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Am J Physiol Gastrointest Liver Physiol 291: G1062-G1070, 2006. First published October 12, 2006; doi:10.1152/ajpgi.00129.2006
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HORMONES AND SIGNALING

Azoxymethane protects intestinal stem cells and reduces crypt epithelial mitosis through a COX-1-dependent mechanism

Terrence E. Riehl, Robert J. George, Mark A. Sturmoski, Randal May, Brian Dieckgraefe, Shrikant Anant, and Courtney W. Houchen

Division of Gastroenterology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri

Submitted 22 March 2006 ; accepted in final form 13 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Azoxymethane (AOM) is a potent DNA-damaging agent and carcinogen that induces intestinal and colonic tumors in rodents. Evaluation of the stem cell population by colony formation assay reveals that, within 8 h after treatment, AOM (10 mg/kg) elicited a prosurvival response. In wild-type (WT) mice, AOM treatment induced a 2.5-fold increase in intestinal crypt stem cell survival. AOM treatment increased stem cell survival in cyclooxygenase (COX)-2–/– but not COX-1–/– mice, confirming a role of COX-1 in the AOM-induced increase in stem cell survival. COX-1 mRNA and protein expression as well as COX-1-derived PGE2 synthesis were increased 8 h after AOM treatment. Immunohistochemical staining of COX-1 demonstrated expression of the enzyme in the crypt epithelial cells, especially in the columnar epithelial cells between the Paneth cells adjacent to the stem cell zone. WT mice receiving AOM exhibited increased intestinal apoptosis and a simultaneous reduction in crypt mitotic figures within 8 h of injection. There were no significant differences in baseline or AOM-induced intestinal epithelial apoptosis between WT and COX-1–/– mice, but there was a complete reversal of the AOM-mediated reduction in mitosis in COX-1–/– mice. This suggests that COX-1-derived PGE2 may play a key role in the early phase of intestinal tumorigenesis in response to DNA damage and suggests that COX-1 may be a potential therapeutic target in this model of colon cancer.

Paneth cells; apoptosis; tumorigenesis; colorectal cancer; crypt survival

COLORECTAL CANCER IS THE SECOND leading cause of cancer death in the Western world (24). It is characterized by distinct genetic, morphological, and cellular events. The transformation of a normal mucosal epithelial cell to an invasive colorectal carcinoma occurs via a well-coordinated accumulation of mutations in a series of critical genes (29).

The time span between the initiation and gross development of tumors creates an enormous challenge in dissecting the critical molecular mechanisms that regulate neoplastic change. In the gut, tumorigenesis is thought to arise specifically in the stem cell (29) population located at or near the base of intestinal and colonic crypts. Transit cell populations become fully differentiated and are eventually sloughed into the lumen. Any deleterious effects of mutation in these cells are limited because of the short life span of these transitional zone cells in the intestinal or colonic crypt (16, 29). Identifying and assaying resident intestinal stem cells is quite difficult and contentious, because no definitive specific gut stem cell markers have been identified. However, the microcolony assay following {gamma}-irradiation is by definition a functional assay of intestinal stem cell fate (44) and can provide a mechanism for examining the early events of tumorigenesis. The effects of mutagens on crypt regeneration and stem cell survival can be examined in this well-characterized model, allowing for insights into the molecular events surrounding genotoxic insult. Defining the mechanisms that regulate stem cell fate has important implications in increasing our understanding of the neoplastic process.

Several epidemiological studies have demonstrated a 40–50% reduction in the relative risk of colon cancer and in cancer-related mortality in individuals taking NSAIDs (9, 25, 42). Inhibition of cyclooxygenase (COX)-2 activity and reduction in PGE2 synthesis is thought to represent a major mechanism by which NSAIDs exert their antineoplastic effects (43). Several animal models have been used to validate this hypothesis. Mice that lack either a functional COX-1 or COX-2 gene and are heterozygous for the APC{Delta}716 gene exhibit a reduction in the number of intestinal tumors compared with mice with the APC{Delta}716 mutation alone (4, 5). Furthermore, mice lacking the functional PGE2 receptor EP2 similarly exhibit reduced tumorigenesis in APC{Delta}716 mice (40). More recently, PGE2 has been shown to directly increase motility and enhance the invasiveness of colorectal cancer cells via activation of the EGF receptor (3, 38). These data support a major role for PGE2-mediated signaling in the regulation of the neoplastic process.

