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We previously described an enhanced sensitivity for cell killing and γ-H2AX focus induction after both high-dose-rate and continuous low-dose-rate γ irradiation in 14 primary fibroblast strains derived from hereditary-type retinoblastoma family members (both affected RB1+/− probands and unaffected RB1+/+ parents). Here we present G2-phase chromosomal radiosensitivity assay data for primary fibroblasts derived from these RB family members and five Coriell cell bank controls (four apparently normal individuals and one bilateral RB patient). The RB family members and two normal Coriell strains had significantly higher (~1.5-fold, P < 0.05) chromatid-type aberration frequencies in the first postirradiation mitosis after doses of 50 cGy and 1 Gy of 137Cs γ radiation compared to the remaining Coriell strains. The induction of chromatid-type aberrations by high-dose-rate G2-phase γ irradiation is significantly correlated to the proliferative ability of these cells exposed to continuous low-dose-rate γ irradiation (reported in Wilson et al., Radiat. Res. 169, 483–494, 2008). Our results suggest that these moderately radiosensitive individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes or may carry epigenetic changes involving genes that more broadly modulate such systems, including G2-phase-specific DNA damage responses.
Individual genetic variation in cellular DNA damage response pathways can influence DNA damage signaling thresholds, rates of DNA repair, and in vitro radiosensitivity after exposure to low-dose and low-dose-rate ionizing radiation (1). Such genetic variants presumably play an important role in determining an individual's predisposition to spontaneous and DNA-damaging agent-induced cancers (2–5), and sensitivity to the genotoxic effects of mutagens has been shown to be highly heritable in studies examining monozygotic and dizygotic twins and first-degree relatives (6–10). Significantly reduced DNA damage signaling and repair capacity has been documented in vitro for several cancer predisposition and chromosomal instability syndromes, including ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and LIG4 syndrome (11–13), while a broad spectrum of radiation responses has also been observed for cells derived from apparently normal individuals (1, 14–17).
The induction of chromosomal aberrations after exposure to radiation or other genotoxic agents (both exogenous and endogenous) is mediated through the misrepair or lack of repair of DNA double-strand breaks (DSBs), which subsequently determines (to a great extent) the proliferative and carcinogenic potential of surviving cells (18–21). Several cytogenetic assays have been developed to measure radiation-induced chromosomal aberrations during different phases of the cell cycle for use as potential radiation and cancer biomarkers (22–25). One assay, the G2 chromosomal radiosensitivity assay, has been developed to measure the response of cells (typically lymphocytes or fibroblasts) to irradiation in G2 phase (26–29). This G2 assay measures the yield of chromatid-type aberrations (mostly chromatid gaps and breaks) at the first postirradiation mitosis in cells irradiated hours previously during the G2 phase. The repair of radiation-induced DSBs in G2-phase mammalian cells occurs primarily through non-homologous end joining (NHEJ) and homologous recombinational repair (HRR) (20). The relative contribution of NHEJ and HRR to the repair of radiation-induced DSBs in G2-phase human cells has not been fully established, although it is apparent that both repair pathways contribute to the repair of double-stranded DNA damage induced by radiation (19, 30–32).
The G2 assay has been applied to a number of radiation sensitivity and cancer predisposition syndromes, including AT, Bloom syndrome, dysplastic nevus syndrome, familial polyposis, Fanconi anemia, Gardner syndrome, Li-Fraumeni syndrome, Wilms tumor and xeroderma pigmentosum, as well as to patients with prostate, head and neck, breast and other types of cancer (27–29, 33–42). The high frequencies of chromatid-type aberrations observed in cells derived from these patients have been attributed to deficiencies in DNA repair or related signaling pathways (27–29, 43, 44). However, the G2 assay has demonstrated only moderate correlation to the degree of in vivo radiosensitivity observed in the clinic (40–42). G2 hypersensitivity has been studied as a potential marker of heritable low-penetrance predisposition to cancer. Roberts et al. demonstrated G2-phase hypersensitivity in 23 of 37 first-degree relatives of radiosensitive breast cancer patients compared to only one of 15 first-degree relatives of breast cancer patients with normal radiosensitivity (9). A recent large-scale examination of G2 chromosomal radiosensitivity and postirradiation apoptotic responses of peripheral blood lymphocytes from 211 untreated breast cancer patients and 170 matched controls did not reveal any significant differences between the two groups for either end point, although it was suggested that both cases and controls with high familial risk of breast cancer were more radiosensitive (40).
