Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention AACR Conference on Molecular Diagnostics - 2008
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[Cancer Research 64, 6240-6246, September 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Activation of Nuclear Factor {kappa}B In vivo Selectively Protects the Murine Small Intestine against Ionizing Radiation-Induced Damage

Yong Wang1, Aimin Meng1, Hainan Lang2, Stephen A. Brown4,5, Jennifer L. Konopa4, Mark S. Kindy3, Richard A. Schmiedt2, John S. Thompson4,5 and Daohong Zhou1

Departments of 1 Pathology and Laboratory Medicine, 2 Otolaryngology-Head and Neck Surgery, and 3 Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina; 4 Department of Medicine, University of Kentucky, Lexington, Kentucky; and 5 Veterans Administration Medical Center, Lexington, Kentucky


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of mice to total body irradiation induces nuclear factor {kappa}B (NF{kappa}B) activation in a tissue-specific manner. In addition to the spleen, lymph nodes, and bone marrow, the tissues that exhibit NF{kappa}B activation now include the newly identified site of the intestinal epithelial cells. NF{kappa}B activated by total body irradiation mainly consists of NF{kappa}B p50/RelA heterodimers, and genetically targeted disruption of the NF{kappa}B p50 gene in mice significantly decreased the activation. By comparing tissue damage and lethality in wild-type and NF{kappa}B p50 knockout (p50–/–) mice after they were exposed to increasing doses of total body irradiation, we additionally examined the role of NF{kappa}B activation in total body irradiation-induced tissue damage. The results show that p50–/– mice are more sensitive to total body irradiation-induced lethality than wild-type mice (LD50/Day 7: wild-type = 13.12 Gy versus p50–/– = 7.75 Gy and LD50/Day 30: wild-type = 9.31 Gy versus p50–/– = 7.81 Gy). The increased radiosensitivity of p50–/– mice was associated with an elevated level of apoptosis in intestinal epithelial cells and decreased survival of the small intestinal crypts compared with wild-type mice (P < 0.01). In addition, RelA/TNFR1-deficient (RelA/TNFR1–/–) mice also exhibited a significant increase in intestinal epithelial cell apoptosis after they were exposed to total body irradiation as compared with TNFR1-deficient (TNFR1–/–) mice (P < 0.01). In contrast, no significant increase in total body irradiation-induced apoptosis or tissue injury was observed in bone marrow cells, spleen lymphocytes, and the liver, heart, lung, and kidney of p50–/– mice in comparison with wild-type mice. These findings indicate that activation of NF{kappa}B selectively protects the small intestine against ionizing radiation-induced damage.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear factor {kappa}B (NF{kappa}B) is a dimeric DNA binding protein consisting of members of the NF{kappa}B/Rel family, which includes the subunits of NF{kappa}B1 (p50), NF{kappa}B2 (p52), RelA, RelB, and c-Rel (1, 2, 3) . Its expression is ubiquitous in mammalian cells. Normally, NF{kappa}B resides in the cytoplasm in an inactive form in association with inhibitory proteins. These inhibitory proteins, which belong to a family of proteins named inhibitor of NF{kappa}B (4) , prevent NF{kappa}B nuclear translocation by masking the NF{kappa}B nuclear localization signal and thus, inhibit NF{kappa}B DNA binding and transactivational function (1, 2, 3) . Various stimuli activate a large number of distinct signaling pathways that eventually result in the phosphorylation of inhibitor of NF{kappa}B and its subsequent degradation by the proteasome or its dissociation from NF{kappa}B without additional degradation (1, 2, 3) . The released NF{kappa}B then translocates to the nucleus and binds to {kappa}B or {kappa}B-like DNA motifs to initiate gene transcription. The putative target genes of NF{kappa}B are mainly involved in immune and inflammatory responses (1, 2, 3) . These genes encode a variety of inflammatory molecules, including various inflammatory cytokines and adhesion molecules. In addition, NF{kappa}B also regulates the expression of many genes of which the products are involved in the control of cell proliferation and cell death (4, 5, 6, 7) .

Tumor cells usually express high levels of constitutive NF{kappa}B activity (8 , 9) . In addition, exposure of these cells to various cytotoxic agents including ionizing radiation increases NF{kappa}B activity (7, 8, 9, 10, 11, 12, 13) . The role of NF{kappa}B in tumorigenesis and cellular resistance to tumor therapy has been extensively studied. The majority of reported studies have demonstrated that NF{kappa}B activation may give transformed cells a growth and survival advantage and additionally may render tumor cells resistant to ionizing radiation and a variety of cytotoxic agents by induction of antiapoptotic proteins (7, 8, 9, 10, 11, 12, 13) . Therefore, molecularly targeted inhibition of NF{kappa}B has been actively pursued as a potential and novel adjuvant treatment for cancer in conjunction with radiotherapy and chemotherapy (7, 8, 9, 10, 11, 12, 13) .