PGE2 has a wide variety of biological effects in the gut and has been implicated in tumor initiation, promotion, and maintenance. Kawamori and colleagues have shown that PGE2 enhances azoxymethane (AOM)-dependent tumor formation and that the administration of a specific COX-1 inhibitor caused a decrease in tumor formation in rats treated with AOM (17). Furthermore, they report that the mechanism by which PGE2 enhances carcinogenesis involves induction of cell proliferation and inhibition of apoptosis (14). PGE2 is a well-known inhibitor of the apoptotic response in many cell types subjected to various stimuli. We have previously demonstrated that PGE2 protects intestinal crypt epithelial cells from radiation-induced apoptosis via a COX-1-dependent mechanism (13). In this report, we demonstrate that AOM enhances gastrointestinal COX-1-dependent PGE2 production within hours of exposure. Furthermore, AOM pretreatment increases intestinal crypt stem cell survival following radiation injury by subverting apoptotic surveillance and deletion of damaged stem cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. COX-1–/– and COX-2–/– and wild-type (WT) littermates on the C57Bl/6 background were maintained on a 12:12-h light-dark schedule and fed standard laboratory mouse chow ad libitum. Breeding pairs of B6/129 mice carrying the disrupted gene for COX-1 or COX-2 were obtained from R. Langenbach (National Institute of Environmental Health Sciences, Research Triangle Park, NC). (18, 22). COX-1–/– and COX-2–/– males were crossed with female WT C57BL/6 mice. The resulting heterozygous offspring were backcrossed for 10 generations to WT C57BL/6 mice to obtain COX-1 knockout and COX-2 knockout mice on the C57BL/6 background. All animal procedures were performed in accordance with and approved by the Institutional Review Board at Washington University School of Medicine (St. Louis, MO). Mice were irradiated at age 10 wk in a Gamacel 40 cesium irradiator (Atomic Energy of Canada, Ottawa, ON, Canada) at 0.77 cGy/min.

Crypt survival. Crypt survival was measured in animals 3.5 days after irradiation by a modification of the microcolony assay (6, 26, 44). Each mouse received 120 mg/kg 5-bromo-2'-deoxyuridine (BrdUrd) (Sigma, St. Louis, MO) and 12 mg/kg 5-fluoro-2'-deoxyuridine (Sigma) via intraperitoneal injection to label the S-phase cells 2 h before being euthanized. Paraffin sections (5-µm) were prepared from the distal jejunum oriented so that the sections were cut perpendicular to the long axis of the small intestine. For purposes of the microcolony assay, a regenerative crypt was determined to have survived irradiation on the basis of its histological appearance. The viability of each surviving crypt was confirmed by immunohistochemical detection of BrdUrd incorporation into five or more epithelial cells within each regenerative crypt. The number of surviving crypts per cross section was determined for each mouse by scoring the number of surviving crypts in eight complete, well-oriented cross sections. Because differences in the size of regenerating crypts can affect the probability that a crypt will appear in a cross section (30), we measured 15 representative crypts in longitudinal sections of distal jejunum at their widest point in control and AOM-treated mice. No differences were found in the size of regenerating crypts in COX-1–/–, COX-2–/–, or WT mice before or after AOM treatment (data not shown). Thus differences observed in crypt survival cannot be attributed to variation in the size of regenerating crypts following AOM.

Apoptosis. Mice were euthanized 8 h after AOM injection (10 mg/kg ip), fixed overnight in 10% neutral-buffered formalin, and embedded in paraffin. Sections were prepared from the distal jejunum. Sections were stained with hematoxylin and eosin, and the number of apoptotic cells per crypt was assessed by morphological criteria as previously described (32). Twenty well-oriented crypts were analyzed in each of eight jejunal cross sections for each mouse. Apoptotic cells were also detected in situ by the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay (Boehringer Mannheim, Indianapolis, IN) following the manufacturer’s instructions. The number of apoptotic cells per crypt as assessed with the TUNEL assay was ~30% higher than those assessed by morphological criteria, but the pattern comparing test groups was identical. AOM injections were routinely administered in the morning.

