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 (
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.) Joiner et 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 responseoff the shoulder) induced an almost doubling in bothincremental (increased D0) 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 Santier et 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 Beam et 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 (1925) 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 cells afterplating (22). Using the latter technique, Marples and Joiner(26) and Marples et 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 (2835).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 slopes as 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-
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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: SF2 5 0.63)and SiHa cervix (SF2 5 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/IRR in vivoThere is evidence that hypersensitivity in 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 a rapid decay of adaptive resistance in thesemammalian systems over the period between fractions. Inthese studies, this interval was 78 h. Recent work on threehuman glioma cell lines in 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 parameter as/ar shown in Fig. 2, butalso on the rate at which the transition from ar to as occurswith decreasing dose. The