Evasion of Early Cellular Response Mechanisms following Low Level Radiation-induced DNA Damage

DNA damage that is not repaired with high fidelity can lead to chromosomal aberrations or mitotic cell death. To date, it is unclear what factors control the ultimate fate of a cell receiving low levels of DNA damage (i.e. survival at the risk of increased mutation or cell death). We investigated whether DNA damage could be introduced into human cells at a level and frequency that could evade detection by cellular sensors of DNA damage. To achieve this, we exposed cells to equivalent doses of ionizing radiation delivered at either a high dose rate (HDR) or a continuous low dose rate (LDR). We observed reduced activation of the DNA damage sensor ataxia-telangiectasia mutated (ATM) and its downstream target histone H2A variant (H2AX) following LDR compared with HDR exposures in both cancerous and normal human cells. This lack of DNA damage signaling was associated with increased amounts of cell killing following LDR exposures. Increased killing by LDR radiation has been previously termed the “inverse dose rate effect,” an effect for which no clear molecular processes have been described. These LDR effects could be abrogated by the preactivation of ATM or simulated in HDR-treated cells by inhibiting ATM function. These data are the first to demonstrate that DNA damage introduced at a reduced rate does not activate the DNA damage sensor ATM and that failure to activate ATM-associated repair pathways contributes to the increased lethality of continuous LDR radiation exposures. This inactivation may reflect one strategy by which cells avoid accumulating mutations as a result of error-prone DNA repair and may have a broad range of implications for carcinogenesis and, potentially, the clinical treatment of solid tumors. DNA damage that is not repaired with high fidelity can lead to chromosomal aberrations or mitotic cell death. To date, it is unclear what factors control the ultimate fate of a cell receiving low levels of DNA damage (i.e. survival at the risk of increased mutation or cell death). We investigated whether DNA damage could be introduced into human cells at a level and frequency that could evade detection by cellular sensors of DNA damage. To achieve this, we exposed cells to equivalent doses of ionizing radiation delivered at either a high dose rate (HDR) or a continuous low dose rate (LDR). We observed reduced activation of the DNA damage sensor ataxia-telangiectasia mutated (ATM) and its downstream target histone H2A variant (H2AX) following LDR compared with HDR exposures in both cancerous and normal human cells. This lack of DNA damage signaling was associated with increased amounts of cell killing following LDR exposures. Increased killing by LDR radiation has been previously termed the “inverse dose rate effect,” an effect for which no clear molecular processes have been described. These LDR effects could be abrogated by the preactivation of ATM or simulated in HDR-treated cells by inhibiting ATM function. These data are the first to demonstrate that DNA damage introduced at a reduced rate does not activate the DNA damage sensor ATM and that failure to activate ATM-associated repair pathways contributes to the increased lethality of continuous LDR radiation exposures. This inactivation may reflect one strategy by which cells avoid accumulating mutations as a result of error-prone DNA repair and may have a broad range of implications for carcinogenesis and, potentially, the clinical treatment of solid tumors. Ionizing radiation (IR) 1The abbreviations used are: IR, ionizing radiation; DSB, double strand break; H2AX, histone H2A variant; Gy, gray (absorbed dose); LDR, low dose rate; HDR, high dose rate; VLDR, very low dose rate; FACS, fluorescence-activated cell sorter; siRNA, small interfering RNA; ATM, ataxia-telangiectasia mutated. causes numerous types of DNA damage, which occur at different frequencies within the cell (1Ward J.F. Prog. Nucleic Acid Res. Mol. Biol. 1988; 35: 95-125Crossref PubMed Scopus (1234) Google Scholar). However, it is generally accepted that DNA double strand breaks (DSBs) are the most important type of damage with respect to the latent effects of radiation exposure such as chromosomal aberrations, tumorigenesis, and cell death (2Hoeijmakers J.H. Nature. 