to ionizingradiation might be lower than current estimates, if the resultof HRS was to protect the cell population from mutationaland initiating events in some cells by eliminating those cellsfrom the population. Thus HRS would be a protectiveresponse at the organismal level. For this to happen wouldnecessitate that hypersensitivity tomutationfollowing low-dose exposure either did not exist or was present at adifferential between low- and high-dose sensitivity that wasless than for the cell killing HRS, shown in Fig. 2.

Several papers review the dependence of mutation fre-quency on doserate (105–107) and the phenomenon ofmutagenic adaptive response whereby cells pre-exposed tovery small doses of radiation might exhibit less pronouncedsensitivity to mutation following subsequent high-dose ex-posure compared with cells not pre-exposed (108). An in-verse dose-rate effect, with continuous exposures typicallyless than 10 cGy per hour inducing higher mutation fre-quency than similar doses given at higher dose rates, hasbeen reported in both rodent and human cell lines. However,this phenomenon is controversial. It has not been seenuniversally even in studies using the same cell lines andendpoints. Most studies have used either the hprt or tk loci,and it remains to be seen whether any inverse dose-rateeffects can be seen in critical loci associated with oncogenesor tumor suppressor genes. Cell cycle progression cannot beignored in studies with protracted continuous exposures,and where inverse dose-rate effects for mutation have beenreported, the effects of cell cycle progression have not beendefinitively ruled out.

In contrast, evidence for a mutational adaptive response ismore compelling, since cell cycle and progression effectscan be largely ruled out when radiation is given as acute,high-dose-rate exposures. The experimental protocols aresimilar to those used in defining the cell killing adaptiveresponse. As with the inverse dose-rate studies, hprt and tk


endpoints have dominated the work to date. However, whilethere are parallels between HRS/IRR and the adaptive re-sponse in cell killing, there are also marked dissimilarities, andit would not be sensible, based on the existence of mutationaladaption, to simply assume the existence of mutational HRS/IRR in the response to single, acute doses. At present there isno direct evidence supporting the existence of single-dosemutational HRS/IRR in mammalian cells, which contrasts withthe situation for the cell lethality endpoints where the existenceof HRS/IRR is incontrovertible.

In the region of HRS, cell survival falls rapidly withincreasing dose (D) over the first;10 cGy, at a rate

corresponding to a value ofa equal to.1 Gy21 (i.e., as)in the relationshipSurviving Fraction5 eaD. Because ofthis exquisite sensitivity to lethality, mutation (based oncurrent estimates in the absence of mutational HRS)would be more than compensated for by cell kill at dosesless than 10 cGy, which would lead to the overall trans-formational risk for the cell population being similar oreven lower than the value for unirradiated cells. There-fore, based on our present knowledge of HRS, currentestimates of risk from exposure to low, acute radiationdoses may represent the upper boundary of the truecancer risk in some cases.

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