Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Administration of ON 01210.Na after exposure to ionizing radiation protects bone marrow cells by attenuating DNA damage response

Administration of ON 01210.Na after exposure to ionizing radiation protects bone marrow cells by... Background: Ionizing radiation-induced hematopoietic injury could occur either due to accidental exposure or due to diagnostic and therapeutic interventions. Currently there is no approved drug to mitigate radiation toxicity in hematopoietic cells. This study investigates the potential of ON 01210.Na, a chlorobenzylsulfone derivative, in ameliorating radiation-induced hematopoietic toxicity when administered after exposure to radiation. We also investigate the molecular mechanisms underlying this activity. Methods: Male C3H/HeN mice (n = 5 mice per group; 6-8 weeks old) were exposed to a sub-lethal dose (5 Gy) of g radiation using a Cs source at a dose rate of 0.77 Gy/min. Two doses of ON 01210.Na (500 mg/kg body weight) were administered subcutaneously at 24 h and 36 h after radiation exposure. Mitigation of hematopoietic toxicity by ON 01210.Na was investigated by peripheral white blood cell (WBC) and platelet counts at 3, 7, 21, and 28 d after radiation exposure. Granulocyte macrophage colony forming unit (GM-CFU) assay was done using isolated bone marrow cells, and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) was performed on bone marrow sections at 7 d post-exposure. The DNA damage response pathway involving ataxia telangiectasia mutated (ATM) and p53 was investigated by Western blot in bone marrow cells at 7 d post-exposure. Results: Compared to the vehicle, ON 01210.Na treated mice showed accelerated recovery of peripheral WBC and platelet counts. Post-irradiation treatment of mice with ON 01210.Na also resulted in higher GM-CFU counts. The mitigation effects were accompanied by attenuation of ATM-p53-dependent DNA damage response in the bone marrow cells of ON 01210.Na treated mice. Both phospho-ATM and phospho-p53 were significantly lower in the bone marrow cells of ON 01210.Na treated than in vehicle treated mice. Furthermore, the Bcl2:Bax ratio was higher in the drug treated mice than the vehicle treated groups. Conclusions: ON 01210.Na treatment significantly mitigated the hematopoietic toxicity induced by a sub-lethal radiation dose. Mechanistically, attenuation of ATM-p53 mediated DNA damage response by ON 01210.Na is contributing to the mitigation of radiation-induced hematopoietic toxicity. Keywords: Radiation toxicity, hematopoietic toxicity, ON 01210.Na, Ex-RAD, radiation mitigation, DNA damage. Background area exposed, and the dose received, radiation exposure In addition to therapeutic and diagnostic interventions, in the immediate aftermath could lead to a myriad of exposure to sub-lethal doses of radiation to civilian popu- deleterious effects, including acute radiation syndrome lation may occur during radiological accidents or terror (ARS) [3]. ARS is a well-defined dose-dependent pattern attacks [1,2]. Depending on the duration of exposure, the of organ damage, mainly affecting tissues with rapidly proliferating cells [4,5]. ARS includes hematopoietic syn- drome (1 Gy to 8 Gy), gastrointestinal syndrome (> 8 Gy) * Correspondence: datta_k2003@yahoo.com and cardiovascular/CNS syndrome (> 20 Gy) and follows Department of Biochemistry and Molecular & Cell Biology, Georgetown well defined pathologies [5,6]. Acute radiation exposure University Medical Center, Research Building, Room E518, 3970 Reservoir Rd., NW, Washington, DC 20057-1468, USA in the range between 1 and 8 Gy leads to a drop in Full list of author information is available at the end of the article © 2012 Suman et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Suman et al. Radiation Oncology 2012, 7:6 Page 2 of 9 http://www.ro-journal.com/content/7/1/6 circulating blood cells [7]. At the higher end of this radia- Methods tion dose range, blood cell counts continue to decline Mice and radiation due to the demise of bone marrow stem/progenitor cells Six to eight week old C3H/HeN male mice were pur- leading to lethality. Blood cell counts also decline at chased from Charles River Laboratories (Wilmington, lower doses, until surviving precursor cells proliferate to MA, USA) and were housed in the Georgetown Univer- restore homeostasis [5]. During this period of declining sity’s (GU) AAALACI (Association for Assessment and bone marrow cells, individuals are at an increased risk of Accreditation of Laboratory and Animal Care Interna- infection and hemorrhage [8]. A number of plant/herbal tional) accredited facility. All the animal procedures were products, like Hippophae rhamnoides and Mentha arven- performed according to protocols approved by the Geor- sis and chemical entities, like amifostin and vitamin E getown University Animal Care and Use Committee related products, have been shown to modulate radiation (GUACUC), and terminal anesthesia with CO was used toxicity. However, very few have been shown to have for collection of tissue and blood samples from these radiomitigator properties and fewer have been shown to mice. For irradiation, mice were placed in a circular pie improve hematological parameters [9,10]. Therefore, shaped, well-ventilated plastic mouse holder, and a Cs therapeutic agents mitigating radiation-induced decrease source (dose rate 0.77 Gy/min) was used as g radiation in bone marrow cells could play an important role not source. Effects in irradiated+ON 01210.Na treated groups only in emergency situations but also in minimizing the were compared to those in the radiation only and the radiation toxicity to bone marrow during radiation ther- radiation+vehicle treated groups and are shown in the apy or diagnostic procedures. results. The unirradiated control, drug only group, and The tumor suppressor p53 plays a vital role in radia- vehicle only group acted as additional control groups. All tion-induced DNA damage response (DDR) and cell irradiation groups were exposed to 5 Gy of g radiation, death [11]. The DDR starts with the sensing of the control groups were sham irradiated and irradiation and DNA damage, which is then conveyed to effectors via experiments were repeated two times. signal transducers. The ataxia telangiectasia mutated (ATM) protein is a well-established sensor of DNA ON 01210.Na formulation and administration damage and gets activated by autophosphorylation on ON 01210.Na (Ex-RAD), a chlorobenzylsulfone deriva- sensing DNA damage [12]. Activated ATM in turn tive developed by Onconova Theraputics (Newtown, PA, phosphorylates its downstream target p53, which is con- USA) as a radioprotector and mitigator, was described sidered a signal transducer of DDR, to effectors like earlier [18-20]. ON 01210.Na (500 mg/kg) was adminis- Bcl2-associated X protein (Bax),p21,and B-cell lym- tered subcutaneously (SC), using 1 mL sterile syringe phoma 2 (Bcl2), to either induce cell cycle arrest for with a 25G needle at 24 and 36 h after 5 Gy radiation repair or initiate programmed cell death if the damage exposure. is beyond repair. It is important to note here that phos- phorylation of p53 leads not only to its activation but Peripheral white blood cell (WBC) and platelet counts also to its stabilization by minimizing its interaction Blood samples (n = 5 mice per group per time point) with murine double minute 2 (MDM2), a negative regu- were collected by cardiac puncture in ethylenediamine- lator of p53 [13]. DNA damage-induced activation of tetra acetic acid (EDTA) tubes after terminal CO p53 is known to regulate the transcription of two major anesthesia at 3, 7, 21, and 28 d after radiation exposure cell death regulators: anti-apoptotic Bcl2 and pro-apop- and subjected to complete blood count. White blood totic Bax via down-regulation and trans-activation, cell (WBC) counts, neutrophil counts, and monocyte respectively [11,14-16]. Bcl2 inhibits apoptosis by