Measurements of G2-phase chromosomal radiosensitivity in retinoblastoma (RB) patients and first-degree family members were reported by Chaum et al. (45) and in two reports by Sanford et al. (26, 46) using the radiomimetic agent bleomycin and radiation, respectively. The spontaneous and bleomycin-induced aberration frequencies in bilateral (hereditary-type) and unilateral (sporadic) RB lymphocytes reported in the study of Chaum et al. (45) did not differ significantly from those of normal control lymphocytes. Similarly, a retinoblastoma tumor cell line examined by Darroudi et al. (47) did not demonstrate an increased aberration induction after X irradiation in G2 phase compared to normal untransformed fibroblasts. On the other hand, the first study of Sanford et al. reported a mean frequency of 3.2 chromatid breaks/cell (range 1.0–5.1) for eight bilateral (including one familial unilateral) RB fibroblast strains and 4.1 breaks/cell for a sporadic unilateral RB fibroblast strain compared to 0.4 breaks/cell (range 0.2–2.0) for 29 normal fibroblast strains after an X-ray dose of 53 cGy (26). In the second study of Sanford et al., the authors reported mean frequencies of 2.2 breaks/cell (range 1.3–3.2) for 13 additional bilateral RB fibroblast strains, 0.7 breaks/cell (range 0.4–2.1) for six additional unilateral RB fibroblast strains, and 0.4 breaks/cell (range 0.3–0.5) for eight additional normal fibroblast strains after 53 cGy (46). Some of the unaffected first-degree relatives displayed high aberration frequencies (≥1.1 breaks per cell) similar to those of the bilateral RB patients. In a later study by Scott et al., evaluation of the same collection of fibroblast strains demonstrated higher aberration frequencies in the normal strains (average of ~2.2 breaks/cell, range 1.1–3.3), different kinetics of repair, and more interexperimental variability, with 12 of the 53 normal strains (23%) being designated as moderately radiosensitive (33).
We previously described an enhanced sensitivity for cell killing and γ-H2AX focus induction after both high-dose-rate and continuous low-dose-rate γ irradiation in primary fibroblast strains derived from 14 hereditary-type (bilateral) RB family members for both the affected RB1+/− probands and the unaffected RB1+/+ parents (1, 48, 49). In the present study, we present data on the G2-phase chromosomal radiosensitivity of fibroblasts derived from these RB family members and five Coriell controls (four normal strains and one RB1+/− strain) after acute doses of 50 cGy and 1 Gy high-dose-rate 137Cs γ radiation. The relative radiosensitivity of the Coriell control strains examined in this study was established by previous survival and cytogenetic assays performed in this laboratory2 (1, 49, 50).
The protocol employed in this study was modified slightly from the standardized National Cancer Institute (NCI) and Paterson Institute for Cancer Research (PICR) G2 chromosomal radiosensitivity assay protocol (28). The fibroblast strains used in this study (identified in Tables 1 and and2)2) have been described previously (1, 48–50). All cultures were grown in α-MEM (GIBCO/Invitrogen) supplemented with 15% FBS, 100 μg/ml streptomycin sulfate, 100 U/ml penicillin G (all Sigma), and 1× GlutaMAX™-I (l-alanyl-l-glutamine, Gibco/Invitrogen) in a 37°C incubator with 95% air/5% CO2. A single lot of FBS was used in all experiments to reduce experimental variability. Cultures were routinely passaged prior to contact inhibition and maintained in exponential growth for a minimum of 5 days until sufficient cell numbers were available for experimental analysis. Cultures were prepared for the G2 assay by replacing the culture medium with fresh medium 24 h prior to irradiation. Asynchronous cultures were irradiated at 37°C with 0 (sham) or 50 cGy or 1 Gy of 662 keV γ rays supplied by a J. L. Shepherd and Associates Mark I-68A cesium-137 beam irradiator at a dose rate of ~2.5 Gy/min.