Immunohistochemical analysis. For detection of incorporated BrdUrd into S-phase cells, deparaffinized sections were incubated with PBS containing 2% bovine serum albumin, 0.2% nonfat dry milk, and 0.3% Triton X-100 to block nonspecific protein binding sites and were subsequently incubated with a 1:2,000 dilution of affinity-purified goat anti-BrdUrd (Accurate Chemical) at 4°C overnight. Bound anti-BrdUrd was subsequently visualized with a biotin-labeled rat anti-goat IgG (Accurate Chemical, Westbury, NY). For immunohistochemical localization of mouse COX-1, deparaffinized sections of Bouin’s fixed tissue were incubated with a 1:2,000 dilution of rabbit anti-mouse COX-1 (a gift from J. L. Masferrer, Pfizer, St. Louis, MO) following quenching of endogenous peroxidase activity with 1% hydrogen peroxide. Sections were incubated with biotin-labeled donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories) and subsequently with streptavidin-horseradish peroxidase. Immune complexes were visualized by tyramide signal amplification (TSA direct; Dupont NEN Life Science Products, Boston, MA) according to the manufacturer’s directions.

COX inhibitors. Indomethacin (Sigma) was dissolved in ethanol and diluted into sterile 5% sodium bicarbonate immediately before use. Indomethacin (1.5 mg/kg) was administered by intraperitoneal injection. The selective COX-2 inhibitor NS-398 (BioMol Research Laboratories, Plymouth Meeting, PA) (19) was dissolved in Methocel and administered intraperitoneally at the indicated times before irradiation. The dosage of NS-398 (1 mg/kg) was based on the dose that inhibited PGE2 production in vivo in COX-2-dependent animal models (19) without affecting synthesis of PGE2 through COX-1.

RNA detection. For expression of COX-1 and COX-2 mRNA, total RNA samples were extracted from the distal jejunum as described above by using TRIAZOL (GIBCO-BRL, Bethesda, MD) according to the manufacturer’s directions. Total RNA was subjected to reverse transcriptase followed by real-time quantitative RT-PCR analyses. As a control, GAPDH expression was determined. The final results were expressed as fold differences in COX-1 or COX-2 gene expression relative to the GAPDH gene. Experiments were performed in triplicate for each data point. For all experiments, controls without templates were included.

Measurement of PGE2 levels. Lipids were extracted by homogenizing flash-frozen tissue in cold ethanol-0.1 M sodium phosphate [pH 4.0, 70:30 (vol/vol)] followed by shaking incubation at room temperature. An aliquot of the extract was dried down under a stream of nitrogen, and the PGE2 concentration was determined by a PGE2-specific ELISA (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s directions.

