[CANCER RESEARCH 54, 3939â€”3946, July 15, 19941 Lack of a Correlation between Radiosensitivity and DNA Double-Strand Induction or Rejoining in Six Human Tumor Cell Lines' Break Peggy L Olive,2Judit P. BanÃ¤th,and H. Susan MacPhail British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada V5Z 1L3 ABSTRACT to remove proteins, when subjected to an electric field. This method is similar to conventional pulsed-field gel electrophoresis methods The neutral filter elution method and the neutral comet assay have been which measure the amount of DNA remaining in the plug after used to analyze radiation-Induced DNA damage and repair in 6 human electrophoresis, but this method has the important advantage of mea tumor cell lines: HT-144 melanoma; DU-145 prostate carcinoma; U-87 suring DNA damage in individual cells. Gel electrophoresis methods glioma; WiDr and HT-29 colon adenocarcinomas; and SIHa cervical are as sensitive as and less technically demanding than neutral-filter carcinoma. In spite of large differences in intrinsic radiosensitivity mess elution, although results using both methods are complicated by ured using a clonogenic assay, double-strand break induction, rejoining retardation,â€•the much slower migration and elution of DNA rate, and amount of residual DNA damage 4 h after Irradintion were â€œS-phase from S-phase cells (14, 32â€”34),apparently as a result of the presence similar among these cell lines when measured using the neutral comet assay. Differences in initial numbers of double-strand breaks were ob of replication forks which retard DNA migration or elution (35). The served using the neutral filter citation method; however, there was no ability of the comet assay to measure not only DNA damage but also correlation with radiosensitivity, nor did the rejoining rate or amount of DNA content of the same cell (19, 35) provides a simple way of residual DNA damage measured using filter elutlon correlate with mdi identifying cells in various phases of the cycle, thereby avoiding this ation sensitivity. We conclude that neither double-strand break assay is problem. While none of the studies summarized in Table 1 report able to reliably rank cells according to clonogenic survival following differences in DSB induction using neutral gel electrophoresis, it irradiatIon. would appear that some radiosensitive cell types can be identified on the basis of a slower DSB rejoining rate. INTRODUCTION Because of the inconsistencies apparent in Table 1, we have exam med DNA damage using both the neutral filter elution and the neutral Initial numbers of DNA DSBs3 induced by ionizing radiation comet assays using 6 established human tumor cell lines. DNA and/or rates of rejoining of DSBs have been shown to correlate in some studies with tumor cell radiosensitivity (Table 1, Refs. 1â€”28). induction, rejoining, and residual damage were measured and com pared with the accepted standard of cell radiosensitivity and clono These results suggest that it may be possible to predict the intrinsic genic potential. radiosensitivity of cells from individual human tumors using one or more assays that detect DSBs. Knowledge of intrinsic radiosensitivity of cells from tumor biopsies, if obtained in a timely fashion, could be MATERIALS AND METhODS used to optimize radiotherapy protocols with the goal of improving Cell CultUresand IrradiationProcedure.All tumorcell lines were individual tumor response to treatment. obtained from American Type Culture Collection, Rockville, MD. Cells were While (unrepaired) DSBs are believed to be critical to radiation adapted and maintained for 4â€”6months prior to experimentation as exponen induced cell death, the ability to use either numbers of DSBs induced tially growing monolayers in minimal essential medium with Earle's salts by a given dose of radiation or rate/extent of rejoining to predict (GIBCO Canada, Burlington, Ontario, Canada) supplemented with 10% fetal intrinsic radiosensitivity remains controversial. For the neutral filter bovine serum and antibiotics (Sigma Chemical Co., St. Louis, MO). For cell elution method, radiation sensitivity has been shown to correlate with survival experiments, exponentially growing cells were removed from dishes initial DNA damage, with DSB rejoining rate, with both, or with using 0.1% trypsin from GIBCO and resuspended at a density of 5 X [email protected] neither (Table 1). There appears to be a reasonable consensus, how cells/mI. Cells were irradiated on ice and then plated at a density appropriate to give about 300 surviving cells/9-cm tissue culture dish. Lethally irradiated ever, that neutral ifiter elution detects, in addition to DSBs, other properties of DNA organization intrinsic to the cell. Differences in DNA â€œpackagingâ€• appear to influence the amount of DNA able to elute through the filter in a given period of time, and these