antag- counts are presented as absolute count, and platelet onizing Bax oligomerization, which is indispensible for counts are presented as percent of normal count (1.2 × Bax-mediated apoptosis [17]. In previous in vitro studies 10 per μL of blood, ± 16.1 standard error of mean). ON 01210.Na has been reported to reduce the level of p53 and its target p21 and Bax, when given prophylacti- Bone marrow histopathology cally before radiation exposure [18]. Here we report that Bone marrow (n = 5 mice per group) was used for his- administration of 2 doses of ON 01210.Na at 24 and 36 topathologic analysis. Femurs were surgically removed h after radiation exposure augmented hematopoietic cell from each mouse at 7 d after 5 Gy radiation exposure, survival through reduction in DNA damage and damage fixed in 10% buffered formalin for 48 h, decalcified, par- response. Our results also indicate that increased clono- affin embedded, and 5 μm thick sections were stained genic survival of bone marrow cells observed in ON with hematoxylin and eosin (H&E) using standard pro- 01210.Na treated mice was due to attenuation of p53- cedures. Unstained sections were used for terminal mediated apoptotic response. deoxynucleotidyl transferase dUTP nick end-labeling Suman et al. Radiation Oncology 2012, 7:6 Page 3 of 9 http://www.ro-journal.com/content/7/1/6 (TUNEL) assay. Bone marrow cellularity in H&E stained Western blot sections was semiquantitatively scored (in 5 mice from Bone marrow cells were isolated, as per the protocol each group) by counting nucleated cells in a 16-square described in the previous section, and cells from 5 mice (1 cm each) grid in randomly chosen 5 high-power were pooled for the Western blot analysis. Cells were (40×) microscopic fields for each section (3 sections lysed in ice-cold protein extraction buffer (0.5% Sodium deoxycholate, 0.5% NP-40, 10 mM EDTA in PBS) con- from each mouse) as described earlier [21]. While plot- taining protease inhibitor cocktail (Sigma, St. Louis, ting the results, control section cellularity was consid- MO, USA). The homogenate was centrifuged at 12000 ered 100 percent. Megakaryocytes were also evaluated xg at 4°C for 10 min and supernatant was collected. The by a semi-quantitative analysis of three adjacent high- power (40×) microscopic fields (n = 5 and 3 sections Bradford protein assay was used to quantify the protein from each mouse were scored). concentration in respective samples. Equal amounts of protein samples were mixed with the appropriate Granulocyte Macrophage-Colony Forming Unit (GM-CFU) volume of Laemmli’s sample buffer (6× solution: 375 assay mM Tris-HCl (pH = 6.8), 6% sodium dodecyl sulphate Mice (n = 5 mice per group) were euthanized at 7 d after (SDS), 48% Glycerol, 9% b-Mercapto-ethanol and 0.03% radiation exposure. Under aseptic conditions femurs were bromophenol blue), heated at 95°C for 5 min, and were excised, ends opened, and bone marrow cells collected by resolved on SDS- polyacrylamide gel electrophoresis flushing. For flushing, Iscove’s modified Dulbecco’smed- (PAGE). Proteins were transferred onto a polyvinylidine ium (IMDM) (StemCell Technologies, Vancouver, BC, fluoride (PVDF) membrane, blocked with 5% milk in Canada) supplemented with 5% fetal bovine serum (FBS) tris-buffered saline with 0.1% Tween (TBST), and incu- was used with sterile syringes and 25G needles. Flushed bated with appropriate primary antibody (p-ATM bone marrow from each femur was pipetted up and down (1:100, Sc-47739, Clone-10H11-E12); p53 (1:500, Sc-98, to prepare a single cell suspension and passed through 70 Clone-1801); p-p53 (1:200, Sc-18078, Clone-mSer20); micron nylon meshes (BD Biosciences, Sparks, MD, USA). Bcl-2 (1:250, Sc-7382, Clone-C-2); Bax (1:250, Sc-7480, Isolated cells were counted using a cell counter (Beckman Clone-B-9) and b-actin (1:2000, Sc-47778, Clone-C4) from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Coulter, Brea, CA, USA), and from each femur 2.5 × 10 Western blot membranes were developed with horserad- cells/mL were plated in triplicate in ultra-low attachment ish peroxidase (HRP) conjugated secondary antibody 60 mm dishes (Corning, NY, USA) using methocult and enhanced chemiluminescence (ECL) detection sys- (M3534, StemCell Technologies) medium supplemented tem (Cat# 34080, Thermo Fisher Scientific, Rockford, with 10 ng/mL granulocyte macrophage colony stimulat- IL, USA). Images were captured on photographic films ing factor (GM-CSF) (StemCell Technologies). The plates were incubated at 37°C in 5% CO and ≥ 95% humidity and scanned. Results from a representative experiment for 7 d, and colonies were counted using a dissecting are displayed. Scanned images of the Western blots microscope (Leica, Wetzlar, Germany). were quantified by ImageJ v4.44 software using the pre- viously described protocol [22]. Briefly, scanned images Apoptosis detection in bone marrow and spleen were opened in ImageJ, and bands were selected using DNA damage and cell death in bone marrow and spleen the rectangular selection tool to generate band profile cells (n = 5 mice per group) were detected, using the plots. Normalized band intensity was generated using b- ApopTag Plus Peroxidase in situ apoptosis detection kit actin band intensity in respective columns. (S7101, Millipore, Billerica, MA, USA) according to manu- facturer’s instruction. Briefly, tissue sections were deparaf- Statistical analysis finized and pretreated with Proteinase-K solution (20 μg/ Statistical analysis to find significance between two mL) at room temperature for 15 min. The endogenous groups was performed using two tailed paired Stu- peroxidase activity was quenched using 3% hydrogen per- dent’s t-test, and p < 0.05 was taken as statistically sig- oxide in phosphate buffered saline (PBS) at room tempera- nificant. Error bars represent ± standard error of mean ture. Following incubation with terminal deoxynucleotidal (SEM). transferase (TdT) at 37°C for 1 h, the apoptotic cells were visualized under a bright field microscope by a diamino- Results benzidine (DAB) based detection system supplied with the Accelerated recovery of peripheral blood cell count and increased clonogenic survival of bone marrow kit, and sections were counterstained using methyl green progenitors in ON 01210.Na treated group (Trevigen, Gaithersburg, MD, USA) nuclear stain. TUNEL The WBC count at 3 d showed uniform reduction in all positive cells were counted in 5 randomly chosen high the irradiated groups. However, at 7 and 21 d signifi- power fields (40×), and counts from 3 sections from each cantly higher counts were observed in mice treated with mouse were used for statistical analysis. Suman et al. Radiation Oncology 2012, 7:6 Page 4 of 9 http://www.ro-journal.com/content/7/1/6 ON 01210.Na (compared to respective radiation+vehicle 01210.Na than in the vehicle treated mice (Figure 2A, B, groups p < 0.04 for 7 d and p < 0.01 for 21 d) (Figure and 2C). When given 24 h and 36 h after radiation 1A). A significant difference in absolute neutrophil exposure, the ON 01210.Na treated group also showed count (ANC) was also observed between the ON 01210. significantly enhanced clonogenic survival of bone mar- Na treated and the vehicle treated groups at 7 and 21 d row cells (p < 0.0005 compared to the radiation+vehicle (p < 0.05 for both of the time points) (Figure 1B). When group) (Figure 3). absolute monocyte counts (AMC) were compared between the ON 01210.Na and the vehicle treated ON 01210.Na treatment showed reduction in apoptotic groups, a significant difference was observed at 21 d (p cells in bone marrow and spleen < 0.05) (Figure 1C). In contrast to WBC count, platelet TUNEL assay on bone marrow and spleen sections from count was decreased at 7 d and recovery was observed the ON 01210.Na treated groups showed fewer apopto- at 21 d. However, at 7 d the count was significantly tic cells than the vehicle treated group (Figure 4A and higher in the ON 01210.Na treated group