2001; 411: 366-374Crossref PubMed Scopus (3216) Google Scholar, 3van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (979) Google Scholar, 4Jackson S. Carcinogenesis. 2002; 23: 687-696Crossref PubMed Scopus (942) Google Scholar). For a cell to survive the potentially lethal damage conferred by radiation exposure, DNA DSBs must be rapidly detected, and DNA repair mechanisms must be initiated. However, in humans, the primary DSB repair pathway is somewhat errorprone, and therefore, the fidelity of the genome is not always maintained (2Hoeijmakers J.H. Nature. 2001; 411: 366-374Crossref PubMed Scopus (3216) Google Scholar, 3van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (979) Google Scholar, 4Jackson S. Carcinogenesis. 2002; 23: 687-696Crossref PubMed Scopus (942) Google Scholar, 5Vilenchik M.M. Knudson A.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12871-12876Crossref PubMed Scopus (515) Google Scholar). This may be particularly important following the production of low levels of DNA damage in which cellular detection mechanisms may not be fully elicited (5Vilenchik M.M. Knudson A.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12871-12876Crossref PubMed Scopus (515) Google Scholar, 6Vilenchik M.M. Knudson Jr., A.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5381-5386Crossref PubMed Scopus (128) Google Scholar). Evidence exists (7Joiner M.C. Marples B. Lambin P. Short S.C. Turesson I. Int. J. Radiat. Oncol. Biol. Phys. 2001; 49: 379-389Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar) that increased cell death in the absence of repair following low level DNA damage may offer a way by which the cell prevents potentially promutagenic lesions from being passed on to progeny. It has been shown recently (8Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2750) Google Scholar, 9Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (854) Google Scholar, 10Horejsi Z. Falck J. Bakkenist C.J. Kastan M.B. Lukas J. Bartek J. Oncogene. 2004; 23: 3122-3127Crossref PubMed Scopus (107) Google Scholar, 11Lee J.H. Paull T.T. Science. 2004; 304: 93-96Crossref PubMed Scopus (592) Google Scholar) that within minutes of incurring small amounts of DNA damage, the critical damage sensor molecule ATM is activated by an autophosphorylation event at Ser-1981, which might require the NBS1 protein. Once activated, ATM is responsible for initiating several signaling cascades, which are essential to halt cell cycle progression that allows the DNA damage to be repaired (see Ref. 12Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2178) Google Scholar and references therein). One of the earliest detectable downstream targets of ATM is the histone H2A variant, H2AX, which is phosphorylated by ATM at Ser-139 (13Burma S. Chen B.P. Murphy M. Kurimasa A. Chen D.J. J. Biol. Chem. 2001; 276: 42462-42467Abstract Full Text Full Text PDF PubMed Scopus (1529) Google Scholar). It appears that phosphorylated H2AX (termed γ-H2AX) encompasses a region of several thousand base pairs around the damage sites, forming foci within the nucleus that act as a molecular beacon signaling for the recruitment of DNA repair factors (14Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1722) Google Scholar, 15Celeste A. Petersen S. Romanienko P.J. Fernandez-Capetillo O. Chen H.T. Sedelnikova O.A. Reina-San-Martin B. Coppola V. Meffre E. Difilippantonio M.J. Redon C. Pilch D.R. Olaru A. Eckhaus M. Camerini-Otero R.D. Tessarollo L. Livak F. Manova K. Bonner W.M. Nussenzweig M.C. Nussenzweig A. Science. 2002; 296: 922-927Crossref PubMed Scopus (1158) Google Scholar, 16Downs J.A. Jackson S.P. Nature. 2003; 424: 732-734Crossref PubMed Scopus (19) Google Scholar). Indeed, mice and cells lacking H2AX show chromosomal instability and defective DNA repair (15Celeste A. Petersen S. Romanienko P.J. Fernandez-Capetillo O. Chen H.T. Sedelnikova O.A. Reina-San-Martin B. Coppola V. Meffre E. Difilippantonio M.J. Redon C. Pilch D.R. Olaru A. Eckhaus M. Camerini-Otero R.D. Tessarollo L. Livak F. Manova K. Bonner W.M. Nussenzweig M.C. Nussenzweig A. Science. 