The purpose of using NF{kappa}B inhibitors as an adjuvant therapy for cancer is to increase the therapeutic index of radiotherapy and chemotherapy. The success of this approach relies on its ability to promote tumor cell killing by ionizing radiation or chemotherapy but to spare normal tissues from enhanced damage. Therefore, it is critical to determine the effects of NF{kappa}B inhibition on normal tissue function in response to ionizing radiation, because activation of NF{kappa}B by ionizing radiation has been documented not only in various tumor cells but also in different types of cultured normal cells in vitro (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) . Previously using a mouse model, we investigated the tissue specificity of ionizing radiation-induced NF{kappa}B activation in vivo and found that total body irradiation induces NF{kappa}B activation in a tissue-specific manner (26) . The activation was observed in the bone marrow and the peripheral lymphoid tissues of the spleen and mesenteric lymph nodes shortly after mice were exposed to a lethal dose of total body irradiation but was absent in all of the other tissues examined, including the liver, lung, colon, thymus, and brain (26) . Now, we have discovered that exposure of mice to total body irradiation also activates NF{kappa}B in intestinal epithelial cells of the small intestine, a prime target of ionizing radiation damage. The NF{kappa}B activated by total body irradiation in various tissues mainly consists of NF{kappa}B p50/RelA heterodimers and genetically targeted disruption of the NF{kappa}B p50 gene in mice significantly decreased the activation. Therefore, in the present study we compared the tissue damage and lethality in wild-type and NF{kappa}B p50 knockout (p50–/–) mice after they were exposed to increasing doses of total body irradiation to determine the role of NF{kappa}B activation in ionizing radiation-induced normal tissue damage. The results of this study have important clinical implications in cancer therapy using NF{kappa}B inhibitors as an adjuvant therapeutic agent in conjunction with ionizing radiation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Normal male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Inbred B6,129PF2 (p50 wild-type or wild-type), B6,129P-Nfkb1 (p50 knockout or p50–/–), RelA/TNFR1-deficient (RelA/TNFR1–/–), and TNFR1-deficient (TNFR1–/–) mice were bred at the Medical University of South Carolina, the Association for the Assessment and Accreditation of Laboratory Animal Care-certified animal facility. The homozygous breeding pairs of wild-type and p50–/– mice were obtained from the Jackson Laboratory and those of RelA/TNFR1–/– and TNFR1–/– mice were kindly provided by Dr. David Baltimore (California Institute of Technology, Pasadena, CA). All of the mice received food and water ad libitum and were maintained in pathogen-free microisolator cages (4 per cage). They were used at approximately 8–12 weeks of age. The Institutional Animal Care and Use Committee of Medical University of South Carolina approved all of the experimental procedures used in this study.

Ionizing Radiation.
Mice were exposed to various doses of ionizing radiation in a JL Shepherd Model 143 137Cesium {gamma}-irradiator (JL Shepherd, Glendale, CA) at a rate of 2.4 Gy/min. Mice were irradiated on a rotating platform.

Tissue Collection.
After exposure to different doses of ionizing radiation, mice were euthanized at various times as indicated in individual experiments by CO2 suffocation followed by cervical dislocation. A group of unirradiated mice was euthanized similarly as control. The spleen, liver, lung, kidney, and heart were harvested, weighed, and fixed with 10% neutral-buffered formalin for histopathological examination. The small intestines were collected. Some of them were processed as described later for epithelial cell apoptosis and crypt survival assays. The others were used for the preparation of nuclear extracts. In addition, the femoral bones were isolated for the preparation of bone marrow-mononuclear cells as reported previously (27 , 28) .