same differences could be relevant to the efficiency of DNA cells ofthe same celltype (7 X [email protected]'/9cm-dish)were as feeders, and colonies were stained approximately Survival data were fitted to the linear-quadratic equation repair ln S= (2, 29â€”31). To supplement the neutral filter elution method, we have developed the neutral comet assay to detect DSBs (19). The comet assay mea sures the mobility of DNA from cells, embedded in agarose and lysed added to each dish to serve 2 weeks later and counted. [email protected]@2 where S is the surviving fraction and D is the radiation dose. Irradiation was performed with cells at 4Â°Cusing an X-ray machine oper ated at 250 kV at a dose rate of 3.2 Gy/min. Rejoining studies were performed by irradiating cells on ice and then diluting cells in medium at 37Â°C for the specified repair time. For repair times longer than 10 mm, cultures were Received 3/25/94; accepted 5/17/94. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the National Cancer Institute of whom requests for reprints should be addressed, at the Medical Biophysics Department, British Columbia Cancer Research Centre, 601 W. 10th Ave., Vancouver, British Columbia, Canada, V5Z 1L3. 3 The abbreviations used are: DSB, double-strand break; SDS, sodium dodecyl sulfate; a and f3, constants of the linear and quadratic terms in the linear-quadratic equation S = describing survival (5) as a function of radiation dose (D). time. Neutral Comet Assay. Single cells were centrifuged and resuspended in ice-cold phosphate-buffered saline at a concentration of 3 X [email protected]/mi. Following irradiation or drug treatment, 0.5 ml of cell suspension was placed Canada with funds provided by the Canadian Cancer Society. 2 To returned to tissue culture dishes and trypsinized for 5 mm at the stated repair in a 5-mi disposable (Sigma tube, and 1.5 ml of 1% low-gelling type VII dissolved in distilled water temperature agarose and held at 40Â°C) were added to the tube. The contents were mixed and 1.5 ml were pipetted quickly onto a fully frosted microscope slide and allowed to gel for about 30 s on a cold surface. Slides were carefully submersed in lysing solution consisting of 30 mMEDTA and 0.5% SDS (pH 8.3), and the temperature was raised to 50Â°C for 4 h by placing containers with slides into an oven. Slides were then 3939 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research. RADIOSENSITIVITY AND DNA DOUBLE-STRAND BREAKS electrophoresisCorrelation Table 1 Comparisons between ceII lines varying in radiosensitivity using nesaral filter elution and neutrci gel between radiosensitivity and double-strand breakCell linesRef.Induction RejoiningNeutral elutionYes filter No (28)a not Yes L5178Y-R and -S mouse lymphoblast Wlodek and Hittelman (6) Yes Yes Yes Yes Yes 6 human squamous cell carcinoma 5 human cervix carcinoma cell lines 2 human neuroblastoma cell lines (HX142, 138) L5178Y-S and -R Schwartz et al. (7) Kellend et aL (8) McMillan ci aL (9) Evans et aL (10) CHO, xrs-1, xrs-5 5 mammalian cell lines Dahm-Daphi et aL (11) Eguchi-Kasai et aL (12) Yes Yes Yes Yes No No No Yes No No Yes ND No No No No ND ND No No2 6 human B-lymphoblast cell lines Evans et aL (13) Mouse mammary 66, 67 cell lines 9 human tumor cell lines Sweigert et al. (14) McMillan et aL (15) CHO and xrs-5 Iliakiset aL (16) 3 CHO cell lines (AA8, EM9, NM2) (18)Neutral 3 hamster cell lines (V79, irs-i, irs-2)Oshita Jones et aL et aL (19) No No V79, CHO, L5178Y-R 2 squamous cell carcinomas No No H-ras/V-myc No No V79, irs-i, irs-2, irs-3 No No No No Yes Yes V79 and Caski human squamous cell carcinoma L5178Y-S and -R No No No No Yes Yes Yes YesHamster CHO, Chinese hamster fliakis et al. (21) Cheong et aL (22) Flentje et aL (23) Olive et aL (19) Giaccia et aL (24) Kysela et aL (25) Waiters et al. (26) Iliakis et aL (27) 5 human squamous carcinoma cell lines V79 cells and a sensitive subline Mouse L-cells and 2 sublines CHO and xrs-5 Human colorectal cells: HT29, SW48Olive Lambin et aL ovary. overnight followed by electrophoresis mM boric acid at 0.55 Smeets et aL (20) transformed rat embryo cells washed free of detergent by transferring them to a container with a large volume of 90 mM Tris-2 mM EDTA-90 mM boric acid buffer (pH 8.5) buffer Van Ankeren et aL (17) gel No done; Radfordand Hodgson(2) Wiodek and Olive (3) Schwartz et aL (4) Green et aL (5) NDÂ° ND ND ND electrophoresisNo @ et aL (1) human lung cancer cell lines Hamster V79, mouse L-cells V79, CHO, mouse L5178Y-R, L5178Y-S 8 human squamous cell carcinoma cell lines 2 CHO cell lines (EM9, AA8) Yes Yes Yes Yes V/cm in 90 [email protected], 2 [email protected], and 90 for 25 mm. Slides were rinsed and (pH 9.6). Elution was performed for 10 h with 2 mMEDTA, 20 simsTris, and 0.2% SDS using a flow rate of 0.04 mi/mm. The radioactivity of the samples was measured in an LKB 1219 liquid