SDS-PAGE and Western blot analysis of COX-1 and COX-2. Distal jejunum from 10-wk-old untreated and AOM-treated mice were homogenized in ice-cold lysis buffer (1 ml; PBS + 10 mM EDTA, 1% Triton X-100, 0.5% deoxycholic acid, and 1 mM diethyldithiocarbamic acid) containing leupeptin (10 µM), pepstatin A (1.5 µM), and aprotinin (0.2 U/ml). Equal amounts of proteins (30 µg) from the controls and AOM-treated mouse intestinal lysates were separated by SDS-PAGE on 7.5% polyacrylamide gels and transferred to an Immobilon membrane (Millipore, Bedford, MA). COX-1 and COX-2 proteins were detected with rabbit antibodies and visualized by using a donkey anti-rabbit IgG linked to horseradish peroxidase and ECL reagent (Amersham) with fluorographic detection on Kodak BioMax MR film. To evaluate protein loading on the gels, blots were reprobed with antibody to beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Statistical methods. Pairwise t-tests using the pooled estimate of variance and Bonferroni’s correction of the P values for multiple comparisons were used for analysis of the effects of COX genotypes on crypt survival and AOM-induced apoptosis. Pairwise Wilcoxon Mann-Whitney tests with Bonferroni’s adjustment of P values for multiple comparisons were used for analysis of the effects of AOM and COX genotype on intestinal PGE2 levels.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
AOM is radioprotective in intestinal crypts. This study explored the role of AOM in intestinal crypt dynamics by testing the effect of AOM on intestinal crypt number and stem cell replication in adult WT, COX-1–/–, and COX-2–/– C57BL/6 mice (Fig. 1). In WT mice, treatment with AOM (10 mg/kg ip) 8 h before euthanasia had no effect on the total number of crypts per cross section or the proportion of S-phase cells during the 2-h pulse-labeling period (Fig. 1, A and B). Irradiating mice with 12 Gy resulted in the majority of crypt epithelial cells being destroyed, and the surviving stem cells proliferated to form regenerative crypts by 3.5 days (Fig. 1, C and D). Treatment by AOM alone had no effect on crypt number or mucosal integrity at 3.5 days (data not shown). AOM treatment of WT mice 8 h before irradiation resulted in an increased number of surviving crypts (Fig. 1D), as shown by BrdUrd staining in regenerative crypts (Fig. 1F). To define the mechanism by which AOM exerts its effect on intestinal crypt stem cell survival, we examined its interaction with prostaglandins. We had previously demonstrated that prostaglandins were able to confer radioprotection on the intestinal epithelium. The experimental dosing schedule is illustrated in Fig. 2A. Crypt survival was determined in irradiated WT mice pretreated with AOM and either with the nonspecific COX inhibitor indomethacin or with the COX-2 specific inhibitor NS-398 (Fig. 2B). Mice treated with AOM 8 h before irradiation had 2.5-fold more surviving crypts per cross section than untreated animals. When indomethacin (1.5 mg/kg) was given before AOM, there was no increase in the number of surviving crypts. Animals treated with NS-398 (1 mg/kg) and AOM, however, still exhibited an increase in crypt survival similar to AOM alone. These data suggest that COX-2-specific inhibition failed to attenuate AOM-mediated changes that conferred radioprotection in the cell, whereas inhibition of both COX-1 and COX-2 with indomethacin abolished the AOM-mediated increase in stem cell survival. A dose of 1 mg/kg NS-398 has been shown previously to block LPS-induced increase in COX-2-derived PGE2 in vivo (33).


Figure 1
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  		Fig. 1. Azoxymethane (AOM) increases intestinal transit cell replication and crypt stem cell survival following 12 Gy {gamma}-irradiation. Unirradiated mice were treated with vehicle (A) or AOM (10 mg/kg) (B) at 8 h before being euthanized. Irradiated mice were treated with vehicle (C) or AOM (D and F) at 8 h before receiving 12 Gy and were euthanized at 3.5 days after irradiation. Animals received 5'-bromo-2'-deoxyuridine (BrdUrd) 2 h before being euthanized to label S-phase cells. BrdUrd-incorporating cells were detected by goat anti-BrdUrd and visualized by use of a biotin-labeled rat anti-goat IgG. BrdUrd was incorporated into replicative cells in all crypts of unirradiated mice, and AOM had no apparent effect on the number of S-phase cells of intestinal crypts in unirradiated mice. The histological appearance of regenerative crypts 3.5 days after irradiation is shown in C and D. Note the substantial increase in the number of surviving crypts in irradiated mice that received AOM (D) compared with controls (C). Higher power magnification (E and F) (x400) demonstrates intense BrdUrd staining in the regenerative crypts, confirming the viability of these surviving crypts (original magnification: A and B, x100; C and D, x200; E and F, x400).

 

Figure 2
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  		Fig. 2. A: Cyclooxygenase (COX) inhibitor dosing schedules in crypt survival experiments. Mice received 5% sodium bicarbonate or AOM (10 mg/kg ip) 8 h before 12 Gy {gamma}-irradiation. Animals that received indomethacin or NS-398 were given a single dose 1 h before vehicle or AOM. All mice were euthanized at 3.5 days after being irradiated and received BrdUrd 2 h before death. B: effect of indomethacin and NS-398 on AOM-mediated crypt survival. AOM (10 mg/kg) treatment resulted in a 2.5-fold increase in crypt survival at 3.5 days after 12 Gy. The nonspecific COX inhibitor indomethacin (Indo), but not the COX-2 specific inhibitor NS-398, blocked the AOM induced increase in crypt survival. Data are shown as the mean ± SE (n = 4 mice/group). *P < 0.05 for AOM compared with control. **P < 0.05 AOM plus NS-398 compared with control. C: effect of AOM on crypt survival in COX-1–/– and COX-2–/– mice. COX-1–/–, COX-2–/–, and wild-type (WT) littermates received AOM 8 h before 12 Gy {gamma}-irradiation and were killed 3.5 days later. AOM induced an increase in crypt survival in WT and COX-2–/– mice but had no radioprotective effect in COX-1–/– mice. Data are the mean number of surviving crypts per cross section ± SE (n = 4 mice per group). *P < 0.04 for AOM compared with control in WT mice. **P < 0.005 AOM compared with control in COX-2–/– mice.