than the vehi- 4C). Quantification of TUNEL positive cells indicating cle treated group (p < 0.05), and at 21 d the ON 01210. apoptosis showed significantly lower counts in the ON Na treated group showed greater recovery than the vehi- 01210.Na treated group than the vehicle treated group cle group (p < 0.05) (Figure 1D). Furthermore, total cel- in both the bone marrow and spleen samples (Figure 4B lularity, as well as megakaryocyte counts in bone and 4D; p < 0.01 for bone marrow and p < 0.05 for marrow sections, was significantly higher in the ON spleen compared to radiation+vehicle groups). Figure 1 Post-exposure peripheral blood cell count with or without ON 01210.Na treatment.A)AbsoluteperipheralWBC counts. B) Absolute neutrophil count (ANC). C) Absolute monocyte counts (AMC). D) Platelet count expressed as percent of normal count. R+V: 5 Gy radiation+vehicle; R: 5 Gy radiation; R+D: 5 Gy radiation+drug (ON 01210.Na). *p < 0.05 compared to radiation+vehicle. Suman et al. Radiation Oncology 2012, 7:6 Page 5 of 9 http://www.ro-journal.com/content/7/1/6 Figure 2 Quantification of bone marrow cellularity in H&E stained sections at 7 d post-radiation. A) Photomicrograph showing H&E stained sections of bone marrow at 40× magnification. B) Relative quantification of total bone marrow cellularity. *p < 0.004 compared to radiation+vehicle (R+V). C) Megakaryocyte counts in H&E stained bone marrow sections. *p < 0.02 compared to radiation+vehicle. Control: no radiation, ON 01210.Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation+vehicle; R+D: 5 Gy radiation+drug (ON 01210.Na). Attenuated DNA damage response in bone marrow cells of ON 01210.Na treated mice The DNA damage response pathway involving ATM and p53 was assessed by Western blot analysis. Com- pared to vehicle treated groups, we observed a signifi- cant reduction of total p53, phospho-ATM, and phospho-p53 levels in ON 01210.Na treated bone mar- row cells at 7 d post-radiation (Figure 5A and 5D). Decrease of the p53 level in ON 01210.Na treated mice was associated with increasein anti-apoptoticBcl2 and decrease in pro-apoptotic Bax (Figure 5A). Quantifica- tion of Western blots showed significant decrease in total p53 and Bax but increase in Bcl2 (Figure 5B; p < 0.002 for p53, < 0.001 for Bax, and < 0.007 for Bcl2 compared to radiation+vehicle groups). Interestingly, the Bcl2:Bax ratio was markedly greater in the ON 01210. Na treated than the vehicle treated mice (Figure 5C). Furthermore, quantification of phospho-ATM and phos- Figure 3 Granulocyte-macrophage colony forming units (GM- CFU) assay of bone marrow cells at 7 d post-radiation. *p < 0.005 pho-p53 showed significant decrease in the ON 01210. compared to radiation+vehicle (R+V). Control: no radiation, ON 01210. Na treated mice (Figure 5E; p < 0.0007 for p-ATM and Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation+vehicle; R+D: 5 Gy < 0.0002 for the p-p53 compared to the radiation+vehi- radiation+drug (ON 01210.Na). cle treated mice). Suman et al. Radiation Oncology 2012, 7:6 Page 6 of 9 http://www.ro-journal.com/content/7/1/6 Figure 4 TUNEL staining of bone marrow and spleen sections at 7 d post-radiation. A) Photomicrograph showing TUNEL staining of bone marrow sections at 20× magnification. B) Quantification of TUNEL positive cells in bone marrow sections. *p < 0.01 compared to radiation +vehicle (R+V). C) Photomicrograph showing TUNEL staining of spleen sections at 20× magnification. D) Quantification of TUNEL positive cells in spleen sections. *p < 0.05 compared to radiation+vehicle. Control: no radiation, ON 01210.Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation +vehicle; R+D: 5 Gy radiation+drug (ON 01210.Na). radiation-induced DDR and apoptosis. Overall, ON Discussion 01210.Na was able to accelerate the post-exposure Terrorist threats and recent accidents in nuclear instal- hematopoietic recovery process as evidenced by a lations emphasize the need to develop strategies to miti- greater increase in peripheral WBC and platelet counts, gate radiation toxicity [23]. Mitigation of radiation higher bone marrow cellularity, and higher number of toxicity also has implications for radiation therapy [3]. GM-CFUs in the drug treated groups than in the vehicle In this initial investigation we have shown that two groups. doses of ON 01210.Na, administered 24 h and 36 h Most of the post-radiation hematopoietic morbidity after radiation exposure, could significantly mitigate and mortality is attributed to infection and hemorrhage radiation-induced hematopoietic toxicity. Mechanisti- due to leukopenia and thrombocytopenia resulting from cally, ON 01210.Na treatment resulted in diminished Suman et al. Radiation Oncology 2012, 7:6 Page 7 of 9 http://www.ro-journal.com/content/7/1/6 Figure 5 Western blot analysis of DNA damage response in bone marrow cells at 7 d post-radiation. A) Scanned images showing Western blot of p53, Bcl2, and Bax. B) Densitometric quantification of p53, Bcl2, and Bax. *p < 0.002 for p53, < 0.001 for Bax, and < 0.007 for Bcl2 compared to respective radiation+vehicle groups C) Bcl2/Bax ratio. *p < 0.005 compared to radiation+vehicle. D) Western blot images of phospho-ATM and phospho-p53. E) Densitometric quantification of phospho-ATM and phospho-p53. *p < 0.0007 for p-ATM and < 0.0002 for p- p53 compared to radiation+vehicle treated mice. R+V: 5 Gy radiation+vehicle; R: 5 Gy radiation; R+D: 5 Gy radiation+drug (ON 01210.Na). loss of bone marrow cells. Our time course study ranging Bone marrow progenitor cells with an average lower radiosensitivity than the more primitive hematopoietic from 3 to 28 d demonstrated faster recovery of radiation- induced WBC cell loss in the ON 01210.Na than in the precursor cells are a rapidly cycling cell population and vehicle treated groups. Higher numbers of peripheral are capable of forming colonies in culture (reviewed by WBC counts in the ON 01210.Na treated animals led us [24]). Our results from the GM-CFU assay support the to believe that the drug is aiding in quicker regeneration notion that ON 01210.Na is enhancing the recovery and and turnover of bone marrow cells than the vehicle regeneration of the progenitor cells, which survived groups through proliferation of surviving precursor cells. initial radiation-induced damage. Mechanistic investiga- Importantly, at 7 d and at 21 d in the ON 01210.Na tion further supports our hypothesis that ON 01210.Na groups, the absolute neutrophil counts were significantly is accelerating recovery/regeneration of bone marrow higher which would equip these animals to resist any cells due to attenuation of DDR. The ON 01210.Na, potential post-radiation infection better than the vehicle through lowering of phospho-ATM and p53, along with treated groups. Furthermore, the ON 01210.Na treated a higher Bcl2/Bax ratio, could be aiding in the prolifera- groups at both these time points showed greater percent tion of the surviving cells. Our results showing diminu- of platelets than the vehicle groups, which would mini- tion of p53 level, along with increase in Bcl2 and mize not only post-radiation infection but would also decrease in Bax level, demonstrate the mechanism of reduce the risk of hemorrhagic events. ON 01210.Na mediated radiation mitigation and is Suman et al. Radiation Oncology 2012, 7:6 Page 8 of 9 http://www.ro-journal.com/content/7/1/6 NW, Washington, DC 20057-1468, USA. Onconova Therapeutics Inc., important for the management of radiation exposure to Newton, PA 18940, USA. healthy tissues. Bone marrow and spleen are important in maintaining Authors’ contributions SS: executed experiments and analyzed and organized results; KD: planned peripheral blood cell pool and proper functioning of the and executed experiments, analyzed results, and prepared the manuscript; immune system. Thus, radiation damage to these vital AJF: participated in preparing the manuscript; MM: planned experiments and organs can affect hematopoiesis as