After irradiation, the cultures were returned to the 37°C incubator and 0.1 μg/ml colcemid (Gibco/Invitrogen) was added 30 min later. Ninety minutes postirradiation, the cultures were collected and processed by standard cytogenetic techniques (51). Briefly, the culture medium was transferred to conical tubes to ensure collection of any detached mitotic cells and the remaining adherent cells were detached with 0.25% trypsin/EDTA (Gibco/Invitrogen) in Ca2+/Mg2+-free PBS (Dulbecco's PBS-A). The single cell suspensions were centrifuged at 300g. After the supernatant was aspirated, the cell pellet was gently broken up, resuspended in 8 ml of 37°C 75 mM KCl hypotonic buffer, and incubated for 7 min at 37°C, after which 2 ml of freshly prepared 3:1 methanol:acetic acid (Carnoy's fixative) was added. The suspensions were incubated for 2 min at room temperature and centrifuged at 200g. After the supernatant was aspirated, the cell suspensions were then fixed by the dropwise addition of fresh fixative. After an additional centrifugation, the fixation process was repeated twice (for a total of three fixative rinses). Cell suspensions were dropped onto clean, wet microscope slides, air-dried and desiccated for 24 h at 37°C. Slides were stained with a 10% Giemsa solution (Gurr® R66, BDH Chemicals, Ltd.) prepared in a pH 6.8-buffered solution (Sorenson's buffer), washed vigorously in McIlvaine's rinsing buffer (approximately 18 mM citric acid/16 mM disodium phosphate prepared in distilled water) and distilled water, and air-dried. The slides were sealed with Cytoseal™ 60 mounting medium (Microm International) and a glass cover slip (VWR).
A total of 150–250 diploid metaphase spreads per dose were examined for each fibroblast strain in two or three independent experiments using a Nikon Microphot microscope equipped with a 100× oil-immersion objective and 2× optivar lens (strains GM04505 and GM08447 were examined in one experiment only). Only metaphase spreads with good chromosome morphology with minimal overlap and cytoplasmic interference were scored. The modal chromosome number for all strains was 46 and ranged from 45–47. The classification scheme used in this study for scoring chromosomal (chromatid-type) aberrations was similar to that described by Savage (52), Sanford and coworkers (26, 46), and Scott and coworkers (9, 29, 33–35). Chromatid gaps were defined as fully discontinuous chromatid fragments detached at a distance less than the width of the chromatid arm. Chromatid breaks were defined as fully discontinuous chromatid fragments that either were displaced at a distance greater than the width of the chromatid arm or were no longer aligned with the axis of the chromatid arm. Small achromatic chromatid fragments that did not appear to be completely severed from the chromatid arm were not scored. While some subjectivity in the scoring criteria for gaps and breaks exists among different research groups (53–55), the sum of chromatid gaps and breaks minimizes variation in scoring and interlaboratory comparisons. In this case, a single individual (PFW) scored the metaphase spreads on coded slides so the aberration scoring criteria were applied consistently. Symmetrical and asymmetrical chromatid-type exchanges were observed at much lower frequencies and were also scored.
The standard errors of the mean (SEM) reported in Tables 1 and and22 were calculated by combining the standard deviations of the aberration frequencies for two or three independent experiments by the square root of the sum-of-the-squares method and dividing this value by the square root of the number of experiments conducted per strain. Statistically significant differences between the aberration frequencies (chromatid breaks and total chromatid-type aberrations) for individual strains or groups of strains were tested using Student's t tests for two independent sample distributions (SigmaPlot, Systat Software, Inc.). Correlations between aberration frequencies and the continuous low-dose-rate survival parameters reported in ref. (1) were assessed using the Pearson product-moment correlation coefficient fit by the least-squares method (SigmaPlot).
Tables 1 and and22 report the mean spontaneous and radiation-induced chromatid-type aberration frequencies (± SEM) after irradiation in G2 phase for the five Coriell control and 14 RB family member primary fibroblast strains examined in this study. The radiation-induced aberration frequencies in Tables 1 and and22 have been corrected for the spontaneous (0 cGy) levels of aberrations (chromatid-type exchanges were not observed in unirradiated cultures). Significant differences (P < 0.05 by Student's t test) in aberration frequencies between individual strains and the average for the three Coriell strains with normal radiosensitivity (AG01521, GM02149 and GM06419) are denoted with asterisks. The mean spontaneous chromatid break frequency was 0.12 per cell (range 0.07–0.17 per cell) and the mean spontaneous total chromatid-type aberration frequency was 0.4 per cell (range 0.3–0.6 per cell) for the 19 strains examined and did not differ significantly among them. There were also no significant differences in the radiation-induced chromatid-type exchange frequencies among these strains (despite an approximately twofold increase observed in five of the 14 RB family member strains after 1 Gy).