Preparation of Nuclear Extracts.
About 20-cm length of the proximal jejunum was immediately removed after mice were euthanized and flushed with ice-cold PBS. After the jejunum was cut open on a ice-chilled Petri dish, intestinal mucosa was scraped in ice-cold PBS with 1 mol/L dithiothreitol as reported elsewhere (29) . The intestinal epithelial cells were collected by centrifugation. The nuclear extracts of intestinal epithelial cells were prepared using a method as published previously (26 , 27 , 30) , and the protein concentrations of the nuclear extracts were accurately quantified using the Bio-Rad Dc protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Analysis of NF{kappa}B Activities by Gel Shift and Supershift Assay.
The double-stranded oligonucleotides containing a consensus {kappa}B sequence (5'-AGTTGAGGGACTTTCCCAGGC-3'; Integrated DNA Technologies, Inc., Coralville, IA) were labeled using the Biotin 3' End DNA Labeling kit (Pierce, Rockford, IL) according to the manufacturer’s protocol. The gel shift assay was performed using the LightShift Chemiluminescent electrophoretic mobility shift analysis kit (Pierce) following the manufacturer’s instructions. Briefly, an aliquot of nuclear extracts containing 5 µg of protein was incubated with 2 µl of electrophoretic mobility shift analysis binding buffer, 20 fmoles of biotin-labeled NF{kappa}B probe and 1 µl of poly(deoxyinosinic-deoxycytidylic acid) in a total volume of 20 µl for 20 min at room temperature. The reaction mixtures were separated on a 6% native polyacrylamide gel by electrophoresis and then transferred to Biodyne B Nylon Membrane (Pierce). After the membrane was incubated with LightShift Stabilized Streptavidin-Horseradish Peroxidase Conjugate and the Luminol/Enhancer and Stable Peroxide Solution, the NF{kappa}B DNA complexes were detected by exposure of the membrane to X-ray film. The relative nuclear NF{kappa}B DNA binding activities were quantified by scanning densitometry. The specificity of the identified NF{kappa}B DNA binding activity in the nuclear extracts was confirmed by using 200-fold excess of unlabeled NF{kappa}B, mutated NF{kappa}B (Santa Cruz Biotechnology, Santa Cruz, CA), or activator protein-1 (Promega, Madison, WI) oligonucleotides. The addition of the excess unlabeled NF{kappa}B oligonucleotides into the gel shift reaction resulted in elimination of the relative NF{kappa}B DNA binding activities, whereas that of the excess unlabeled mutated NF{kappa}B or activator protein-1 oligonucleotides did not affect the assay (Fig. 1C)Citation . For gel supershift analysis, extracted nuclear proteins (5 µg) were incubated with 2 µg of the polyclonal antibodies specifically against the p50, p52, RelA, and/or Erg-2 proteins (Santa Cruz Biotechnology) for 20 min before their incubations with biotin-labeled NF{kappa}B probe in the gel shift assay described above.



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  		Fig. 1. Ionizing radiation induces NF{kappa}B activation in intestinal epithelial cells in a dose- and time-dependent manner. Wild-type mice were euthanized 2 hours after receiving 0.5, 1, 2, 4, 8, or 12 Gy total body ionizing radiation (A) or euthanized at 0.5, 1, 2, 4, 8, 16 or 24 hours after 8 Gy total body irradiation (B). Control mice (0) were euthanized without exposure to total body irradiation. Nuclear proteins were extracted from isolated intestinal epithelial cells and analyzed for NF{kappa}B DNA binding activity using gel shift assay. The molecular composition of ionizing radiation-activated NF{kappa}B in intestinal epithelial cells from wild-type mice was determined by gel supershift assay (C). N, no sample; TBI, total body irradiation; ab, antibody.

 
Analysis of Bone Marrow-Mononuclear Cell Apoptosis and the Hematopoietic Function of Bone Marrow Stem Cells and Progenitors.
Bone marrow-mononuclear cell apoptosis was determined by measuring sub-G0/1 cells after DNA staining with propidium iodide and the hematopoietic function of bone marrow stem cells and progenitors was measured by cobblestone area-forming cell assay. These assays were performed as described previously (27 , 28) . The frequencies of cobblestone area-forming cell were determined at weekly intervals (on days 7, 14, 21, 28, and 35). Wells were scored positive if at least one dark-phase hematopoietic clone (or cobblestone area containing ≥5 cells) was seen. The frequency of cobblestone area-forming cell was then calculated by using Poisson statistics as described previously (31 , 32) . This assay provides an estimate of the hematopoietic function of a spectrum of cobblestone area-forming cell day-types that correspond to various stages of stem cells and progenitors. Specifically, day-7 cobblestone area-forming cell and day-14 cobblestone area-forming cell correspond to colony forming unit-granulocytes and monocytes and day-12 colony forming unit-spleen, respectively. The primitive stem cells with long-term repopulating ability correspond to day-28 and -35 cobblestone area-forming cell (32) .