well as immune defense, participated in preparing the manuscript. All authors read and approved this manuscript. which are critical determinant of post-exposure morbidity and mortality [25,26]. Although chemical entities and her- Competing interests notification bal preparations have been investigated for their radiopro- Dr. Manoj Maniar is employed at Onconova Therapeutics, Inc. No other authors have financial obligations to Onconova Therapeutics, Inc. tection and radiomitigation properties, we are yet to have an approved pharmacological agent to counter radiation Received: 24 October 2011 Accepted: 20 January 2012 damage to critical tissues, like bone marrow, and to reduce Published: 20 January 2012 subsequent morbidity and mortality [27]. Compounds like References statins and palifermin has been shown to mitigate radiation 1. Nenot JC: Radiation accidents over the last 60 years. J Radiol Prot 2009, mucositis and enteropathy, and herbal preparations from 29:301-320. Hippophae rhamnoides and Mentha arvensis have shown 2. Niazi AK, Niazi SK: Endocrine effects of Fukushima: Radiation-induced endocrinopathy. Indian J Endocrinol Metab 2011, 15:91-95. significant hematopoietic protection [3,9,10]. However, 3. Citrin D, Cotrim AP, Hyodo F, Baum BJ, Krishna MC, Mitchell JB: there are presently no approved agents available to mitigate Radioprotectors and mitigators of radiation-induced normal tissue radiation-induced hematopoietic toxicity. In a post-expo- injury. Oncologist 2010, 15:360-371. 4. Andrews GA: Radiation accidents and their management. Radiat Res Suppl sure scenario, administration of ON 01210.Na was able to 1967, 7:390-397. reduce the number of apoptotic cells (TUNEL positive) in 5. Hall EJ, Giaccia AJ: Philadelphia: Lippincott Williams & Wilkins; 2006. both bone marrow and spleen, indicating lower cell death 6. Moulder JE, Cohen EP: Future strategies for mitigation and treatment of chronic radiation-induced normal tissue injury. Semin Radiat Oncol 2007, than that with vehicle treatment. Our Western blot results 17:141-148. suggest that lessening of cell death in bone marrow and 7. Fliedner TM, Friesecke I, Graessle D, Paulsen C, Weiss M: Hematopoietic cell spleen has to be p53 mediated and is dependent on the renewal as the limiting factor in low-level radiation exposure: diagnostic implications and therapeutic options. Mil Med 2002, 167:46-48. activated ATM. Although we did not observe any altera- 8. Williams JP, McBride WH: After the bomb drops: A new look at radiation- tions in total ATM levels (data not shown), we did find sig- induced multiple organ dysfunction syndrome (MODS). Int J Radiat Biol 2011. nificantly lower levels of activated phospho-ATM and, 9. Maurya DK, Devasagayam TP, Nair CK: Some novel approaches for radioprotection and the beneficial effect of natural products. Indian J Exp consequently, significantly reduced phospho-p53 in the Biol 2006, 44:93-114. ON 01210.Na treated bone marrow cells. Stability of p53 is 10. Nair CK, Parida DK, Nomura T: Radioprotectors in radiotherapy. J Radiat dependent on its phosphorylation status [12]. We believe Res (Tokyo) 2001, 42:21-37. 11. Zhou BB, Elledge SJ: The DNA damage response: putting checkpoints in that decreased total p53 in bone marrow cells is due to perspective. Nature 2000, 408:433-439. reduced phosphorylation of p53, allowing enhanced inter- 12. Tanaka T, Huang X, Jorgensen E, Gietl D, Traganos F, Darzynkiewicz Z, action with MDM2, leading to increased ubiquitinylation Albino AP: ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke. BMC Cell Biol 2007, 8:26. and subsequent degradation. Pro-apoptotic Bax and anti- 13. Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O, Moas M, apoptotic Bcl2 are under the regulation of tumor suppres- Buschmann T, Ronai Z, Shiloh Y, Kastan MB, Katzir E, Oren M: ATM- sor p53. Antagonizing Bax and enhancing Bcl2 has been dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev 2001, 15:1067-1077. shown to confer resistance on cells to ionizing radiation 14. Bache M, Pigorsch S, Dunst J, Wurl P, Meye A, Bartel F, Schmidt H, Rath FW, [28,29]. Reduction in activated ATM and p53 levels leading Taubert H: Loss of G2/M arrest correlates with radiosensitization in two to a decrease in DDR signal, is playing a role in enhanced human sarcoma cell lines with mutant p53. Int J Cancer 2001, 96:110-117. 15. Mazzatti DJ, Lee YJ, Helt CE, O’Reilly MA: p53 modulates radiation hematopoietic recovery in ON 01210.Na treated mice. sensitivity independent of p21 transcriptional activation. Am J Clin Oncol 2005, 28:43-50. Conclusions 16. Zhan Q, Kontny U, Iglesias M, Alamo IJ, Yu K, Hollander MC, Woodworth CD, Fornace AJJ: Inhibitory effect of Bcl-2 on p53-mediated Taken together, we conclude that the post-exposure transactivation following genotoxic stress. Oncogene 1999, 18:297-304. administration of ON 01210.Na enhances the recovery 17. Dlugosz PJ, Billen LP, Annis MG, Zhu W, Zhang Z, Lin J, Leber B, of hematopoietic cells by employing a mechanism that Andrews DW: Bcl-2 changes conformation to inhibit Bax oligomerization. EMBO J 2006, 25:2287-2296. not only attenuates DNA damage sensing and damage 18. Ghosh SP, Perkins MW, Hieber K, Kulkarni S, Kao TC, Reddy EP, Reddy MV, signal transduction but also alters levels of effectors like Maniar M, Seed T, Kumar KS: Radiation protection by a new chemical Bax and Bcl2. entity Ex-Rad: efficacy and mechanisms. Radiat Res 2009, 171:173-179. 19. Chun AW, Cosenza SC, Taft DR, Maniar M: Preclinical pharmacokinetics and in vitro activity of ON 01910.Na, a novel anti-cancer agent. Cancer Chemother Pharmacol 2009, 65:177-186. Author details 20. Chun AW, Freshwater RE, Taft DR, Gillum AM, Maniar M: Effects of Department of Biochemistry and Molecular & Cell Biology, Georgetown formulation and route of administration on the systemic availability of University Medical Center, Research Building, Room E518, 3970 Reservoir Rd., Suman et al. Radiation Oncology 2012, 7:6 Page 9 of 9 http://www.ro-journal.com/content/7/1/6 Ex-RAD((R)), a new radioprotectant, in preclinical species. Biopharm Drug Dispos 2011, 32:99-111. 21. Singh VK, Parekh VI, Brown DS, Kao TC, Mog SR: Tocopherol succinate: modulation of antioxidant enzymes and hematopoietic recovery. Int J Radiat Oncol Biol Phys 2011, 79:571-578. 22. Western blot analysis protocol. [http://rsb.info.nih.gov/ij/docs/menus/ analyze.html#gels]. 23. Pellmar TC, Rockwell S: Priority list of research areas for radiological nuclear threat countermeasures. Radiat Res 2005, 163:115-123. 24. Yang FT, Lord BI, Hendry JH: Gamma irradiation of the fetus damages the developing hemopoietic microenvironment rather than the hemopoietic progenitor cells. Radiat Res 1995, 141:309-313. 25. Gridley DS, Pecaut MJ: Whole-body irradiation and long-term modification of bone marrow-derived cell populations by low- and high- LET radiation. In Vivo 2006, 20:781-789. 26. Gridley DS, Pecaut MJ, Miller GM, Moyers MF, Nelson GA: Dose and dose rate effects of whole-body gamma-irradiation: II. Hematological variables and cytokines. In Vivo 2001, 15:209-216. 27. Weiss JF, Landauer MR: History and development of radiation-protective agents. Int J Radiat Biol 2009, 85:539-573. 28. Domen J, Gandy KL, Weissman IL: Systemic overexpression of BCL-2 in the hematopoietic system protects transgenic mice from the consequences of lethal irradiation. Blood 1998, 91:2272-2282. 29. Xiang J, Chao DT, Korsmeyer SJ: BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci USA 1996, 93:14559-14563. doi:10.1186/1748-717X-7-6 Cite this article as: Suman et al.: Administration of ON 01210.Na after exposure to ionizing radiation protects bone marrow cells by attenuating DNA damage response. Radiation Oncology 2012 7:6. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Oncology Springer Journals

Administration of ON 01210.Na after exposure to ionizing radiation protects bone marrow cells by attenuating DNA damage response

Loading next page...