2002; 296: 922-927Crossref PubMed Scopus (1158) Google Scholar, 17Bassing C.H. Chua K.F. Sekiguchi J. Suh H. Whitlow S.R. Fleming J.C. Monroe B.C. Ciccone D.N. Yan C. Vlasakova K. Livingston D.M. Ferguson D.O. Scully R. Alt F.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8173-8178Crossref PubMed Scopus (464) Google Scholar). Recently, γ-H2AX has been shown (18Banath J.P. Olive P.L. Cancer Res. 2003; 63: 4347-4350PubMed Google Scholar, 19MacPhail S.H. Banath J.P. Yu T.Y. Chu E.H. Lambur H. Olive P.L. Int. J. Radiat. Biol. 2003; 79: 351-358Crossref PubMed Scopus (290) Google Scholar, 20Rothkamm K. Löbrich M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5057-5062Crossref PubMed Scopus (1366) Google Scholar) to be a reliable marker of the number of DNA DSBs produced in a cell following exposure to DNA damaging agents such as IR. Furthermore, Rothkamm and Löbrich (20Rothkamm K. Löbrich M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5057-5062Crossref PubMed Scopus (1366) Google Scholar) recently demonstrated that this rapid activation of H2AX occurs following even very low doses (1 mGy) of IR given at high dose rates. They also showed that the surviving fraction of cell cultures exposed to very low doses of IR was lower than would be predicted from dose-response curves, suggesting that a threshold of damage is reached before cellular detection mechanisms are efficiently activated. The existence of such a DNA damage threshold is supported by the phenomenon known as low dose hyperradiosensitivity, which is the default response of the majority of cell cultures exposed to either low doses of IR (7Joiner M.C. Marples B. Lambin P. Short S.C. Turesson I. Int. J. Radiat. Oncol. Biol. Phys. 2001; 49: 379-389Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar) or to radiation delivered at a low dose rate (i.e. a reduced amount of radiation dose/unit of time but amounting to the same total dose of radiation) (21Mitchell C.R. Folkard M. Joiner M.C. Radiat. Res. 2002; 158: 311-318Crossref PubMed Scopus (80) Google Scholar). Given these previous studies, we were interested in investigating whether low levels of DNA damage were capable of evading these early cellular DNA damage detection mechanisms, which would predictably lead to enhanced cell death. To achieve continuous low levels of DNA damage, we irradiated cells at low dose rates (LDR) of IR, which produces ∼4–5 DNA DSBs/h. These dose rates are ∼450 times less than the high dose rates (HDRs) typically employed in conducting in vitro and in vivo experimental research (which produces ∼1800 DNA DSBs/h) and are used in the HDR experiments reported here. Although the same total dose of IR is delivered to the cell in both HDR and LDR exposures, LDR radiation exposure is generally accepted as a potentially less damaging modality given that the cells are exposed to a lower radiation dose/unit of time (6Vilenchik M.M. Knudson Jr., A.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5381-5386Crossref PubMed Scopus (128) Google Scholar, 22Ruiz de Almodovar J.M. Bush C. Peacock J.H. Steel G.G. Whitaker S.J. McMillan T.J. Radiat. Res. 1994; 138: S93-S96Crossref PubMed Scopus (39) Google Scholar). In all four of the different human cancer cell lines we used, we observed greater amounts of cell killing (reduced clonogenic capacity) following 2 Gy delivered at a LDR compared with cells given the same total dose at a HDR. Importantly, we demonstrate that the increased cell killing of LDR-treated cells is a consequence of inefficient activation of the DNA damage sensor ATM and its downstream target H2AX, which acts as a cellular signal of DNA damage. Similar results were also seen in normal human primary fibroblasts, and thus inefficient activation may represent a general method by which cellular DNA damage mechanisms can be evaded and potential mutations avoided. These findings give insight into how low levels of DNA damage are detected within the cell and may aid in further understanding such early cellular DNA damage response mechanisms. These findings also have broad implications for carcinogenesis mechanisms and, potentially, for the clinical treatment of solid tumors. Cell Lines and