Intestinal Apoptosis Assay.
The small intestine was rapidly removed from the abdomen after mice were euthanized at various times as described in individual experimental plans. The intestinal contents were removed, and then the intestinal tube was flushed with saline and cut into ~10 1-cm lengths, which were bundled together with micropore tape before fixation with 10% neutral-buffered formalin overnight. The tissues were embedded in paraffin, and sections (5 µm) were cut perpendicular to the long axis of the intestine and stained with hematoxylin and eosin. The number of apoptotic cells per crypt was assessed by morphological criteria in a blind fashion as described previously by Potten et al. (33) . Only well-oriented crypts (50 crypts/animal) in longitudinal sections containing Paneth cells, a crypt lumen, and an uninterrupted column of epithelial cells extending to the crypt-villus junction were scored. In addition, ionizing radiation-induced intestinal epithelial cell apoptosis was examined by immunohistochemistry of active caspase 3 and terminal deoxynucleotidyl transferase-mediated nick end labeling assay (34) . For active caspase 3 immunohistochemistry, intestinal tissue sections were deparaffinized and rehydrated. After blocking endogenous peroxidase with 0.3% hydrogen peroxide solution for 15 min, the sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 min and then cooled for 20 min to enhance antigen exposure. Nonspecific binding was blocked by incubation of the sections in 10% normal goat serum for 30 min. The sections were incubated with 1:500 rabbit polyclonal antiactive caspase 3 antibody (R&D Systems, Minneapolis, MN) for 18 h at 4°C, extensively washed, and then incubated with 1:200 biotinylated goat antirabbit secondary antibody for 45 min at room temperature. The immunostaining was developed using Vectastain ABC reagents (Vector Laboratories, Inc., Burlingame, CA), 3,3'-diaminobenzidine, and hydrogen peroxide. The sections were counterstained with hematoxylin. Terminal deoxynucleotidyl transferase-mediated nick end labeling assay was performed using the ApopTag fluorescent in situ apoptosis detection kit (Serologicals, Norcross, GA). Briefly, deparaffinized and rehydrated intestinal tissue sections were permeabilized with proteinase K (20 µg/ml) for 20 min, washed, and then incubated with digoxygenin-deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase at 37°C for 1 h. The sections were immersed in stop/wash buffer for 10 min to terminate the reaction and then incubated with fluorescent-conjugated antidigoxigenin antibody for 30 min. Antifade mounting medium was used for fluorescence coverslipping.

Crypt Survival Assay.
Three days after exposure to ionizing radiation (12 Gy), each mouse received i.p. injection of 120 mg/kg BrdUrd (Sigma, St. Louis, MO) and 12 mg/kg 5-fluoro-2'-deoxyuridine (Sigma) to label the S-phase cells. Two hours after the injection, mice were euthanized, and their proximal jejunum were collected, prepared, fixed, embedded, and sectioned at 5 µm as described above. Cells incorporating bromodeoxyuridine were detected by mouse antibromodeoxyuridine antibody and visualized by immunofluorescence using Texas red-labeled goat antimouse IgG (red) and Hoechst 33342 (blue, for nuclear counter staining). A surviving crypt is defined as one containing ≥5 bromodeoxyuridine-positive cells as described previously (35) . The number of surviving crypts per cross-section was determined for each mouse by scoring the number of surviving crypts in 10 complete, well-oriented cross-sections in a blind manner and dividing the total by the number of cross-sections scored.

Statistical Analysis.
The data were analyzed by analysis of variance. If analysis of variance justified post hoc comparisons between group means, these were conducted using the Student-Newman-Keuls test for multiple comparisons. For experiments in which only single experimental and control groups were used, group differences were examined by unpaired Student’s t test. Differences were considered significant at P < 0.05. All of these analyses were done using GraphPad Prism from GraphPad Software, Inc. (San Diego, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ionizing Radiation Induces NF{kappa}B Activation in Intestinal Epithelial Cells in a Dose- and Time-Dependent Manner.
We demonstrated previously that mice responded to total body irradiation with increased NF{kappa}B activity in a tissue-specific manner (26) . Specifically, the increase in NF{kappa}B activity was found in the bone marrow, spleen, and lymph node, but was not seen in the liver, lung, colon, thymus, and brain (26) . Now, we demonstrate that exposure of mice to total body irradiation also activated NF{kappa}B in intestinal epithelial cells in a dose-dependent manner after mice were exposed to increasing doses of total body irradiation (from 0.5 to 12 Gy; Fig. 1ACitation ). In addition, the increase in NF{kappa}B activity in intestinal epithelial cells was time dependent, because the increase occurred within 30 minutes, peaked at 2 hours, and then gradually declined thereafter but remained elevated for up to 24 hours after exposure to 8 Gy total body irradiation (Fig. 1B)Citation .