scintillation counter after adding 6 ml of Hydrofluor scintillation fluid (National Diagnostics, Manville, NJ). Results are stained for 1 h in 2.5 @.tg/ml propidium iodide. To prevent detachment of the presented as absolute radioactivity remaining on the filter or as radioactivity agarose after lysis, slides were often placed for about 2 mm in the 50Â°C relative to an internal standard. oven and the edges were allowed to dry. Individual cells or â€œcometsâ€• were viewed using a Zeiss epifluorescence microscope and image analysis system (36). Double-strand damage was quantified as an increase in tail RESULTS moment, the product of the amount of DNA (fluorescence) in the tail and the distance between the means of the head and tail fluorescence distribu Cell Survival. Radiationsurvival curves for the six humantumor lions. For each comet, tail moment and total DNA content (total fluores cell lines are shown in Fig. 1; plating efficiency and survival param cence per comet in arbitrary units) were recorded. In some experiments, the eters, a, ,3, and the surviving fraction after 2 Gy irradiation are given response of [email protected] 02 cells only was determined on the basis of DNA in Table 2. These cell lines encompass a wide range of radiosensitiv fluorescence by using data from comets with relative DNA content values ities with HT29 and its derivative, WiDr, representing the most greater than 20 and less than 10. radioresistant and HT-144 representing the most radiosensitive cell Nondenaturing Filter Elutlon. Nondenaturing filter elution was a modi fication of the method radiolabeled of Bradley with 0.74 kBq/ml and Kohn (37). [â€˜4Cjthymidine DNA (specific of test cells activity, lines. was NeutralCometAssay.Resultsusingthecometassayshowfairly 2.07 GBq/ mmol; Amersham) for two cell doublings (24â€”48h). Following radiolabeling, fresh medium was added to the cultures for 4 h. Cells were irradiated and then mixed with irradiated â€œinternal standardâ€• cells, Chinese hamster V79 cells that had been radiolabeled for 24 h with 3.7 kBq/ml [3H]thymidine (specific conclusively that there is no significant difference in the initial num ber of DSBs detected for these cell lines (Fig. 2, iâ€”n).Even when analysis was confined to non-S-phase cells, there was no relation Reproducibility between filters was excellent, so that results could be pre sented as absolute counts per fraction as well as elution relative to the internal between the initial numbers of breaks and radiosensitivity (Fig. 2/i). DNA content varied considerably between cell lines but apparently had no influence on amount of damage induced or detected. Bivariate plots of DNA content versus tail moment for DU-145 and HT-144 cells show the dramatic influence of active replication forks on DNA standard. migration activity, 925 GBq/mmol; Amersham). Preliminary studies indicated a coelu tion problem between the test and internal standard cells which could be minimized by limiting the total cell number to less than 5 X [email protected]/filter. The cell mixture (5 x [email protected] total) was layered onto 25 mm (2 @m pore size) Nucleopore polycarbonate filters, washed twice with ice-cold phosphate buffered saline, and lysed with 5 ml of 2 mMEDTA, 1% SDS, and 20 mMTris from cells with S-phase DNA content(Fig. 3). However, for a given dose of radiation, there was no apparent difference in the pattern or amount of damage produced in these two cell lines or in any of the other tumor cell lines examined. 3940 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research. RADIOSENSITWITY AND DNA DOUBLE-STRAND @ I BREAKS â€˜ , I â€˜ â€˜ â€˜ , I 1 @Â° 0 .â€˜@ [email protected] C) CD UD) @ Fig. 1. Clonogenicity of six human tumor cell -2 â€¢ lines following exposure to X-rays. Symbols, means Â±SD (bars) for 4â€”6independent experi- 1 0 â€¢U87 . ments. > C/) io-3 V HT29 V WiDr 0 Du145 U HT144 [email protected] SiHa [email protected] 0 5 10 Radiation Rejoining of DSBs was also independent of cell type, as shown in Fig. 2. Less than 10% of the initial number of breaks remained 4 h after 50 Gy, and this value was not significantly different for the six tumor cell lines. Of the rejoinable breaks, approximately 15% were therefore accumulate more rapidly in G2 following irradiation. The dotted lines for G1 cells only in Fig. 2, aâ€”findicate the effect is small and may become important only when assessing residual DNA dam age 4 h after treatment. still present 1 h following 50 Gy. The time to rejoin one-half of the breaks averaged about 14 mm (Table 2) and did not differ significantly among the cell lines examined. Bivariate plots indicated that Dose (Gy) Results shown in Fig. 2g and the values for residual damage in Table 2 therefore represent the response of the G1 and G2 cells only. Neutral Filter Elution. Two methods were used for quantifying DSB rejoining rates were