 
To confirm the role of COX-1 in mediating the AOM-dependent increase in intestinal stem cell survival, COX-1–/– and COX-2–/– mice were treated with 10 mg/kg AOM before irradiation (Fig. 2C). Irradiated control COX-1–/– mice had lower baseline crypt survival compared with WT mice as previously described (13), whereas COX-2–/– mice had baseline crypt survival similar to WT mice. Treatment of COX-1–/– mice with AOM failed to induce an increase in intestinal stem cell survival (Fig. 2C), whereas AOM treatment of COX-2–/– mice resulted in a fourfold increase in number of surviving crypts after 12 Gy (Fig. 2C). These data demonstrate that the AOM enhancement of stem cell survival was dependent on functional COX-1 expression.

AOM induces synthesis of intestinal PGE2. To define the role of endogenous PGE2 synthesis in the AOM-induced increases in intestinal stem cell survival, PGE2 was measured by ELISA in the proximal jejunum of mice 8 h after AOM treatment. Baseline intestinal PGE2 levels in control mice were ~40 pg/mg, which is consistent with levels observed in our previous studies. At 8 h, the time at which AOM conferred radioprotection on the stem population in the intestine, PGE2 was increased fourfold (Fig. 3).


Figure 3
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  		Fig. 3. Effect of COX inhibition on AOM-induced intestinal PGE2 levels in WT and COX-1–/– mice. Mice received either vehicle (controls) or AOM (10 mg/kg) 8 h before death. The nonspecific COX inhibitor Indo (1.5 mg/kg), and the COX-2-specific inhibitor NS-398 (1 mg/kg ip) was given at 1 h before AOM. AOM induced a 4-fold increase in PGE2 at 8 h in the jejunum of WT mice, and this increase was blocked by Indo but not by NS-398. AOM had no effect in COX-1–/– mice. Data are means ± SE (n = 6 mice/group). PGE2 levels are expressed as means ± SE; n = 6 mice/group. *P < 0.05 AOM WT vs. control WT, **P < 0.05 NS-398+AOM vs. control WT.

 
AOM-mediated increases in PGE2 levels in the intestine are COX-1 dependent. To determine whether the increased PGE2 synthesis following AOM administration in mice is COX-1 or COX-2 mediated, we measured intestinal PGE2 levels in WT and COX-1–/– mice (Fig. 3). In the distal jejunum, baseline PGE2 levels in COX-1–/– mice were about half that of WT mice. AOM treatment failed to cause an increase in PGE2 levels in COX-1–/– mice by 8 h, suggesting that the AOM-mediated increases in intestinal PGE2 synthesis are COX-1 dependent.

Because AOM has been shown to cause an increased synthesis of COX-2 in AOM tumor models (7, 21) and in view of the reported effects of COX-2 inhibitors on AOM-induced tumorigenesis (7, 8), we sought to examine the role of COX-2 in PGE2 synthesis induced 8 h after treatment with AOM. In adult WT mice, NS-398 given before AOM administration failed to inhibit the increase in PGE2 synthesis at this early time point (Fig. 3). Indomethacin, however, completely blocked AOM induction of PGE2 synthesis in the intestine (Fig. 3). These data taken together support our hypothesis that the immediate AOM-induced increases in intestinal PGE2 synthesis are COX-1 derived.

Levels of COX-1 mRNA and protein are increased following AOM administration. The increase in PGE2 synthesis following AOM treatment was COX-1 dependent and may result from increased synthesis via endogenous COX-1 or increased arachidonate availability, or it may be a result of upregulation of COX-1. To determine whether COX-1 was induced at 8 h by AOM, mRNA and protein levels of COX-1 and COX-2 were determined in the proximal jejunum of control and AOM-treated mice. Relative COX-1 and COX-2 mRNA was determined by real-time PCR. AOM treatment resulted in a threefold increase in COX-1 mRNA in the intestine (Fig. 4A) but no increase in COX-2 mRNA. It should be noted that at baseline there was ~30-fold more COX-1 than COX-2 mRNA in the intestine (data not shown). By Western blot analysis, there was a 2.0-fold increase in COX-1 protein 8 h after AOM administration (Fig. 4B).