 
/lp/springer-journals/administration-of-on-01210-na-after-exposure-to-ionizing-radiation-qvMYZMtrT3

References (56)

Publisher
Springer Journals
Copyright
Copyright © 2012 by Suman et al; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Oncology; Radiotherapy
eISSN
1748-717X
DOI
10.1186/1748-717X-7-6
pmid
22264334
Publisher site
See Article on Publisher Site

Abstract

Background: Ionizing radiation-induced hematopoietic injury could occur either due to accidental exposure or due to diagnostic and therapeutic interventions. Currently there is no approved drug to mitigate radiation toxicity in hematopoietic cells. This study investigates the potential of ON 01210.Na, a chlorobenzylsulfone derivative, in ameliorating radiation-induced hematopoietic toxicity when administered after exposure to radiation. We also investigate the molecular mechanisms underlying this activity. Methods: Male C3H/HeN mice (n = 5 mice per group; 6-8 weeks old) were exposed to a sub-lethal dose (5 Gy) of g radiation using a Cs source at a dose rate of 0.77 Gy/min. Two doses of ON 01210.Na (500 mg/kg body weight) were administered subcutaneously at 24 h and 36 h after radiation exposure. Mitigation of hematopoietic toxicity by ON 01210.Na was investigated by peripheral white blood cell (WBC) and platelet counts at 3, 7, 21, and 28 d after radiation exposure. Granulocyte macrophage colony forming unit (GM-CFU) assay was done using isolated bone marrow cells, and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) was performed on bone marrow sections at 7 d post-exposure. The DNA damage response pathway involving ataxia telangiectasia mutated (ATM) and p53 was investigated by Western blot in bone marrow cells at 7 d post-exposure. Results: Compared to the vehicle, ON 01210.Na treated mice showed accelerated recovery of peripheral WBC and platelet counts. Post-irradiation treatment of mice with ON 01210.Na also resulted in higher GM-CFU counts. The mitigation effects were accompanied by attenuation of ATM-p53-dependent DNA damage response in the bone marrow cells of ON 01210.Na treated mice. Both phospho-ATM and phospho-p53 were significantly lower in the bone marrow cells of ON 01210.Na treated than in vehicle treated mice. Furthermore, the Bcl2:Bax ratio was higher in the drug treated mice than the vehicle treated groups. Conclusions: ON 01210.Na treatment significantly mitigated the hematopoietic toxicity induced by a sub-lethal radiation dose. Mechanistically, attenuation of ATM-p53 mediated DNA damage response by ON 01210.Na is contributing to the mitigation of radiation-induced hematopoietic toxicity. Keywords: Radiation toxicity, hematopoietic toxicity, ON 01210.Na, Ex-RAD, radiation mitigation, DNA damage. Background area exposed, and the dose received, radiation exposure In addition to therapeutic and diagnostic interventions, in the immediate aftermath could lead to a myriad of exposure to sub-lethal doses of radiation to civilian popu- deleterious effects, including acute radiation syndrome lation may occur during radiological accidents or terror (ARS) [3]. ARS is a well-defined dose-dependent pattern attacks [1,2]. Depending on the duration of exposure, the of organ damage, mainly affecting tissues with rapidly proliferating cells [4,5]. ARS includes hematopoietic syn- drome (1 Gy to 8 Gy), gastrointestinal syndrome (> 8 Gy) * Correspondence: datta_k2003@yahoo.com and cardiovascular/CNS syndrome (> 20 Gy) and follows Department of Biochemistry and Molecular & Cell Biology, Georgetown well defined pathologies [5,6]. Acute radiation exposure University Medical Center, Research Building, Room E518, 3970 Reservoir Rd., NW, Washington, DC 20057-1468, USA in the range between 1 and 8 Gy leads to a drop in Full list of author information is available at the end of the article © 2012 Suman et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Suman et al. Radiation Oncology 2012, 7:6 Page 2 of 9 http://www.ro-journal.com/content/7/1/6 circulating blood cells [7]. At the higher end of this radia- Methods tion dose range, blood cell counts continue to decline Mice and radiation due to the demise of bone marrow stem/progenitor cells Six to eight week old C3H/HeN male mice were pur- leading to lethality. Blood cell counts also decline at chased from Charles River Laboratories (Wilmington, lower doses, until surviving precursor cells proliferate to MA, USA) and were housed in the Georgetown Univer- restore homeostasis [5]. During this period of declining sity’s (GU) AAALACI (Association for Assessment and bone marrow cells, individuals are at an increased risk of Accreditation of Laboratory and Animal Care Interna- infection and hemorrhage [8]. A number of plant/herbal tional) accredited facility. All the animal procedures were products, like Hippophae rhamnoides and Mentha arven- performed according to protocols approved by the Geor- sis and chemical entities, like amifostin and vitamin E getown University Animal Care and Use Committee related products, have been shown to modulate radiation (GUACUC), and terminal anesthesia with CO was used toxicity. However, very few have been shown to have for collection of tissue and blood samples from these radiomitigator properties and fewer have been shown to mice. For irradiation, mice were placed in a circular pie improve hematological parameters [9,10]. Therefore, shaped, well-ventilated plastic mouse holder, and a Cs therapeutic agents mitigating radiation-induced decrease source (dose rate 0.77 Gy/min) was used as g radiation in bone marrow cells could play an important role not source. Effects in irradiated+ON 01210.Na treated groups only in emergency situations but also in minimizing the were compared to those in the radiation only and the radiation toxicity to bone marrow during radiation ther- radiation+vehicle treated groups and are shown in the apy or diagnostic procedures. results. The unirradiated control, drug only group, and The tumor suppressor p53 plays a vital role in radia- vehicle only group acted as additional control groups. All tion-induced DNA damage response (DDR) and cell irradiation groups were exposed to 5 Gy of g radiation, death [11]. The DDR starts with the sensing of the control groups were sham irradiated and irradiation and DNA damage, which is then conveyed to effectors via experiments were repeated two times. signal transducers. The ataxia telangiectasia mutated (ATM) protein is a well-established sensor of DNA ON 01210.Na formulation and administration damage and gets activated by autophosphorylation on ON 01210.Na (Ex-RAD), a chlorobenzylsulfone deriva- sensing DNA damage [12]. Activated ATM in turn tive developed by Onconova Theraputics (Newtown, PA, phosphorylates its downstream target p53, which is con- USA) as a radioprotector and mitigator, was described sidered a signal transducer of DDR, to effectors like earlier [18-20]. ON 01210.Na (500 mg/kg) was adminis- Bcl2-associated X protein (Bax),p21,and B-cell lym- tered subcutaneously (SC), using 1 mL sterile syringe phoma 2 (Bcl2), to either induce cell cycle arrest for with a 25G needle at 24 and 36 h after 5 Gy radiation repair or initiate programmed cell death if the damage exposure. is beyond repair. It is important to note here that phos- phorylation of p53 leads not only to its activation but Peripheral white blood cell (WBC) and platelet counts also to its stabilization by minimizing its interaction Blood samples (n = 5 mice per group per time point) with murine double minute 2 (MDM2), a negative regu- were collected by cardiac puncture in ethylenediamine- lator of p53 [13]. DNA damage-induced activation of tetra acetic acid (EDTA) tubes after terminal CO p53 is known to regulate the transcription of two major anesthesia at 3, 7, 21, and 28 d after radiation exposure cell death regulators: anti-apoptotic Bcl2 and pro-apop- and subjected to complete blood count. White blood totic Bax via down-regulation and trans-activation, cell (WBC) counts, neutrophil