Culture Techniques—All cell lines were obtained from the American Type Culture Collection and maintained as adherent monolayer cultures in appropriate media as outlined in their respective product data sheets. All cultures were grown at 37 °C in a humidified atmosphere of 5% carbon dioxide, fed every 5 days (every 3 days for fibroblasts) with complete medium, and subcultured when confluence was reached. Transfection with siRNA plasmids and subsequent FACS procedures were carried out as described previously (23Collis S.J. Swartz M.J. Nelson W.G. DeWeese T.L. Cancer Res. 2003; 63: 1550-1554PubMed Google Scholar). For all chloroquine experiments, subconfluent cell cultures were pretreated 4 h prior to radiation exposure with 128 μg/ml chloroquine, which was removed prior to trypsinization and replating into 10-cm culture dishes (clonogenic survival assays) or fixing (H2AX assays). Clonogenic Survival Assays—For HDR exposures, cell monolayers were trypsinized, counted, and diluted to the appropriate cell density and loaded into 100-mm culture dishes to yield at least 50 colonies/dish following irradiation. Cells were then irradiated at ∼4500 cGy/h to the desired dose using a Gammacell 40 137Cs irradiator (Atomic Energy of Canada, Ltd., Ottawa). At 7–14 days after irradiation, colonies consisting of at least 50 cells were stained with 50% crystal violet (Sigma) and counted. Cell survival was plotted as a function of dose and fitted using the linear quadratic model, S = e (–αD–βD2), where e is exponent, S is the cell survival, D is the dose of radiation, and α and β are constants. For LDR exposures, subconfluent cell monolayers were irradiated using a custom-built low dose rate irradiator (24Marin L.A. Smith C.E. Langston M.Y. Quashie D. Dillehay L.E. Int. J. Radiat. Oncol. Biol. Phys. 1991; 21: 397-402Abstract Full Text PDF PubMed Scopus (57) Google Scholar) at a LDR of 9.4 cGy/h or a very low dose rate (VLDR) of 2 cGy/h to the desired final dose. For low level radiation exposures, sealed flasks were maintained at 37 °C in the low dose rate irradiator for the desired time. As controls, unirradiated flasks were also sealed and incubated at 37 °C for an equivalent amount of time. Following IR exposures, cells were trypsinized, counted, and diluted to the appropriate cell density into 10-cm culture dishes to give at least 50 colonies/dish following 7–14 days of growth after plating. Surviving fractions were calculated as for HDR experiments and then corrected for cell loss during the protracted radiation exposures as described previously (21Mitchell C.R. Folkard M. Joiner M.C. Radiat. Res. 2002; 158: 311-318Crossref PubMed Scopus (80) Google Scholar). All statistical analyses (two-tailed independent t-tests) were performed using Microsoft Excel. FACS Analysis of γ-H2AX Activation—Subconfluent cell monolayers were irradiated as described above and fixed in 70% cold ethanol for 45 min following the completion of irradiation. Cells were then stained for activated γ-H2AX with an antibody specific for phosphorylated Ser-139 (Upstate Biotechnology, Waltham, MA) using the protocol described by MacPhail et al. (19MacPhail S.H. Banath J.P. Yu T.Y. Chu E.H. Lambur H. Olive P.L. Int. J. Radiat. Biol. 2003; 79: 351-358Crossref PubMed Scopus (290) Google Scholar). Stained cells were analyzed on a LSR flow cytometer (BD Biosciences), and the relative amount of fluorescence in each cell population was determined using the BD Biosciences CellQuest program. Immunoblots—Whole cell extracts were collected at 15 min postradiation exposure and separated on 4–15% acrylamide gels (Bio-Rad) using standard SDS-PAGE techniques. Antibodies for ATM, phospho-specific ATM (Ser-1981), phospho-specific NBS1 (Ser-343), NBS1, and β-actin were obtained from Dr. Michael Kastan (St. Jude Children's Research Hospital, Memphis, TN), Upstate Biotechnology, Novus Biologicals (Littleton, CO), and Sigma, respectively. A total of 10–20 μg of protein extracted from each transfected cell population was loaded onto each gel, electrophoresed at 100 V for 3 h at 4 °C, and then transferred overnight at 50 mA onto polyvinylidene difluoride membranes (Bio-Rad) at 4 °C. Membranes were probed with both primary