independentof cell cycle position for the the amountof DNA eluted. The absoluteamountof DNA eluted from tumor cell lines (Fig. 4). However, radiation-induced increases in the duration of S phase (inhibition of replicon initiation and chain dongation) can lead to an apparent increase in the rate of rejoining. This is more obvious in the data for V79 cells which cycle faster and the filter 10 h after irradiation was not significantly different among the human tumor cell lines tested, with the possible exception of the SiHa cells which appeared to elute more slowly (Fig. 5a). However, averaging results between experiments tends to mask small differ Table 2 Radiation survival cutee parameters, DSB induction rates, rejoiningkineticsDamage and DSB inductionRepair half-timeResidualdamageCell @eCome1NFE1CometDU145 lineaSF21'PECCometâ€• prostatic carcinoma SiHacervicalcarcinomaâ€”0.06 (0.04)1ff144 (0.06) (0.013) â€”0.31 â€”0.025 (0.14)â€”0.055 (0.016)0.61 (0.08) 0.41 (0.07)0.50 (0.11) 0.59 (0.06)0.15 (0.13â€”0.16) 0.15 (0.12â€”0.17) (0.15)â€”0.010 (0.020)0.09 (0.02)0.38 (0.07)0.16 (0.13â€”0.16) (0.04)â€”0.043(0.008)0.76 (0.08)0.70 (0.06)0.15 (0.12â€”0.16) (0.014)0.84 (0.04)0.61 (0.09)0.14 (0.12â€”0.15) (2.8) 13.0 (3.1)9.2 (4.1) 12.6 (3.6)0.06 (0.03) 0.04 (0.05)0.07 (0.12)13.2 1.41 (0.08)14.5 1.35 (0.06)13.1 1.22 (0.05) (4.4)6.9 (0.4)0.05 (0.02)0.03 (1.6)7.0 (2.5)0.05 (0.04)0.07 (2.6)6.9 (0.6)0.08 (0.02)0.08 (4.4) (0.5) 1.0015.7 13.36.7 (0.06) 0.030.03 (0.05) 0.14 1.23 melanomaâ€”1.20 (0.01)WiDr colon carcinomaâ€”0.04 (0.03)HT29 colon carcinomaâ€”0.04 (0.02)0.045 (0.05)U87 glioma (0.03) (0.11) (0.008) 0.07(Chinese â€”0.16â€”0.023 â€”0.0180.43 0.680.17 V79lungfibroblastâ€”0.37 hamster)(0.08)(0.009)(0.06)(0.06)(0.15â€”0.18)(2.5)(1.8)(0.02)(0.03) @ 1.12 (0.09) 1.09 (0.09)9.8 (0.07) (0.11â€”0.15) 0.16 0.790.13 8.00.10 (0.02) a and valuesdetermined fromthelinear-quadratic equation describing clonogemc survival asafunction ofradiation dose.Mean(SD)for4â€”6 survival curves. [email protected] @sthe fraction of cells surviving exposure to 2 Gy. Mean (SD) for 6â€”10experiments. CPE, plating efficiency mean (SD) for8â€”10 experiments. d Slope a Relative of dose-response amount of DNA curve eluted (95% from confidence filters limits) by from 10 h after Fig. exposure 2k of cells to 75 Gy. Mean (SD) for 12 filters. Elution is relative to the internal standard V79 cells which also received75 Gy.NFE,neutralfilterelution. â€œTime (mm) required to rejoin one-half of the initial number of double-strand breaks after exposure to 50 Gy for the comet assay or 25 Gy for NFE. Values represent the mean (SD) of at least 3 independent experiments for rejoining rates calculated for the first 15 min of repair. Rejoining data are shown in Figs. 2 and 5. g Residual damage is the fraction of initial damage remaining in cells 4 h after exposure to 50 Gy for the comet assay or 4 h after 25 Gy for NFE. The mean (SD) of 3 independent experiments is shown. For the comet data, S-phase cells were excluded from analysis. 3941 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research. RADIOSENSITIVITYAND DNA DOUBLE-STRAND BREAKS Q) E 0 F-0 0 2 40 2 40 2 40 2 40 2 40 2 4 Time After Irradiation (h) C) C h. 15 C C a, E E a, 10 0 a, C) a, a, Iâ€” 5 a, 0 0 1 2 3 4 . I . , 0 Time After Irradiation (h) @ . . I . 25 . . . I . . . . 50 I. 75 Dose (Gy) 10 Iâ€” 0 25 50 75 0 25 50 75 0 25 50 75 Radiation 0 25 50 75 0 25 50 75 0 25 50 75 Dose (Gy) Fig. 2. DSB induction and rejoining after 50 Gy for 6 human tumor cell lines measured using the comet assay. Average tail moment, proportional to the number of DSBs per cell, was calculated from 100â€”200comets/point using a fluorescence image processing system. aâ€”fresponse of asynchronous cells [symbols, mean Â±SD (bars) for 3 experiments, response of G1 and G2 cells onlyj. g compares the normalized rejoining response of G, and G2 cells from the different cell lines. h, DSB induction for G1 and 02 cells only. iâ€”n, means Â±SD (bars) for asynchronous cells from 3 independent experiments. ences in elution. Therefore, to improve sensitivity, each cell line was first compared to an internal standard of Chinese hamster V79 cells irradiated with the same dose as the test cells. To verify accurate detection of the 3H- and â€˜4C-labeledDNA, cross-over experiments were performed where each cell line was labeled with [â€˜4C]thy midine and [3H]thymidine. Samples were then mixed (3H-labeled test cells mixed with â€˜4C-labeled internal standard cells and vice versa), eluted, and counted. As shown in Fig. 6, WiDr cells eluted significantly more rapidly than the V79 cells and slightly more rapidly than the U87 cells, but SiHa cells showed a similar rate of elution as V79 cells. Based on similar analyses, Table 1 compares the ratio of amount of DNA eluted in 10 h for the test cells relative to the V79 cells. While the internal standard