The molecular composition of the NF{kappa}B activated by ionizing radiation in intestinal epithelial cells was determined by gel supershift assay. As shown in Fig. 1CCitation , a single retarded band that represented the specific NF{kappa}B DNA binding activity in the nuclear extracts of irradiated intestinal epithelial cells was abrogated by the addition of excess unlabeled NF{kappa}B oligonucleotides but was not affected by that of unlabeled activator protein-1 or mutated NF{kappa}B oligonucleotides, demonstrating the specificity of the assay. The retarded band was supershifted by the addition of anti-p50 and/or anti-RelA antibodies, but was not changed by the addition of anti-Erg-1 antibody, indicating that ionizing radiation-activated NF{kappa}B in intestinal epithelial cells mainly consisted of p50/RelA heterodimers. A similar molecular composition of ionizing radiation-activated NF{kappa}B was also found in the spleen, lymph node, and bone marrow in our studies reported previously (26 , 27 , 30) .

Targeted Disruption of the p50 Gene in Mice Attenuated Ionizing Radiation-Induced NF{kappa}B Activation in Intestinal Epithelial Cells.
Because the NF{kappa}B complex activated by ionizing radiation in murine intestinal epithelial cells mainly consists of p50/RelA heterodimers, we examined whether the gene-targeted disruption of the p50 gene could attenuate ionizing radiation-induced NF{kappa}B activation in intestinal epithelial cells. As shown in Fig. 2Citation , intestinal epithelial cells from both unirradiated wild-type and p50–/– mice expressed a barely detectable level of NF{kappa}B activity. Exposure of wild-type mice to 8 Gy total body irradiation resulted in a 219-fold increase in intestinal epithelial NF{kappa}B activity, whereas only a 43-fold increase in NF{kappa}B activity was observed in the intestinal epithelium of p50–/– mice after the same dose of total body irradiation (P < 0.001). The residual NF{kappa}B activity activated by ionizing radiation in p50–/– intestinal epithelium mainly consists of p52/RelA heterodimers (Fig. 2C)Citation . This finding is in agreement with our previous observations in the spleen, lymph node, and bone marrow (27 , 30) , demonstrating that the p50 NF{kappa}B subunit is an essential component of the NF{kappa}B complexes activated by ionizing radiation in vivo and that the lack of p50 cannot be fully replaced by the other members of the NF{kappa}B/Rel family.



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  		Fig. 2. Targeted disruption of the p50 gene attenuated ionizing radiation-induced NF{kappa}B activation in intestinal epithelial cells. Wild-type (WT) and p50–/– mice were exposed to 8 Gy total body irradiation or unirradiated (C). Two hours after total body irradiation, mice were euthanized, and nuclear proteins were extracted from isolated intestinal epithelial cells and analyzed for NF{kappa}B DNA binding activity using gel shift assay (A). The increase in NF{kappa}B activity in irradiated intestinal epithelial cells from individual animals was quantified by densitometry and expressed as mean (n = 3) of fold increase from the baseline value obtained from unirradiated intestinal epithelial cells (B); bars, ±SE. The molecular composition of ionizing radiation-activated NF{kappa}B in intestinal epithelial cells from p50–/– mice was determined by gel supershift assay (C). N, no sample; TBI, total body irradiation; ab, antibody.

 
Activation of NF{kappa}B Selectively Protects Intestinal Epithelial Cells of the Small Intestinal Crypts from Ionizing Radiation-Induced Damage.
NF{kappa}B can induce the expression of many proapoptotic and antiapoptotic proteins that regulate cell survival, and its activation has been implicated in inhibition or promotion of apoptosis in a cell type- and stimulus-dependent manner (4, 5, 6, 7) . The role of NF{kappa}B activation in ionizing radiation-induced normal tissue damage has not been well established. However, our recent studies have showed that activation of NF{kappa}B by ionizing radiation has no significant effect on ionizing radiation-induced splenic lymphocyte apoptosis (27) . More recently, Egan et al. (36) have also reported that activation NF{kappa}B protects intestinal epithelial cells from ionizing radiation-induced apoptosis. Therefore, we additionally examined the role of NF{kappa}B in ionizing radiation-induced normal tissue damage in various nonlymphoid tissues. We compared total body irradiation-induced tissue damage and apoptosis in wild-type versus p50–/– mice, because p50–/– mice exhibited a significant reduction in ionizing radiation-induced NF{kappa}B activation (Fig. 2Citation ; refs. 27 , 30 ).

As shown in Fig. 3ACitation , exposure of wild-type and p50–/– mice to total body irradiation induced bone marrow-mononuclear cell apoptosis in a dose- and time-dependent manner. Similarly, total body irradiation (4 Gy) also significantly decreased the frequency of various day types of cobblestone area-forming cell, probably due to the induction of apoptosis (Fig. 3BCitation ; refs. 28 , 37 ). However, there was no significant difference between wild-type animals and p50–/– mice in their response to ionizing radiation-induced bone marrow-mononuclear cell apoptosis and in their decrease in cobblestone area-forming cell frequency (P > 0.05), suggesting that activation of NF{kappa}B by ionizing radiation had no significant effect on ionizing radiation-induced bone marrow toxicity.