The mean radiation-induced chromatid break and total chromatid-type aberration frequencies (± SD) for groups of these strains are summarized in Table 3 (weighted per unit dose and averaged for the data for 50 cGy and 1 Gy). The strains are grouped by RB1 genotype for the RB family members and by radiosensitivity group for the Coriell controls. The average radiation-induced chromatid break and total chromatid-type aberration frequencies for the 14 RB family member strains and the two radiosensitive Coriell strains were approximately 37–55% and 26–39% higher, respectively, than those measured in the three Coriell strains with normal radiosensitivity. Separating the RB family members by RB1 genotype, the average radiation-induced chromatid break and total aberration frequencies were 44% and 34% higher, respectively, for the RB1+/+ strains and 37% and 26% higher, respectively, for the RB1+/− strains compared to controls. The unaffected RB family member fibroblast strains had slightly higher aberration frequencies compared to the affected RB family member strains, although this difference was not statistically significant. The two radiosensitive Coriell strains and all of the RB family member fibroblast strains, except the affected proband MF-14R in RB family IV, had significantly higher radiation-induced chromatid-type aberration frequencies than the three Coriell strains with normal radiosensitivity. A histogram of total chromatid-type aberration frequencies averaged for the data for 50 cGy and 1 Gy is shown in Fig 1.
In Fig. 2A, the total chromatid-type aberration frequencies (spontaneous plus radiation-induced) measured for the doses in this study are plotted as a function of the dose rates required to reduce relative survival to 10% or 1% during continuous low-dose-rate c irradiation (1). The low-dose-rate survival assay described in ref. (1) measures the proliferative capacity (colony-forming ability) of cells exposed continuously to low-dose-rate (0.5–8.4 cGy/h) 137Cs γ radiation for 2 weeks and provides a means to accentuate minor differences in radiosensitivity observed after acute, high-dose-rate exposures. The continuous-irradiation dose rates required for 10% and 1% relative survival are significantly correlated with the G2 assay aberration frequencies (R2 = 0.33–0.39 for 50 cGy, R2 = 0.62–0.64 for 1 Gy, P < 10−8). No significant correlation between the spontaneous (0 cGy) aberration frequencies and survival after continuous low-dose-rate irradiation was observed (R2 = 0.05). In Fig. 2B, the 19 fibroblast strains are identified individually using the same legend symbols from Fig. 1; this arrangement of strains is very similar for the data sets plotted in Fig. 2A. This correlation between high-dose-rate G2-phase chromosomal radiosensitivity and the capacity of cells to proliferate under continuous low-dose-rate irradiation was not entirely unexpected. Previous studies from this laboratory reported that the inability of cells to proliferate during continuous low-dose-rate irradiation at higher dose rates (i.e., those approaching the proliferation-limiting low dose rate) correlated with cell cycle redistribution and checkpoint-induced arrest in the radiosensitive G2 phase of the cell cycle2 (56, 57).
The G2-phase chromosomal radiosensitivity assay data presented here clearly support the assignment of a phenotype of moderate in vitro radiosensitivity in cells derived from these RB family members, as we reported previously using other radiobiological assays (1, 48, 49). G2-phase irradiation of the three Coriell strains with normal radiosensitivity yielded mean values of ~3.2 chromatid breaks and ~7.3 total chromatid-type aberrations per cell/Gy. The RB family and the two radiosensitive Coriell strains had ~30–50% higher radiation-induced aberration frequencies, having mean values of 4.5–4.9 chromatid breaks and 9.5–10.1 total chromatid-type aberrations/cell/Gy, respectively. Interestingly, GM06419, a bilateral RB1+/− strain from the Coriell cell bank, demonstrated the lowest radiation-induced aberration frequencies of all the strains examined (~2.8 chromatid breaks and ~7.1 total chromatid-type aberrations/cell/Gy). A similar radioresistant response for GM06419 in the G2 assay was reported by the group of Sanford (26, 46) and was the only RB1+/− strain examined in common with this study. These results provide further evidence that RB1 mutation alone is not directly correlated with radiosensitivity.