method increases sensitivity, there was no apparent correlation between initial dam age and surviving fraction for these 6 cell lines (Table 1). Rejoining rates measured using neutral filter elution were com parable among the cell lines with the exception of the SiHa cells which showed less rapid rejoining and more residual damage than the other cell lines (Fig. 5b). On average, about 7% of the initial damage remained 4 h after 25 Gy and the fraction of unrejoined breaks was not correlated with radiosensitivity (Table 2). DISCUSSION Several properties of a tumor contribute to its response to radio therapy. One of the most important is the sensitivity of the tumor cell, removed from its environment, to killing by ionizing radiation. The conventional colony formation assay used to estimate intrinsic radi osensitivity of human tumor cell lines is limited by several factors including, the number of weeks required to obtain an estimate of tumor cell radiosensitivity and the relatively small proportion of cells that will survive and grow in vitro. These and other limitations of clonogenic assays have stimulated an interest in developing new methods which would provide a measure of intrinsic radiosensitivity in a much shorter period of time so that results could be considered in the planning of treatment (38). Of course, any assay suggested to fill this role must provide results which correlate well with relevant clonogenic end points such as the surviving fraction after 2 Gy irradiation. Our earlier work led us to believe that the neutral filter elution assay, but not the comet assay, might provide an indication of intrinsic differences in radiosensitivity. It is clear from a number of studies that results obtained using neutral filter elution can be influenced by many 3942 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research. RADIOSENSITIVITY @ 0 Gy BREAKS 25 Gy 50 Gy 75 Gy HT 30 C 20 E 10 0 10 Gy AND DNA DOUBLE-STRAND 0 30 â€˜â€˜â€˜â€˜I''''I''' -â€˜1. I I.â€¢@,@[email protected]:%@ F.â€˜â€˜â€˜I''.,I_.,',I,,,J,',G.I.â€¢ H.â€˜â€˜â€˜â€˜I''''l'''' DU-145 20 Co 10 @ ,,,[email protected]â€¢!!.!,@, 0 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 DNA Content Fig. 3. Bivariate display of tall moment for damage induction versus DNA content (total image fluorescence) measured immediately following irradiation on ice. Results for 100â€”200 comets/done are shown. Note the inhibition of migration in comets with S-phase DNA content. factors, in addition to the number of strand breaks present in the cell (39â€”42).Most of the studies in Table 1 were conducted at pH 9.6, and perhaps the increased sensitivity for detecting DSBs at a slightly alkaline pH may improve the ability to resolve small differences in response between cell lines. One study that was not included in Table 1 showed a correlation between radiosensitivity and levels of DSBs in high-dose rate (43). In these experiments, it is not clear whether the results reflect initial damage or rejoining rates, but this approach may provide an additional degree of sensitivity which could identify some radioresistant cell types. Our data also indicate reproducible and significant differences in DNA elution which cannot be explained on the basis of numbers of initial DSBs produced in a cell. As observed 4 humantumor cell lines irradiatedat low-doserate but not at previously(6), thosedifferencesmaybe magnifiedat low radiation 0 mm i 0 mm 30 mm 60 mm 240 mm 10 V-79 1 C â€˜â€˜â€˜â€˜I''''I'''' I â€˜â€˜â€˜â€˜I''''I'''' I J. E HT-144 0 @ [email protected]@@ [email protected]@ I @[email protected] _______ CO N 10 _______ â€˜a1 â€˜ DU-145 1 0 10 20 [email protected] 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 DNA Content Fig. 4. Bivariate display of tail moment versus DNA content for rejoining of strand breaks following exposure of cells to 50 Gy. Cells were irradiated on ice and then returned to a 37Â°Cincubator for repair. Results for 100â€”200comets/time point are shown. Note the accumulation of cells in G2 by 4 h following irradiation. 3943 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research. [email protected]@@Y AND DNA DOUBLE-STRAND BREAKS C 80 B. After 25 Gy C CD E a) 1 @0 a) 0) CD E CD 0 @ Fig 5. DSB induction (A) and rejoining after 25 Gy (B) measured using the neutral elution method. [email protected] means Â± SD (bars) for 4â€”6damage 0.1 inductionexperimentsand 3 repair experiments. â€˜I , response of Chinese hamster V79 cells. 0 C 0 C-) CD U- 0 @ 0 20 40 60 . I . . 0 80 of DNA which may be related â€œpackagingâ€• and in some cases to radiosensitivity, variations among these human . . . 