counts, and monocyte respectively [11,14-16]. Bcl2 inhibits apoptosis by antag- counts are presented as absolute count, and platelet onizing Bax oligomerization, which is indispensible for counts are presented as percent of normal count (1.2 × Bax-mediated apoptosis [17]. In previous in vitro studies 10 per μL of blood, ± 16.1 standard error of mean). ON 01210.Na has been reported to reduce the level of p53 and its target p21 and Bax, when given prophylacti- Bone marrow histopathology cally before radiation exposure [18]. Here we report that Bone marrow (n = 5 mice per group) was used for his- administration of 2 doses of ON 01210.Na at 24 and 36 topathologic analysis. Femurs were surgically removed h after radiation exposure augmented hematopoietic cell from each mouse at 7 d after 5 Gy radiation exposure, survival through reduction in DNA damage and damage fixed in 10% buffered formalin for 48 h, decalcified, par- response. Our results also indicate that increased clono- affin embedded, and 5 μm thick sections were stained genic survival of bone marrow cells observed in ON with hematoxylin and eosin (H&E) using standard pro- 01210.Na treated mice was due to attenuation of p53- cedures. Unstained sections were used for terminal mediated apoptotic response. deoxynucleotidyl transferase dUTP nick end-labeling Suman et al. Radiation Oncology 2012, 7:6 Page 3 of 9 http://www.ro-journal.com/content/7/1/6 (TUNEL) assay. Bone marrow cellularity in H&E stained Western blot sections was semiquantitatively scored (in 5 mice from Bone marrow cells were isolated, as per the protocol each group) by counting nucleated cells in a 16-square described in the previous section, and cells from 5 mice (1 cm each) grid in randomly chosen 5 high-power were pooled for the Western blot analysis. Cells were (40×) microscopic fields for each section (3 sections lysed in ice-cold protein extraction buffer (0.5% Sodium deoxycholate, 0.5% NP-40, 10 mM EDTA in PBS) con- from each mouse) as described earlier [21]. While plot- taining protease inhibitor cocktail (Sigma, St. Louis, ting the results, control section cellularity was consid- MO, USA). The homogenate was centrifuged at 12000 ered 100 percent. Megakaryocytes were also evaluated xg at 4°C for 10 min and supernatant was collected. The by a semi-quantitative analysis of three adjacent high- power (40×) microscopic fields (n = 5 and 3 sections Bradford protein assay was used to quantify the protein from each mouse were scored). concentration in respective samples. Equal amounts of protein samples were mixed with the appropriate Granulocyte Macrophage-Colony Forming Unit (GM-CFU) volume of Laemmli’s sample buffer (6× solution: 375 assay mM Tris-HCl (pH = 6.8), 6% sodium dodecyl sulphate Mice (n = 5 mice per group) were euthanized at 7 d after (SDS), 48% Glycerol, 9% b-Mercapto-ethanol and 0.03% radiation exposure. Under aseptic conditions femurs were bromophenol blue), heated at 95°C for 5 min, and were excised, ends opened, and bone marrow cells collected by resolved on SDS- polyacrylamide gel electrophoresis flushing. For flushing, Iscove’s modified Dulbecco’smed- (PAGE). Proteins were transferred onto a polyvinylidine ium (IMDM) (StemCell Technologies, Vancouver, BC, fluoride (PVDF) membrane, blocked with 5% milk in Canada) supplemented with 5% fetal bovine serum (FBS) tris-buffered saline with 0.1% Tween (TBST), and incu- was used with sterile syringes and 25G needles. Flushed bated with appropriate primary antibody (p-ATM bone marrow from each femur was pipetted up and down (1:100, Sc-47739, Clone-10H11-E12); p53 (1:500, Sc-98, to prepare a single cell suspension and passed through 70 Clone-1801); p-p53 (1:200, Sc-18078, Clone-mSer20); micron nylon meshes (BD Biosciences, Sparks, MD, USA). Bcl-2 (1:250, Sc-7382, Clone-C-2); Bax (1:250, Sc-7480, Isolated cells were counted using a cell counter (Beckman Clone-B-9) and b-actin (1:2000, Sc-47778, Clone-C4) from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Coulter, Brea, CA, USA), and from each femur 2.5 × 10 Western blot membranes were developed with horserad- cells/mL were plated in triplicate in ultra-low attachment ish peroxidase (HRP) conjugated secondary antibody 60 mm dishes (Corning, NY, USA) using methocult and enhanced chemiluminescence (ECL) detection sys- (M3534, StemCell Technologies) medium supplemented tem (Cat# 34080, Thermo Fisher Scientific, Rockford, with 10 ng/mL granulocyte macrophage colony stimulat- IL, USA). Images were captured on photographic films ing factor (GM-CSF) (StemCell Technologies). The plates were incubated at 37°C in 5% CO and ≥ 95% humidity and scanned. Results from a representative experiment for 7 d, and colonies were counted using a dissecting are displayed. Scanned images of the Western blots microscope (Leica, Wetzlar, Germany). were quantified by ImageJ v4.44 software using the pre- viously described protocol [22]. Briefly, scanned images Apoptosis detection in bone marrow and spleen were opened in ImageJ, and bands were selected using DNA damage and cell death in bone marrow and spleen the rectangular selection tool to generate band profile cells (n = 5 mice per group) were detected, using the plots. Normalized band intensity was generated using b- ApopTag Plus Peroxidase in situ apoptosis detection kit actin band intensity in respective columns. (S7101, Millipore, Billerica, MA, USA) according to manu- facturer’s instruction. Briefly, tissue sections were deparaf- Statistical analysis finized and pretreated with Proteinase-K solution (20 μg/ Statistical analysis to find significance between two mL) at room temperature for 15 min. The endogenous groups was performed using two tailed paired Stu- peroxidase activity was quenched using 3% hydrogen per- dent’s t-test, and p < 0.05 was taken as statistically sig- oxide in phosphate buffered saline (PBS) at room tempera- nificant. Error bars represent ± standard error of mean ture. Following incubation with terminal deoxynucleotidal (SEM). transferase (TdT) at 37°C for 1 h, the apoptotic cells were visualized under a bright field microscope by a diamino- Results benzidine (DAB) based detection system supplied with the Accelerated recovery of peripheral blood cell count and increased clonogenic survival of bone marrow kit, and sections were counterstained using methyl green progenitors in ON 01210.Na treated group (Trevigen, Gaithersburg, MD, USA) nuclear stain. TUNEL The WBC count at 3 d showed uniform reduction in all positive cells were counted in 5 randomly chosen high the irradiated groups. However, at 7 and 21 d signifi- power fields (40×), and counts from 3 sections from each cantly higher counts were observed in mice treated with mouse were used for statistical analysis. Suman et al. Radiation Oncology 2012, 7:6 Page 4 of 9 http://www.ro-journal.com/content/7/1/6 ON 01210.Na (compared to respective radiation+vehicle 01210.Na than in the vehicle treated mice (Figure 2A, B, groups p < 0.04 for 7 d and p < 0.01 for 21 d) (Figure and 2C). When given 24 h and 36 h after radiation 1A). A significant difference in absolute neutrophil exposure, the ON 01210.Na treated group also showed count (ANC) was also observed between the ON 01210. significantly enhanced clonogenic survival of bone mar- Na treated and the vehicle treated groups at 7 and 21 d row cells (p < 0.0005 compared to the radiation+vehicle (p < 0.05 for both of the time points) (Figure 1B). When group) (Figure 3). absolute monocyte counts (AMC) were compared between the ON 01210.Na and the vehicle treated ON 01210.Na treatment showed reduction in apoptotic groups, a significant difference was observed at 21 d (p cells in bone marrow and spleen < 0.05) (Figure 1C). In contrast to WBC count, platelet TUNEL assay on bone marrow and spleen sections from count was decreased at 7 d and recovery was observed the ON 01210.Na treated groups showed fewer apopto- at 21 d. However, at 7 d the count was significantly tic cells than