The chromatid break frequencies in the three Coriell stains with normal radiosensitivity measured in this study are roughly four times higher than those reported for a large collection of apparently normal strains in the two reports by Sanford et al. (~3.2 breaks/cell/Gy for the current study compared to ~0.8 breaks/cell/Gy reported by Sanford et al.) (26, 46). Scott et al. (33) re-evaluated the same collection of normal strains used by Sanford and coworkers and reported a higher average number of breaks (~2.2 breaks/cell/Gy) and a broader range (1.1–3.3 breaks/cell/Gy) for 53 normal strains. Their data are more comparable to this study and others (17). Despite these differences recorded for the normal strains, the radiation-induced aberration frequencies measured for the affected RB patients in this study (~4.3 breaks/cell/Gy, range 3.3–5.2) are consistent with the average of 4.4–6.4 breaks/cell/Gy reported by Sanford et al. and Scott et al. There is some subjectivity regarding the scoring of chromatid “gaps” and “breaks” and even differences in the criteria adopted for distinguishing them, depending on the particular laboratory (53–55). While this could underlie some discrepancies in comparisons of G2 assay results, the reason for the differences in aberration yields reported for the normal strains but not the RB patient strains in this study compared to the studies of Sanford et al. is not immediately obvious.
Recently, this laboratory developed an extension of the G2 chromosomal radiosensitivity assay in which γ-H2AX foci are detected immunocytochemically and measured on cytocentrifuged metaphase chromosome spreads (49). Phosphorylation of histone variant H2AX on serine 139 at sites of DSBs by the DNA-PK, ATM and ATR protein kinases produces cytologically visible γ-H2AX foci and provides a sensitive measure of DSB induction and repair at low radiation doses (58, 59). It should be noted that G2-phase/mitotic cells have 4N DNA content and twice as many radiation-induced γ-H2AX foci per unit dose compared to G0/G1-phase cells (defined as having 2N DNA content), and approximately 20–50% of the initial radiation-induced DSBs are repaired in the 90 min required to perform the G2 assay (60, 61). Using the same irradiation and mitotic collection schedule used in this study, after a dose of 50 cGy, the unaffected RB parents had a mean of 29 radiation-induced γ-H2AX foci/cell (range of 20–40), the radiosensitive Coriell strains GM04505 and GM08447 both had 31 radiation-induced γ-H2AX foci/cell, and the group of Coriell controls with normal radiosensitivity had a mean of 20 radiation-induced γ-H2AX foci/cell (range of 14–26). Thus, for this DNA damage end point, the relative radiosensitivities were qualitatively similar to the results reported in this study. Not unexpectedly, the frequencies of radiation-induced γ-H2AX foci and chromatid-type aberrations after irradiation in G2 phase in these strains are significantly correlated (P < 10−8).
In conclusion, this study supports the utility of the G2 chromosomal radiosensitivity assay as a means to assess the relative cellular radiosensitivity of cells compared to a group of validated “normally responding” controls and is predictive of relative cellular radiosensitivity in the continuous low-dose-rate irradiation colony formation assay we described previously (1). The significantly higher frequencies of chromatid-type aberrations in the unaffected parents of the RB families after irradiation in G2 phase compared to the Coriell control strains suggests that low-penetrance or hypomorphic mutations may affect the integrity of DSB repair (either NHEJ or HRR) or associated cell cycle checkpoint pathways in these cells and possibly underlie their radiosensitivity phenotype. Alternatively, epigenetic changes in genes that could more broadly affect genome maintenance, including the capacity to process radiation-induced DNA damage, may also underlie the mild hypersensitivities we have observed (Liu et al., unpublished results). If such mild genomic maintenance defects likewise affect germline mutation rates, such genetic polymorphisms or epigenetic changes could be important underlying factors in determining the frequency of genetic conditions predisposing to cancer, such as heterozygosity for RB1 or other tumor suppressor genes relevant to the etiology of childhood cancers.
The authors would like to acknowledge Mr. Zane H. Story and Ms. Christy L. Warner for their valued technical assistance. This work was supported in part by grant T32-CA09236 (JSB) from the U.S. Department of Health and Human Services National Cancer Institute and grants DE-FG03-01ER63235 (JSB) and DE-FG02-05ER64089 (JBL) from the U.S. Department of Energy Low Dose Radiation Research Program. A portion of this work (PFW) was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.
2P. F. Wilson, Genetic factors affecting radiation sensitivity and genomic stability. Ph.D. dissertation, Colorado State University, 2006.