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  		Fig. 3. Targeted disruption of the p50 gene fails to affect ionizing radiation-induced apoptosis in bone marrow-mononuclear cells and suppression of hematopoietic function of bone marrow stem cells and progenitors. Six or 24 hours after receiving 0, 2, 4, or 8 Gy of total body irradiation, bone marrow-mononuclear cells were harvested from individual wild-type and p50–/– mice (n = 5). Apoptosis of these cells was measured by quantification of sub-G0/1 population (A). In addition, the hematopoietic function of bone marrow stem cells and progenitors were measured by cobblestone area-forming cell assay after mice (n = 3) were exposed to 4 Gy total body irradiation (B). The day 7 cobblestone area-forming cell and day-14 cobblestone area-forming cell correspond to progenitors that produce colony forming unit-granulocytes and monocytes and day 12 colony forming unit spleen, respectively. The primitive bone marrow stem cells with long-term repopulating ability correspond to day 28 and 35 cobblestone area-forming cell. The data presented are mean; bars, ±SE. A, {square}, wild-type 6 hours; {blacksquare}, p50–/– 6 hours; {circ}, wild-type 24 hours; {bullet}, p50–/– 24 hours; B, {circ}, wild-type control; {bullet}, p50–/– control; {square}, wild-type 4 Gy; {blacksquare}, p50–/– 4 Gy.

 
Exposure of wild-type and p50–/– mice to total body irradiation (8 Gy) also significantly increased intestinal epithelial cell apoptosis in the small intestinal crypts at 6 and 24 hours after ionizing radiation as compared with the baseline levels of apoptosis in unirradiated mice (P < 0.01; Fig. 4ACitation and Fig. 5ACitation ). The increase in intestinal epithelial cell apoptosis then declined to insignificant levels at 72 hours after total body irradiation (P > 0.05; Fig. 4ACitation ). Although the levels of intestinal epithelial cell apoptosis were not significantly different between wild-type and p50–/– mice at 6 hours after ionizing radiation (apoptotic cell/crypt: wild-type 4.3 ± 0.7 versus p50–/– 3.8 ± 0.4, P > 0.05), the level of intestinal epithelial cell apoptosis in p50–/– mice was 1.4-fold higher than that of wild-type at 24 hours after total body irradiation (apoptotic cell/crypt: wild-type 4.1 ± 0.2 versus p50–/– 5.8 ± 0.5, P < 0.01). The increase in ionizing radiation-induced intestinal epithelial cell apoptosis in p50–/– mice at 24 hours after total body irradiation was associated with an enhanced activation of caspase 3 and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated nick end labeling assay; Fig. 5ACitation ). Similarly, RelA/TNFR1–/– mice also showed a 1.5-fold increase in intestinal epithelial cell apoptosis at 24 hours after total body irradiation (8 Gy) as compared with TNFR1–/– mice (apoptotic cell/crypt: TNFR1–/– 3.5 ± 0.2 versus RelA/TNFR1–/– 5.3 ± 0.5, P < 0.01; Fig. 4BCitation ). In addition, p50–/– mice exhibited a diminished survival of the intestinal crypts at 3 days after exposure to 12 Gy total body irradiation when compared with the same dose-irradiated wild-type mice (Fig. 4CCitation and Fig. 5BCitation ). The number of surviving crypts was significantly lower in irradiated p50–/– mice than that of irradiated wild-type mice (surviving crypts/cross-section: wild-type 28.3 ± 2.3 versus p50–/– 20.3 ± 0.9, P = 0.012; Fig. 4CCitation ). In contrast, no significant tissue damage was observed in the liver, heart, lung, and kidney after p50–/– and wild-type mice were exposed to 8 Gy total body irradiation (data not shown). Together, the data presented above suggest that activation of NF{kappa}B selectively protects the small intestine from ionizing radiation-induced damage but has no significant effect on ionizing radiation-induced lymphoid and hematopoietic toxicity.



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  		Fig. 4. Lack of p50 or RelA sensitizes intestinal epithelial cells to ionizing radiation-induced damage. A, wild-type (WT) and p50–/– mice were exposed to 8 Gy total body irradiation or unirradiated (control). Apoptotic intestinal epithelial cells in the small intestinal crypts were determined at 6 (n = 3), 24 (n = 7), and 72 hours (n = 3) after total body irradiation (TBI). The results are expressed as mean; bars, ±SE. a, P < 0.01 versus their respective unirradiated controls (n = 3); b, P < 0.01 versus 24 hours after irradiated wild-type mice. B, TNFR1–/– and RelA/TNFR1–/– mice (n = 4) were exposed to 8 Gy total body irradiation. Apoptotic intestinal epithelial cells in the small intestinal crypts were determined at 24 hours after total body irradiation. The results are expressed as mean; bars, ±SE. C, wild-type and p50–/– mice were exposed to 12 Gy total body irradiation. The number of surviving crypts in the small intestine was determined at 3 days after total body irradiation. The results are expressed as mean; bars, ± SE (n = 7).