the vehicle treated group (Figure 4A and higher in the ON 01210.Na treated group than the vehi- 4C). Quantification of TUNEL positive cells indicating cle treated group (p < 0.05), and at 21 d the ON 01210. apoptosis showed significantly lower counts in the ON Na treated group showed greater recovery than the vehi- 01210.Na treated group than the vehicle treated group cle group (p < 0.05) (Figure 1D). Furthermore, total cel- in both the bone marrow and spleen samples (Figure 4B lularity, as well as megakaryocyte counts in bone and 4D; p < 0.01 for bone marrow and p < 0.05 for marrow sections, was significantly higher in the ON spleen compared to radiation+vehicle groups). Figure 1 Post-exposure peripheral blood cell count with or without ON 01210.Na treatment.A)AbsoluteperipheralWBC counts. B) Absolute neutrophil count (ANC). C) Absolute monocyte counts (AMC). D) Platelet count expressed as percent of normal count. R+V: 5 Gy radiation+vehicle; R: 5 Gy radiation; R+D: 5 Gy radiation+drug (ON 01210.Na). *p < 0.05 compared to radiation+vehicle. Suman et al. Radiation Oncology 2012, 7:6 Page 5 of 9 http://www.ro-journal.com/content/7/1/6 Figure 2 Quantification of bone marrow cellularity in H&E stained sections at 7 d post-radiation. A) Photomicrograph showing H&E stained sections of bone marrow at 40× magnification. B) Relative quantification of total bone marrow cellularity. *p < 0.004 compared to radiation+vehicle (R+V). C) Megakaryocyte counts in H&E stained bone marrow sections. *p < 0.02 compared to radiation+vehicle. Control: no radiation, ON 01210.Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation+vehicle; R+D: 5 Gy radiation+drug (ON 01210.Na). Attenuated DNA damage response in bone marrow cells of ON 01210.Na treated mice The DNA damage response pathway involving ATM and p53 was assessed by Western blot analysis. Com- pared to vehicle treated groups, we observed a signifi- cant reduction of total p53, phospho-ATM, and phospho-p53 levels in ON 01210.Na treated bone mar- row cells at 7 d post-radiation (Figure 5A and 5D). Decrease of the p53 level in ON 01210.Na treated mice was associated with increasein anti-apoptoticBcl2 and decrease in pro-apoptotic Bax (Figure 5A). Quantifica- tion of Western blots showed significant decrease in total p53 and Bax but increase in Bcl2 (Figure 5B; p < 0.002 for p53, < 0.001 for Bax, and < 0.007 for Bcl2 compared to radiation+vehicle groups). Interestingly, the Bcl2:Bax ratio was markedly greater in the ON 01210. Na treated than the vehicle treated mice (Figure 5C). Furthermore, quantification of phospho-ATM and phos- Figure 3 Granulocyte-macrophage colony forming units (GM- CFU) assay of bone marrow cells at 7 d post-radiation. *p < 0.005 pho-p53 showed significant decrease in the ON 01210. compared to radiation+vehicle (R+V). Control: no radiation, ON 01210. Na treated mice (Figure 5E; p < 0.0007 for p-ATM and Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation+vehicle; R+D: 5 Gy < 0.0002 for the p-p53 compared to the radiation+vehi- radiation+drug (ON 01210.Na). cle treated mice). Suman et al. Radiation Oncology 2012, 7:6 Page 6 of 9 http://www.ro-journal.com/content/7/1/6 Figure 4 TUNEL staining of bone marrow and spleen sections at 7 d post-radiation. A) Photomicrograph showing TUNEL staining of bone marrow sections at 20× magnification. B) Quantification of TUNEL positive cells in bone marrow sections. *p < 0.01 compared to radiation +vehicle (R+V). C) Photomicrograph showing TUNEL staining of spleen sections at 20× magnification. D) Quantification of TUNEL positive cells in spleen sections. *p < 0.05 compared to radiation+vehicle. Control: no radiation, ON 01210.Na or vehicle; R: 5 Gy radiation; R+V: 5 Gy radiation +vehicle; R+D: 5 Gy radiation+drug (ON 01210.Na). radiation-induced DDR and apoptosis. Overall, ON Discussion 01210.Na was able to accelerate the post-exposure Terrorist threats and recent accidents in nuclear instal- hematopoietic recovery process as evidenced by a lations emphasize the need to develop strategies to miti- greater increase in peripheral WBC and platelet counts, gate radiation toxicity [23]. Mitigation of radiation higher bone marrow cellularity, and higher number of toxicity also has implications for radiation therapy [3]. GM-CFUs in the drug treated groups than in the vehicle In this initial investigation we have shown that two groups. doses of ON 01210.Na, administered 24 h and 36 h Most of the post-radiation hematopoietic morbidity after radiation exposure, could significantly mitigate and mortality is attributed to infection and hemorrhage radiation-induced hematopoietic toxicity. Mechanisti- due to leukopenia and thrombocytopenia resulting from cally, ON 01210.Na treatment resulted in diminished Suman et al. Radiation Oncology 2012, 7:6 Page 7 of 9 http://www.ro-journal.com/content/7/1/6 Figure 5 Western blot analysis of DNA damage response in bone marrow cells at 7 d post-radiation. A) Scanned images showing Western blot of p53, Bcl2, and Bax. B) Densitometric quantification of p53, Bcl2, and Bax. *p < 0.002 for p53, < 0.001 for Bax, and < 0.007 for Bcl2 compared to respective radiation+vehicle groups C) Bcl2/Bax ratio. *p < 0.005 compared to radiation+vehicle. D) Western blot images of phospho-ATM and phospho-p53. E) Densitometric quantification of phospho-ATM and phospho-p53. *p < 0.0007 for p-ATM and < 0.0002 for p- p53 compared to radiation+vehicle treated mice. R+V: 5 Gy radiation+vehicle; R: 5 Gy radiation; R+D: 5 Gy radiation+drug (ON 01210.Na). loss of bone marrow cells. Our time course study ranging Bone marrow progenitor cells with an average lower radiosensitivity than the more primitive hematopoietic from 3 to 28 d demonstrated faster recovery of radiation- induced WBC cell loss in the ON 01210.Na than in the precursor cells are a rapidly cycling cell population and vehicle treated groups. Higher numbers of peripheral are capable of forming colonies in culture (reviewed by WBC counts in the ON 01210.Na treated animals led us [24]). Our results from the GM-CFU assay support the to believe that the drug is aiding in quicker regeneration notion that ON 01210.Na is enhancing the recovery and and turnover of bone marrow cells than the vehicle regeneration of the progenitor cells, which survived groups through proliferation of surviving precursor cells. initial radiation-induced damage. Mechanistic investiga- Importantly, at 7 d and at 21 d in the ON 01210.Na tion further supports our hypothesis that ON 01210.Na groups, the absolute neutrophil counts were significantly is accelerating recovery/regeneration of bone marrow higher which would equip these animals to resist any cells due to attenuation of DDR. The ON 01210.Na, potential post-radiation infection better than the vehicle through lowering of phospho-ATM and p53, along with treated groups. Furthermore, the ON 01210.Na treated a higher Bcl2/Bax ratio, could be aiding in the prolifera- groups at both these time points showed greater percent tion of the surviving cells. Our results showing diminu- of platelets than the vehicle groups, which would mini- tion of p53 level, along with increase in Bcl2 and mize not only post-radiation infection but would also decrease in Bax level, demonstrate the mechanism of reduce the risk of hemorrhagic events. ON 01210.Na mediated radiation mitigation and is Suman et al. Radiation Oncology 2012, 7:6 Page 8 of 9 http://www.ro-journal.com/content/7/1/6 NW, Washington, DC 20057-1468, USA. Onconova Therapeutics Inc., important for the management of radiation exposure to Newton, PA 18940, USA. healthy tissues. Bone marrow and spleen are important in maintaining Authors’ contributions SS: executed experiments and analyzed and organized results; KD: planned peripheral blood cell pool and proper functioning of the and executed experiments, analyzed results, and prepared the manuscript; immune system. Thus, radiation damage to these vital AJF: participated in preparing the