2 doses. However, elution differences do not correlate with radiosensi tivity for these cell lines. Therefore, while there are reproducible in the elutabiity 1 .1 4 Rejoining Time (h) Dose (Gy) differences , to DNA we have not seen tumor cell lines which would allow us to rank them accurately for radiosensitivity (clonogenicity). Results using the comet assay confirmed our previous work with this method indicating that the initial number of DSBs following irradiation is independent of cell type (19). An advantage of the comet assay over the neutral-filter elution method is that S-phase cells can be omitted from analysis by using total comet fluorescence as a measure of DNA content. Since replicating DNA migrates inefficiently, a cell population with a higher proportion of S-phase cells appears to show less overall DNA damage following irradiation. However, whether or not S-phase cells were included in the analysis, all cell lines showed identical dose-response relationships. DNA rejoining kinetics from comet assays is in good agreement with neutral-filter elution data considering the differences in the initial dose delivered (see below); about one-half of the breaks were rejoined within 15 mm following 50 Gy and only about 5% of the initial damage was detectable 4 h after 50 Gy. DSB rejoining rates measured using the comet assay should be more accurate than population-based 1.0 \[email protected] 4-J 0.5 U- C-SiHa Fig. 6. Neutral filter elution analysis of human @ tumor cells using Chinese hamster V79 cells as an internal control. Each panel shows results from a single filter in which V79 and human tumor test cells, radiolabeled with either [3Hjthymidine or [â€˜4C]thymidine, were irradiated with 75 Gy, lysed, and elutedfrom filtersfor 10 h. The amountof tumor cell DNA eluted by 10 h, relative to the 0 0 amount of V79 cell DNA eluted at this time, is C) shown in Table 2. CO U- i o 0.5 0.0 0 5 10 0 5 10 0 5 ElutionTime (h) 3944 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research. 10 RADIOSENSITIVITYAND DNA DOUBLE-STRAND BREAKS methods like neutral-ifiter elution since degrading cells are not ob served and apoptotic cells can be easily omitted from analysis (44). This may explain the observation that in spite of the higher radiation dose used to induce significant damage for the comet assay, residual damage for this method 4 h after irradiation was as low as or often lower than for the neutral-filter elution assay. None of the cell lines examined here demonstrated apoptosis during the 4 h of repair time following irradiation. Results in Fig. 4 indicate that all cells of the population rejoin breaks at a similar rate and that there was no evidence of a slowly repairing population of cells. Unlike single-strand break rejoining (35), there was no indication that G1 cells rejoined DSBs breaks more rapidly than cells in G2. We did notice, however, that measuring residual damage 4 h after 50 Gy could be influenced by the increase in the percentage of cells in S phase following irradiation; since S-phase DNA migrates inefficiently, there was a reduction in the amount of damage detected leading to a small (but correctable) underestimate in the amount of residual DSBs. Differences in absolute rejoining rates or residual damage between the two assays are likely to be a result of differences in the initial radiation dose (25 Gy for neutral-filter elution versus 50 Gy for the comet assay). Iliakis et a!. (45), using a variety of DSB assays including neutral filter elution, measured a half-time of rejoining of 15â€”20 mm following exposure of Chinese hamster ovary cells to 50 Gy, but only 9â€”12mm following 25 Gy, in good agreement with the results in Table 2. The higher pH of the neutral filter elution method (9.6 versus 8.3 for the comet assay) may also allow detection of more DSBs (e.g., those formed from two single-strand breaks or alkali labile sites which are displaced farther apart on the double-stranded molecule). Since such lesions are likely to be rejoined more easily than frank DSBs, the rejoining rate could appear faster for filter elution than for the comet assay. As concluded by Evans et aL (13), proficiency in DSB rejoining appears to be necessary but not always sufficient for radiation resist ance. Our results support this conclusion and indicate that DSB induction and rejoining, measured using these two DSB assays, are not adequate indicators of intrinsic tumor cell radiosensitivity. It would seem that a subset of radiosensitive cells can be detected because of the slow rejoining of DSBs (e.g., xrs.5). Another subset of cells may be identifiable on the basis of rapid initial elution from filters during neutral filter elution (e.g., mouse L5178Y-S). However misrejoining is responsible undoubtedly for a substantial third subset of radiosensitive cells, and these are unlikely to be detected with either of these DSB assays. Therefore, it appears that some measure of fidelity of repair will be necessary to reliably identify radioresistant tumor cells. radiosensitivity of L5178Y-S and L5178Y-R cells. Radiat. Res., ii2: 146â€”155, 1987. 