 


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  		Fig. 5. Representative photomicrographs of irradiated small intestinal crypts. A, apoptotic cells (arrow-pointed) in the small intestinal crypts of wild-type (WT) and p50–/– mice at 24 hours after exposure to 8 Gy total body irradiation were identified by hematoxylin and eosin (H&E) staining, active caspase 3 immunostaining, and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay (x400). B, surviving crypts (arrow-pointed) in wild-type and p50–/– mouse intestines at 3 days after exposure to 12 Gy total body irradiation. The S-phase cells incorporating bromodeoxyuridine were stained red by mouse antibromodeoxyuridine antibody, and Texas red-labeled goat antimouse IgG and nuclei were stained blue with Hoechst 33342.

 
Targeted Disruption of the p50 Gene Sensitizes Mice to Total Body Irradiation-Induced Lethality.
To determine whether targeted disruption of the p50 gene not only increases ionizing radiation-induced intestinal epithelial cell damage but more importantly sensitizes mice to total body irradiation-induced lethality, wild-type and p50–/– mice were exposed to increasing doses of total body irradiation, and their survival was monitored for 30 days. The LD50 values for day 7 (LD50/7) and day 30 (LD50/30) were calculated for both strains of mice and are presented in Table 1Citation . As shown in Table 1Citation , p50–/– mice had much lower LD50/7 and LD50/30 values than those of wild-type mice (LD50/7: p50–/– = 7.75 Gy versus wild-type = 13.12 Gy; LD50/30: p50–/– = 7.81 Gy versus wild-type = 9.31 Gy), indicating that p50–/– mice were more sensitive to total body irradiation-induced death than wild-type mice. However, the LD50/30 value of p50–/– mice was similar to their LD50/7 value. This suggests that the increased susceptibility of p50–/– mice to total body irradiation-induced lethality is mainly attributable to an enhanced intestinal damage, because it has been well established that the LD50/7 value indicates that ionizing radiation caused intestinal toxicity and LD50/30 value represents ionizing radiation-induced hematopoietic syndrome (38) . This suggestion is in agreement with the observations that p50–/– mice exhibited an increase in intestinal epithelial cell apoptosis and decrease in crypt survival after they were exposed to total body irradiation.


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  		Table 1 LD50 values of TBI in WT and p50–/– mice

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Molecularly targeted inhibition of NF{kappa}B has the potential to be developed as a novel cancer therapy. Particularly, treatment with a combination of NF{kappa}B inhibitors and conventional ionizing radiation or chemotherapeutic agents may dramatically improve the antitumor response. However, the effect of NF{kappa}B inhibition on the normal tissue response to ionizing radiation- and chemotherapy-induced injury must be evaluated before the clinical application of NF{kappa}B inhibition. Therefore, using a mouse model we examined the tissue specificity of ionizing radiation-activated NF{kappa}B and the role of NF{kappa}B in ionizing radiation-induced normal tissue damage. In addition to the spleen, lymph node, and bone marrow identified in our previous studies (26 , 27 , 30) , we now discovered that exposure of mice to a low dose (≤2 Gy) of total body irradiation also activated NF{kappa}B in intestinal epithelial cells of the small intestine. The activation of NF{kappa}B in these tissues was in a dose-dependent fashion when mice were exposed to increasing doses (up to 12 Gy) of total body irradiation. In contrast, no significant activation of NF{kappa}B was observed in the liver, lung, colon, and brain after mice were exposed to total body irradiation in the same dose range (26 , 27 , 30) . These results suggest that when mice are exposed to a clinically relevant dose (≤2 Gy) of total body irradiation, ionizing radiation activates NF{kappa}B in vivo in a tissue-specific manner.

However, when mice were exposed to a super lethal dose (20 Gy) of total body irradiation, a significant activation of NF{kappa}B was found in the liver and kidney (39) . In addition, a delayed activation of NF{kappa}B was also observed in irradiated rat lung after 20 Gy pulmonary irradiation (40) . These findings demonstrate that the tissues that are prone to ionizing radiation toxicity, such as the spleen, lymph node, bone marrow, and small intestine, are more sensitive to ionizing radiation-induced activation of NF{kappa}B, whereas radioresistant tissues are less responsive to ionizing radiation for NF{kappa}B activation. Thus, it appears that different normal tissues possess diverse sensitivity to ionizing radiation-induced activation of NF{kappa}B, which correlates to their susceptibility to ionizing radiation-induced tissue damage.