manuscript; MM: planned experiments and organs can affect hematopoiesis as well as immune defense, participated in preparing the manuscript. All authors read and approved this manuscript. which are critical determinant of post-exposure morbidity and mortality [25,26]. Although chemical entities and her- Competing interests notification bal preparations have been investigated for their radiopro- Dr. Manoj Maniar is employed at Onconova Therapeutics, Inc. No other authors have financial obligations to Onconova Therapeutics, Inc. tection and radiomitigation properties, we are yet to have an approved pharmacological agent to counter radiation Received: 24 October 2011 Accepted: 20 January 2012 damage to critical tissues, like bone marrow, and to reduce Published: 20 January 2012 subsequent morbidity and mortality [27]. Compounds like References statins and palifermin has been shown to mitigate radiation 1. Nenot JC: Radiation accidents over the last 60 years. J Radiol Prot 2009, mucositis and enteropathy, and herbal preparations from 29:301-320. Hippophae rhamnoides and Mentha arvensis have shown 2. Niazi AK, Niazi SK: Endocrine effects of Fukushima: Radiation-induced endocrinopathy. Indian J Endocrinol Metab 2011, 15:91-95. significant hematopoietic protection [3,9,10]. However, 3. Citrin D, Cotrim AP, Hyodo F, Baum BJ, Krishna MC, Mitchell JB: there are presently no approved agents available to mitigate Radioprotectors and mitigators of radiation-induced normal tissue radiation-induced hematopoietic toxicity. In a post-expo- injury. Oncologist 2010, 15:360-371. 4. Andrews GA: Radiation accidents and their management. Radiat Res Suppl sure scenario, administration of ON 01210.Na was able to 1967, 7:390-397. reduce the number of apoptotic cells (TUNEL positive) in 5. Hall EJ, Giaccia AJ: Philadelphia: Lippincott Williams & Wilkins; 2006. both bone marrow and spleen, indicating lower cell death 6. Moulder JE, Cohen EP: Future strategies for mitigation and treatment of chronic radiation-induced normal tissue injury. Semin Radiat Oncol 2007, than that with vehicle treatment. Our Western blot results 17:141-148. suggest that lessening of cell death in bone marrow and 7. Fliedner TM, Friesecke I, Graessle D, Paulsen C, Weiss M: Hematopoietic cell spleen has to be p53 mediated and is dependent on the renewal as the limiting factor in low-level radiation exposure: diagnostic implications and therapeutic options. Mil Med 2002, 167:46-48. activated ATM. Although we did not observe any altera- 8. Williams JP, McBride WH: After the bomb drops: A new look at radiation- tions in total ATM levels (data not shown), we did find sig- induced multiple organ dysfunction syndrome (MODS). Int J Radiat Biol 2011. nificantly lower levels of activated phospho-ATM and, 9. Maurya DK, Devasagayam TP, Nair CK: Some novel approaches for radioprotection and the beneficial effect of natural products. Indian J Exp consequently, significantly reduced phospho-p53 in the Biol 2006, 44:93-114. ON 01210.Na treated bone marrow cells. Stability of p53 is 10. Nair CK, Parida DK, Nomura T: Radioprotectors in radiotherapy. J Radiat dependent on its phosphorylation status [12]. We believe Res (Tokyo) 2001, 42:21-37. 11. Zhou BB, Elledge SJ: The DNA damage response: putting checkpoints in that decreased total p53 in bone marrow cells is due to perspective. Nature 2000, 408:433-439. reduced phosphorylation of p53, allowing enhanced inter- 12. Tanaka T, Huang X, Jorgensen E, Gietl D, Traganos F, Darzynkiewicz Z, action with MDM2, leading to increased ubiquitinylation Albino AP: ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke. BMC Cell Biol 2007, 8:26. and subsequent degradation. Pro-apoptotic Bax and anti- 13. Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O, Moas M, apoptotic Bcl2 are under the regulation of tumor suppres- Buschmann T, Ronai Z, Shiloh Y, Kastan MB, Katzir E, Oren M: ATM- sor p53. Antagonizing Bax and enhancing Bcl2 has been dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev 2001, 15:1067-1077. shown to confer resistance on cells to ionizing radiation 14. Bache M, Pigorsch S, Dunst J, Wurl P, Meye A, Bartel F, Schmidt H, Rath FW, [28,29]. Reduction in activated ATM and p53 levels leading Taubert H: Loss of G2/M arrest correlates with radiosensitization in two to a decrease in DDR signal, is playing a role in enhanced human sarcoma cell lines with mutant p53. Int J Cancer 2001, 96:110-117. 15. Mazzatti DJ, Lee YJ, Helt CE, O’Reilly MA: p53 modulates radiation hematopoietic recovery in ON 01210.Na treated mice. sensitivity independent of p21 transcriptional activation. Am J Clin Oncol 2005, 28:43-50. Conclusions 16. Zhan Q, Kontny U, Iglesias M, Alamo IJ, Yu K, Hollander MC, Woodworth CD, Fornace AJJ: Inhibitory effect of Bcl-2 on p53-mediated Taken together, we conclude that the post-exposure transactivation following genotoxic stress. Oncogene 1999, 18:297-304. administration of ON 01210.Na enhances the recovery 17. Dlugosz PJ, Billen LP, Annis MG, Zhu W, Zhang Z, Lin J, Leber B, of hematopoietic cells by employing a mechanism that Andrews DW: Bcl-2 changes conformation to inhibit Bax oligomerization. EMBO J 2006, 25:2287-2296. not only attenuates DNA damage sensing and damage 18. Ghosh SP, Perkins MW, Hieber K, Kulkarni S, Kao TC, Reddy EP, Reddy MV, signal transduction but also alters levels of effectors like Maniar M, Seed T, Kumar KS: Radiation protection by a new chemical Bax and Bcl2. entity Ex-Rad: efficacy and mechanisms. Radiat Res 2009, 171:173-179. 19. Chun AW, Cosenza SC, Taft DR, Maniar M: Preclinical pharmacokinetics and in vitro activity of ON 01910.Na, a novel anti-cancer agent. Cancer Chemother Pharmacol 2009, 65:177-186. Author details 20. Chun AW, Freshwater RE, Taft DR, Gillum AM, Maniar M: Effects of Department of Biochemistry and Molecular & Cell Biology, Georgetown formulation and route of administration on the systemic availability of University Medical Center, Research Building, Room E518, 3970 Reservoir Rd., Suman et al. Radiation Oncology 2012, 7:6 Page 9 of 9 http://www.ro-journal.com/content/7/1/6 Ex-RAD((R)), a new radioprotectant, in preclinical species. Biopharm Drug Dispos 2011, 32:99-111. 21. Singh VK, Parekh VI, Brown DS, Kao TC, Mog SR: Tocopherol succinate: modulation of antioxidant enzymes and hematopoietic recovery. Int J Radiat Oncol Biol Phys 2011, 79:571-578. 22. Western blot analysis protocol. [http://rsb.info.nih.gov/ij/docs/menus/ analyze.html#gels]. 23. Pellmar TC, Rockwell S: Priority list of research areas for radiological nuclear threat countermeasures. Radiat Res 2005, 163:115-123. 24. Yang FT, Lord BI, Hendry JH: Gamma irradiation of the fetus damages the developing hemopoietic microenvironment rather than the hemopoietic progenitor cells. Radiat Res 1995, 141:309-313. 25. Gridley DS, Pecaut MJ: Whole-body irradiation and long-term modification of bone marrow-derived cell populations by low- and high- LET radiation. In Vivo 2006, 20:781-789. 26. Gridley DS, Pecaut MJ, Miller GM, Moyers MF, Nelson GA: Dose and dose rate effects of whole-body gamma-irradiation: II. Hematological variables and cytokines. In Vivo 2001, 15:209-216. 27. Weiss JF, Landauer MR: History and development of radiation-protective agents. Int J Radiat Biol 2009, 85:539-573. 28. Domen J, Gandy KL, Weissman IL: Systemic overexpression of BCL-2 in the hematopoietic system protects transgenic mice from the consequences of lethal irradiation. Blood 1998, 91:2272-2282. 29. Xiang J, Chao DT, Korsmeyer SJ: BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci USA 1996, 93:14559-14563. doi:10.1186/1748-717X-7-6 Cite this article as: Suman et al.: Administration of ON 01210.Na after exposure to ionizing radiation protects bone marrow cells by attenuating DNA damage response. Radiation Oncology 2012 7:6. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

Journal

Radiation OncologySpringer Journals

Published: Jan 20, 2012

There are no references for this article.