and secondary antibodies at optimized concentrations, and protein expression was visualized using an ECL kit (Amersham Biosciences). Membranes were reprobed for β-actin to normalize for loading errors. Protein expression was quantified using a Versa-Doc gel documentation system (Bio-Rad). For immunoprecipitation Western blots, lysed whole cell extracts were mixed with 0.5 μg of ATM or NBS1-P antibody (courtesy of Dr. Michael Kastan and Upstate Biotechnology, respectively) for 1 h at 4 °C prior to binding to protein G-linked agarose beads (1 h incubation at 4 °C). Samples were then lysed by boiling for 5 min in the presence of 2× SDS loading buffer. Approximately half of each sample was loaded onto a 4–15% acrylamide gel and probed for phospho-specific ATM as described above. Membranes were then stripped and reprobed for ATM or NBS1 expression as described above. The DU145 cell line used for the majority of these studies has been shown previously (9Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (854) Google Scholar, 10Horejsi Z. Falck J. Bakkenist C.J. Kastan M.B. Lukas J. Bartek J. Oncogene. 2004; 23: 3122-3127Crossref PubMed Scopus (107) Google Scholar, 11Lee J.H. Paull T.T. Science. 2004; 304: 93-96Crossref PubMed Scopus (592) Google Scholar) to express all three components of the MRE11-RAD50-NBS1 complex, 2S. J. Collis, J. M. Schwaninger, A. J. Ntambi, T. W. Keller, W. G. Nelson, L. E. Dillehay, and T. L. DeWeese, unpublished data. which has been shown recently to play a role in the activation of ATM following DNA damage. Low Level Radiation-induced DNA Damage Results in Increased Cell Death—To ascertain whether greater amounts of cell killing could be achieved by low level radiation damage, we exposed a panel of human cancer cell lines to 2 Gy of IR delivered at either a HDR of ∼4500 cGy/h, which is typically used in both clinical practice and experimental research, or a LDR of ∼9.4 cGy/h and assessed clonogenic survival. We observed a general trend for greater cell killing following radiation exposures delivered at the LDR compared with the HDR in all cell lines tested (Fig. 1). This was statistically significant (p < 0.05) in two (RKO and DU145) of the four cell lines tested. The observation of enhanced cell killing following LDR radiation exposures compared with the more common HDR is a well known but poorly understood phenomenon in the radiation biology field and is termed the inverse dose rate effect (see Ref. 25Hall E.J. Radiobiology for the Radiologist. 4th Ed. J. B. Lippincott, Philadelphia1994: 45-73Google Scholar). Early DNA Damage Responses Are Abrogated following Low Level Radiation Exposure—We were interested in determining whether attenuation in the recognition of DNA damage could explain the inverse dose rate phenomenon. We hypothesized that the recently identified rapid activation of ATM and its downstream target H2AX following radiation exposure might be reduced or abrogated in cells receiving LDR radiation compared with those receiving HDR exposures. Using the two cancer cell lines that exhibited a statistically significant reduced survival following LDR exposures (Fig. 1), we showed that the activation of ATM (phosphorylation of Ser-1981) was reduced by approximately 40–50% following LDR radiation exposure compared with cells treated with equivalent doses of radiation delivered at the HDR (Fig. 2A). LDR exposure also led to a reduction (∼25%) in levels of phosphorylated NBS1 (Ser-343) and concurrently in a reduced (∼25%) association of ATM with phosphorylated NBS1 (Fig. 2B). Reduced activation of ATM was also seen in the normal human primary fibroblasts (Fig. 2C), which were used by Bakkenist and Kastan (8Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2750) Google Scholar) to identify the rapid phosphorylation of ATM at Ser-1981 following IR exposure. To determine whether LDR exposure would result in the activation of processes that prevent ATM phosphorylation, we exposed DU145 cells to a 2-Gy LDR followed immediately by a 6-Gy HDR. Cells that were preirradiated with LDR IR were capable of further phosphorylation of ATM at Ser-1981 on exposure to HDR radiation (Fig. 2D). This suggests that the low levels of activated