7. Schwartz, J. L, Rotmensch, J., Giovanazzi, S. M., Cohen, M. B., and weichselbaum, R. R. Faster repair of DNA double-strand breaks in radioresistant human tumor cells. hit. J. Radial. Oncol. Biol. Phys., 15: 907â€”912,1988. 8. Kellend, L R., Edwards, S. M., and Steel, 0. 0. Induction and rejoining of DNA double-strand breaks in human cervix carcinoma cell lines of differing radiosensitiv ity. Radiat. Res., ii6: 526â€”538,1988. 9. McMillan, T. 3., Eady, J. J., Holmes, A., Peacock, J. H., and Steel, G. G. The radiosensitivity of human neuroblastoma: a cellular and molecular study. Int. J. Radiat.Biol.,56: 651â€”656, 1989. 10. Evans, H. H., Ricanti, M., and Horng, M. Deficiency in DNA repair in mouse lymphoma strain L5178Y-S. Proc. NatI. Acad. Sci. USA, 84: 7562â€”7566,1987. 11. Dahm-Daphi, I., Dikomey, E., Pyttlik, C., and Jeggo, P. A. Repairable and non repairable DNA strand breaks induced by X-irradiation in CHO K! cells and the radiosensitive mutants xrsi and xrs5. tnt. J. Radiat. Biol., 64; 19â€”26,1993. 12. Eguchi-Kasai, K., Kosaka, T., Sato, K, and Kaneko, I. Repairability of DNA double-strand breaks and radiation sensitivity in five mammalian cell lines. Int. J. Radiat. Biol., 59: 97â€”104,1991. 13. Evans, H. H., Ricanti, M., Horng, M., Jiang, 0., Mend, J., and Olive, P. DNA double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell lines. Radiat. Res., i34: 307â€”315,1993. 14. Sweigert, S. E., Rowley, R., Warters, R. L, and Dethlefsen, L A. Cell cycle effect on the induction of DNA double-strand breaks by X-rays. Radiat. Res., 116: 228â€” 244, 1988. 15. McMillan, T. J., Casoni, A. M., Edwards, S., Holmes, A., and Peacock, J. H. The relationship of DNA double-strand break induction to radiosensitivity in human tumour cell lines. Int. J. Radiat. Biol., 58: 427â€”438,1990. 16. Iliakis, G., Okayasu, R., and Seaner, R. Radiosensitive xrs-5 and parental CHO cells show an identical DNA neutral filter elution dose-response: implications for a relationship between cell radiosensitivity and induction of DNA double-strand breaks. Int. J. Radiat. Biol., 54: 55â€”62,1988. 17. Van Ankeren, S. C., Murray, D., and Meyn, R. E. lnduction and rejoining of y-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells and in two radiosensitive clones. RadiaL Res., ii6: 511â€”525, 1988. 18. Jones, N. J., Stewart, S. A., and Thompson, L H. Biochemical and genetic analysis of Chinese hamster mutants irsi and irs2 and their comparison to cultured ataxia telangiectasia cells. Mutagenesis, 5: 15-23, 1990. 19. Olive, P. L., Wiodek, D., and Banath, J. P. DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. Cancer Res., 51: 4671â€”4676, 1991. 20. Smeets, M. F., Mooren, E. H., and Begg, A. C. Radiation-induced DNA damage and repair in radiosensitive and radioresistant human tumour cells measured by field inversion gel electrophoresis. tnt. J. Radiat. Biol., 63: 703â€”713,1993. 21. Iliakis, U., Metzger, L, Muschel, R. J., and McKema, W. 0. Induction and repair of DNA double-strand breaks in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ms and v-myc. Cancer Rex., 50: 6575-6579,1990. 22. Cheong, N., Wang, Y., Jackson, M., and Iliakis, 0. Radiation-sensitive irs mutants rejoin DNA double-strand breaks with efficiency similar to that of parental V79 cells but show altered response to radiation-induced 02 delay. Mutat. Res., 274: 111â€”122, 19fl 23. Flentje, M., Asadpour, B., Latz, D., and Weber, K. J. Sensitivity of neutral filter elution but not PFGE can be modified by non-dab chromatin damage. tnt. J. Radiat. Biol., 63: 715â€”724,1993. 24. Giaccia, A. J., Schwartz, J., Shieh, J., and Brown, inversion gel electrophoresis to predict tumor cell 24: 231â€”238,1992. 25. Kysela, B. P., Michael, B. D., and Arrand, J. E. initial DNA damage and repair of double-strand J. M. The use of asymmetric-field radiosensitivity. Radiother. Oncol., Relative contributions of levels of breaks to the ionizing radiation sensitivephenotypeoftheChinesehamstercellmutant,XR-V15B.PartI. X-rays.Int. J. Radiat. Biol., 63: 609â€”616, 1993. 26. Warters, R. L, Lyons, B. W., Chen, D. J., and Sato, K. DNA damage processing in a radiation-sensitive mouse cell line. Mutat. Res., 293: 91â€”98,1993. 27. lliakis, G., Mehta, R., and Jackson, M. Level of DNA double-strand break rejoining in Chinese hamster xrs-5 cells is dose-dependent: implications for the mechanism of radiosensitivity. hit. I. Radiat. Biol., Ã³i:315â€”321,1992. 28. REFERENCES Lambin, P., Cedervall, B., Chavaudra, N., Sadie, C., Joiner, M. C., and Malaise, E. P. Initial and residual number of X-ray induced DNA double-strand breaks in radiosen sitive and radioresistant human tumor cell lines. Radiother. OncoL, 25: S5l, 1992. 