Such a correlation implies that NF{kappa}B may function as a sensor that can detect ionizing radiation-induced tissue damage. In turn, activated NF{kappa}B can modulate tissue responses to ionizing radiation damage by stimulating the expression of various genes that are involved in regulation of cell survival, cell proliferation, and tissue inflammation (1, 2, 3) . Depending on the particular tissue involved, activation of NF{kappa}B may confer tissue protection or contribute to tissue injury in response to ionizing radiation (27 , 36 , 40) . In agreement with this hypothesis, we found that down-regulation of NF{kappa}B activation by the targeted disruption of the p50 or RelA gene in mice sensitized intestinal epithelial cells to ionizing radiation-induced damage. A similar finding was reported recently by Egan et al. (36) using mice that had genetically targeted ablation of inhibitor of NF{kappa}B kinase-ß expression in intestinal epithelial cells to specifically block NF{kappa}B activation in these cells. These findings indicate that activation of NF{kappa}B is radioprotective in the small intestine. Correspondingly, p50–/– mice exhibited a higher sensitivity to ionizing radiation-induced intestinal syndrome and had a much lower LD50/7 value than wild-type animals after they were exposed to increasing doses of total body irradiation. However, the inhibition of NF{kappa}B activation by targeted disruption of the p50 gene had no significant effects on ionizing radiation-induced lymphoid and hematopoietic cell damage nor did it affect the responses of the liver, lung, heart, and kidney to ionizing radiation (27) . This suggests that ionizing radiation not only activates NF{kappa}B in vivo in a tissue-specific manner, but more importantly the activation also confers tissue-specific protection against ionizing radiation-induced normal tissue damage.

The mechanisms underlying the tissue-specific protection against ionizing radiation by NF{kappa}B activation are not clear at the present. It may relate to cross-talk among different transcriptional factors activated by ionizing radiation. For example, it is well known that exposure of mice to total body irradiation also induces tissue-specific activation of p53 (41, 42, 43) . Transcriptional induction of certain apoptosis proteins by p53 contributes to ionizing radiation-induced apoptosis in various tissues (41, 42, 43, 44, 45) . NF{kappa}B and p53 share the transcriptional coactivator proteins CREB-binding protein and p300 (46, 47, 48) . Competition between NF{kappa}B and p53 for binding to these transcriptional coactivator proteins may dictate the outcome of the responses in different tissues to ionizing radiation and has yet to be investigated.

The discovery that mice respond to total body irradiation with tissue-specific activation of NF{kappa}B and that this activation can selectively protect the intestine from ionizing radiation-induced damage is intriguing. It suggests that additional investigations are required for a better understanding of the role of NF{kappa}B in radiation biology and cancer therapy to provide guidance to the development of targeted NF{kappa}B inhibition as a novel adjuvant therapy for cancer in combination with radiotherapy. Particularly, it has yet to be determined if activation of NF{kappa}B by a clinical relevant dose of fractionated irradiation confers a similar protection against ionizing radiation-induced normal tissue damage. These investigations will be important to determine whether irradiation therapy and NF{kappa}B inhibition should or should not overlap in sensitive normal tissues to avoid an augmented normal tissue injury. On the basis of our finding, we hypothesize that a combination therapy using a NF{kappa}B inhibitor plus ionizing radiation will be useful in treating tumors that are not in close proximity to the small intestine. However, it may carry a significant risk if it is used to treat abdominal tumors, because NF{kappa}B inhibition may also increase ionizing radiation-induced intestinal damage. Likewise, these studies suggest that combination of NF{kappa}B inhibition with chemotherapy may also carry a substantial risk of normal tissue damage due the overlap of the two therapies within the body, which has yet to be determined. This underscores the importance of evaluating the potential clinical complications using ionizing radiation and chemotherapy in combination with NF{kappa}B inhibitors in cancer treatment.


   FOOTNOTES
 
Grant support: NIH (R01-CA78688 and R01-CA86688 to Dr. Daohong Zhou and R01-AG14748; R. Schmiedt) and Veterans Administration (J. Thompson and S. Brown).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Y. Wang and A. Meng contributed equally to this work.

Requests for reprints: Daohong Zhou, Department of Pathology, Medical University of South Carolina, 165 Ashley Ave., Suite 309, Charleston, SC 29425. Phone: (843) 792-7532; Fax: (843) 792-0368; E-mail: zhoud{at}musc.edu

Received 2/18/04. Revised 5/10/04. Accepted 6/22/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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