ATM seen following LDR radiation exposure are not the consequence of a mutation to the Ser-1981 residue, the presence of a dysfunctional ATM protein, or the activation of inhibitory processes. The histone variant H2AX is considered a marker of DNA damage detected as the result of insults such as ionizing radiation (18Banath J.P. Olive P.L. Cancer Res. 2003; 63: 4347-4350PubMed Google Scholar, 19MacPhail S.H. Banath J.P. Yu T.Y. Chu E.H. Lambur H. Olive P.L. Int. J. Radiat. Biol. 2003; 79: 351-358Crossref PubMed Scopus (290) Google Scholar, 26Olive P.L. Banath J.P. Int. J. Radiat. Oncol. Biol. Phys. 2004; 58: 331-335Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 27Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4297) Google Scholar, 28Taneja N. Davis M. Choy J.S. Beckett M.A. Singh R. Kron S.J. Weichselbaum R.R. J. Biol. Chem. 2004; 279: 2273-2280Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). It is one of the first downstream targets of ATM, becoming phosphorylated on Ser-139 within 1–3 min following DNA damage (13Burma S. Chen B.P. Murphy M. Kurimasa A. Chen D.J. J. Biol. Chem. 2001; 276: 42462-42467Abstract Full Text Full Text PDF PubMed Scopus (1529) Google Scholar, 29Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-916Crossref PubMed Scopus (2010) Google Scholar), thereby promoting DNA repair processes. We were interested in determining whether the low levels of activated ATM seen following LDR radiation also resulted in a reduction in the phosphorylation and activation of H2AX. We performed FACS analyses to quantify γ-H2AX levels at appropriate times after exposing RKO and DU145 cells to 2 Gy of IR, delivered at either the HDR or the LDR. As with the activation of ATM, γ-H2AX levels were statistically lower in cell cultures exposed at the LDR compared with the HDR-treated cells (Fig. 3). Low Level DNA Damage Results in Reduced Signaling across a Range of IR Doses and Is Ubiquitous throughout the Cell Cycle—Although we observed that the surviving fraction of cells following a 2-Gy dose of IR was lower in cells exposed to LDR compared with HDR exposures (Fig. 1), we were interested to see the effects of LDR exposures over a range of IR doses. Exposure of DU145 cells to increasing doses of IR revealed that clonogenic survival was significantly reduced in cell cultures treated with LDR IR compared with HDR IR at doses between 0 and 6 Gy and that this difference increased dramatically as the total dose increased (Fig. 4A). This increased cell killing is thought to be caused by a decrease in the clonogenic capacity of the cells following LDR IR exposures because of the inefficient activation of DNA repair processes. Another possible cause for increased cell killing could be an accumulation of senescent cells following IR exposure before replating for the clonogenic assays. However, analysis of the senescence marker senescence-associated β-galactosidase following HDR- and LDR-treated cells showed no differences in expression levels compared with unirradiated controls or between HDR- and LDR-treated cells (data not shown). To assess the relationship between the detection of DNA double strand breaks and radiation dose rate, we carried out FACS-based analyses to assess levels of γ-H2AX following HDR and LDR IR exposures across a range of doses. Similar to the data shown in Fig. 3, we observed decreased levels of γ-H2AX in cell cultures irradiated between 0 and 6 Gy with a LDR compared with a HDR. As with clonogenic survival (Fig. 4A), this difference became more apparent as the total dose increased (Fig. 4B). Further analysis of H2AX activation revealed that γ-H2AX levels following a LDR was lower in all phases of the cell cycle compared with cells exposed to equivalent radiation doses delivered at the HDR and thus was not cell cycle-dependent (Fig. 4C). These data are consistent with an overall inefficient detection of DNA DSBs caused by the reduced activation of early cellular DNA damage response mechanisms following LDR, leading ultimately to greater amounts of cell killing per radiation dose compared with HDR-treated cells (Fig. 4D). This increased cell killing is likely caused by a failure to sufficiently activate DNA