29. Wlodek, D., Olive, P. L Physical basis for detection of DNA double-strand breaks 1. Oshita, F., Fujiwara, Y., and Saijo, N. Radiation sensitivities in various anticancer drug-resistant human lung cancer cell lines and mechanism of radiation cross resistance in a cisplatin-resistant cell line. J. Cancer Res. Clin. Oncol., ii9: 28â€”34, 1992 2. Redford, I. R., and Hodgson, 0. S. [email protected] DNA double-strand breaks: use in calibration of the neutral filter clution technique and comparison with X-ray induced breaks. Int. J. Radial. Biol., 48: 555-566, 1985. 3. Wlodek, D., and Olive, P. L Neutral ifiter elution detects differences in chromatin organization which can influence cellular radiosensitivity. Radiat. Res., 132: 242247, 1992. 4. Schwartz, J. L, Mustafi, R., Bcckett, M. A., Czyzewski, E. A., Farhangi, E., Ordina, D., Rotmensch, J., and Weichselbaum, R. R. Radiation-induced DNA double-strand break frequencies in human squamous cell carcinoma cell lines of different radiation using neutral filter elution. Radiat. Res., 124: 326â€”333,1990. 30. [email protected], to -y-radiation. Part 1: Intrinsicradiosensitivity.tat. J. Radiat.Biol, 61: 465â€”472, R. w. Detection of ionizing radiation-induced DNA Radiat. Res., i24: 309â€”316,1990. 31. Olive, P. L DNA organization affects cellular radiosensitivity and detection of initial DNA strand breaks. Int. J. Radiat. Biol., 62: 389â€”396,1992. 32. Radford, I. F., and Broadhurst, S. Enhanced induction by X-irradiation of DNA double-strand breakage in mitosis as compared with S-phase V79 cells. Int. J. Radiat. Biol., 49: 909â€”914,1986. 33. iliakis, 0. E., Cicilioni, 0., and Metzger, L Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel sensitivities. hit. J. Radiat. Biol., 59: 1341â€”1352,1991. 5. Green, A., Prager, A., Stoudt, P. M., and Murray, D. Relationships between DNA damage and the survival of radiosensitive mutant Chinese hamster cell lines exposed R. L., and Lyons, double-strandbreaksby filter elutionis affectedby nuclearchromatinstructure. electrophoresis: calibration by means of @I decay. Int. J. Radiat. Biol., 59: 343â€”357, 1991. 34. Okayasu, R., Bloecher, dose-response of DNA D., and Iliakis, 0. neutral filter elution Variation through the cell cycle in the in X-irradiated synchronous CHO cells. Int.J. Radiat.Biol.,53: 729â€”747, 1988. 1992. 6. Wlodek, D., and Hittelman, W. N. The repair of double-strand breaks correlates with 35. Olive, P. L, and Banith, J. P. Induction and rejoining of radiation induced DNA 3945 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research. RADIOSENSITIVITY AND DNA DOUBLE-STRAND BREAKS single-strandbreaksâ€œtail momentâ€• as a functionof positionin thecellcycle.Mutat. Res., 294: 275â€”283,1993. 36. Olive, P. L., and Banath, J. P. Growth fraction measured using the comet assay. Cell Prolif., 25: 447â€”457, 1992. 37. Bradley, M. 0., and Kohn, K. W. X-ray induced DNA double-strand break production and repair in mammalian cells as measured by neutral filter elution. Nucleic Acids Res., 7: 793â€”804,1979. 38. Brock, W. A., Baker, F. L., and Tofilon, P. J. Tumor cell sensitivities to drugs and radiation, In: J. D. Chapman, L. J. Peters, and H. R. withers (eds.), Prediction of Tumor Treatment Response, pp. 139â€”155.Elmsford, NY: Pergamon Press, Inc., strand break analysis by filter elution. Int. J. Radiat. Biol., 54: 739â€”747,1988. 42. Prise, K. M., Davies, S., and Michael, B. C. Non-linear dose-effect curve for DNA double-strand breaks by low LET radiation: the effect of eluting buffer composition on the measurement of breaks by filter elution technique. Int. J. Radiat. Biol., 56: 943â€”950, 1989. 43. Cassoni, A. M., McMillan, T. J., Peacock, J. H., and Steel, G. 0. Differences in the level of DNA double-strand breaks in human tumour cell lines following low dose-rate irradiation. Eur. J. Cancer, 284: 1610â€”1614,1992. 44. 39. Hutchinson, F. On the measurement of DNA double-strand breaks by neutral elution (Letter to the Editor). Radiat. Res., i20: 182â€”186,1989. 40. lliakis, 0. The role of DNA double-strand breaks in ionizing radiation-induced killing of eukaryotic cells. Bioessays, i3: 641â€”648,1991. 41. Koval, T. M., and Kazmar, E. R. Eluting solution composition affects DNA double Olive, P. L., Fraser, G., and BanÃ¡th, J. P. Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay. Radiat. Res., i36: 130â€”136, 1989. 1993. 45. Iliakis, 0., Blocher, D., Metzger, L., and Pantelias, 0. Comparison of DNA double strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose gradient centrifugation, and non-unwinding filter elution in irradiated plateau-phase CHO cells. Int. J. Radial. Biol., 59: 927â€”939,1991. 3946 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1994 American Association for Cancer Research. Lack of a Correlation between Radiosensitivity and DNA Double-Strand Break Induction or Rejoining in Six Human Tumor Cell Lines Peggy L. Olive, Judit P. Banáth and H. Susan MacPhail Cancer Res 1994;54:3939-3946. 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