damage checkpoints and DNA repair mechanisms before cell division takes place. The Role of ATM in Early Cellular Responses to Low Level DNA Damage and Modulation of ATM Activity in LDR- and HDR-treated Cells—To further investigate the role of ATM in cellular responses to low level DNA damage following LDR radiation exposures, we pretreated DU145 cells with chloroquine 4 h prior to the initiation of radiation exposures. Chloroquine has been shown previously (8Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2750) Google Scholar) to activate ATM without the production of DNA DSBs and thus does not affect γ-H2AX levels (data not shown). Cells pretreated with chloroquine prior to LDR radiation exposure have demonstrated both clonogenic survival and γ-H2AX levels on a par with HDR-treated cells (Fig. 5, A and B), that is at levels representing “normal” cellular responses to a 2-Gy IR exposure. In accordance with these data, we noticed that the increased amount of cell killing of DU145 cells seen in LDR-treated cultures mimicked that observed in HDR-treated DU145 cells in which ATM had been knocked down by ∼80–90% following siRNA-mediated inhibition (23Collis S.J. Swartz M.J. Nelson W.G. DeWeese T.L. Cancer Res. 2003; 63: 1550-1554PubMed Google Scholar). We were therefore interested to see whether we could reproduce these data and whether siRNA-mediated inhibition of ATM prior to HDR radiation exposure would yield reduced amounts of γ-H2AX, as observed in LDR-treated cells. To achieve this, we assessed clonogenic survival and γ-H2AX levels following 2-Gy HDR and LDR exposures in DU145 cells, which had been transfected prior to the start of the radiation exposures with an anti-ATM siRNA-encoding plasmid as described previously (23Collis S.J. Swartz M.J. Nelson W.G. DeWeese T.L. Cancer Res. 2003; 63: 1550-1554PubMed Google Scholar). As predicted, cells transfected with the anti-ATM siRNA plasmid showed statistically significant reductions in clonogenic survival and H2AX activation following a 2-Gy HDR exposure compared with untransfected controls (Fig. 6, A and B). Both clonogenic survival and levels of γ-H2AX in HDR-irradiated siRNA-treated cells were similar to those seen in untransfected LDR-treated cells (Fig. 6, A and B), highlighting the importance of ATM in early cellular responses to radiation-mediated damage. The amounts of γ-H2AX observed in ATM siRNA-treated cells were similar to the residual levels reported (13Burma S. Chen B.P. Murphy M. Kurimasa A. Chen D.J. J. Biol. Chem. 2001; 276: 42462-42467Abstract Full Text Full Text PDF PubMed Scopus (1529) Google Scholar, 30Fernandez-Capetillo O. Chen H.T. Celeste A. Ward I. Romanienko P.J. Morales J.C. Naka K. Xia Z. Camerini-Otero R.D. Motoyama N. Carpenter P.B. Bonner W.M. Chen J. Nussenzweig A. Nat. Cell Biol. 2002; 4: 993-997Crossref PubMed Scopus (581) Google Scholar) for irradiated mutant ATM cells and were likely caused by small amounts (∼10%) of ATM still present in the cell following siRNA treatment (23Collis S.J. Swartz M.J. Nelson W.G. DeWeese T.L. Cancer Res. 2003; 63: 1550-1554PubMed Google Scholar). Finally, we were interested in determining whether reducing the dose rate further would result in lower levels of activated ATM and H2AX, culminating in greater amounts of cell killing compared with that seen at 9.4 cGy/h. To study this, we irradiated DU145 cells at a VLDR of 2 cGy/h, producing ∼1 DNA DSB/h, and observed an increased amount of cell killing in these cells compared with HDR-treated cells (Fig. 7A). Importantly, the surviving fraction of VLDR-treated cells was lower than that seen for LDR-treated cells (p = 0.03). Western blot analyses showed drastically reduced activated ATM levels following a 2-Gy VLDR radiation exposure (Fig. 7B). Finally, we carried out FACS analyses for activated H2AX in VLDR-treated cells. Although γ-H2AX levels were much lower in VLDR-treated compared with HDR-treated cells, we did not see any significant reduction in γ-H2AX levels in VLDR-treated compared with LDR-treated cells (Figs. 3 and 7C). This observation may reflect a limitation of the FACS assay used, or it may be that H2AX can be activated in ATM-independent mechanisms, for example, by eith