Figure 4
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  		Fig. 4. A: AOM induced COX-1 mRNA in mouse intestine. WT mice were treated with AOM (10 mg/kg) 8 h before death, and mRNA for COX-1 and COX-2 were by real-time PCR. AOM induced a 3-fold increase in COX-1 message, but there was no induction of COX-2. Data are means ± SE (n = 3 mice/group). B: Western blot analysis of COX-1 protein expression in the AOM-treated mice. Lysates from jejunum were prepared from unirradiated control and AOM-treated mice at 8 h after a single dose of AOM (10 mg/kg ip). Equal amounts of protein from control and AOM-treated mouse intestinal lysates were separated by electrophoresis on 10% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes, and bands corresponding to COX-1 and actin were detected by enhanced chemiluminescence by using rabbit antibodies against COX-1 and actin. C: immunohistochemical localization of COX-1 in the distal jejunum of control and AOM-treated mice. The cellular localization of COX-1 in epithelial cells of crypts and lower regions of the villi was the same in control (a) and AOM-treated mice (c). Higher power views of the intestines from control and AOM-treated mice show cytoplasmic staining of crypt epithelial cells with sparing of the Paneth cells. Arrows in b and d point to staining of columnar epithelial cells interspersed between Paneth cells. Original magnification: a and b, x200; c and d, x400.

 
COX-1 is expressed in crypt epithelial cells. Immunohistochemistry for COX-1 was carried out to identify the cell types involved in the AOM-induced radioprotection in the intestine. Figure 4 shows that COX-1 protein (Fig. 4C) was expressed in intestinal crypt epithelial cells both in controls and in AOM-treated WT mice. Prominent staining was present throughout the crypt and extended to cells in the lower portion of the villi (Fig. 4Cd). Paneth cells were almost completely devoid of COX-1. At higher power (Fig. 4Cc–d), COX-1 staining in columnar epithelial cells immediately adjacent to Paneth cells was evident (Fig. 4Cc–d arrows). WT mice had only scattered COX-2 expression either at baseline or after AOM (data not shown), consistent with data published previously (34).

Azoxymethane induces crypt epithelial apoptosis in the small intestine. The short-term effects of AOM on intestinal epithelial apoptosis were examined in WT adult C57/BL6 mice. Apoptotic cells were determined by using morphological criteria on hematoxylin and eosin-stained sections and confirmed by TUNEL staining (Fig. 5A) (20). In WT and COX-1–/– control mice, baseline apoptosis was extremely rare in the small intestine (0.2 apoptotic cells per crypt). At 8 h after AOM administration, the number of apoptotic cells increased by 3.5-fold. To determine whether endogenously produced prostaglandins prevented AOM-induced apoptosis, we examined crypt epithelial apoptosis at 8 h after AOM in COX-1–/– mice (Fig. 5A). PGE2 has been previously demonstrated to be protective against various apoptotic stimuli including radiation injury (13). COX-1–/– mice displayed the same degree of apoptosis as WT mice (0.732 vs. 0.786 apoptotic cells/crypt, P = 0.26), indicative that COX-1-mediated prostaglandins do not protect against AOM-induced apoptosis in crypt epithelial transit cells in the small intestine.


Figure 5
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  		Fig. 5. A: AOM-induced intestinal (proximal jejunum) apoptosis in WT and COX-1–/– mice. Unirradiated mice received either vehicle or AOM (10 mg/kg) and were euthanized 8 h later. Baseline apoptosis was the same in WT and Cox-1–/– mice. AOM induced a 3.5-fold increase in intestinal apoptosis in both WT and Cox-1–/– mice. Thirty well-oriented crypts were analyzed in each of eight jejunal cross sections for each mouse. Data are expressed as means ± SE (n = 3 mice/group *P < 0.002 AOM vs. WT control, **P < 0.001 AOM vs. COX-1–/– control mice). B: AOM-induced intestinal (jejunum) apoptosis in WT mice. Representative section from a WT mouse that received AOM and was killed 8 h later. Apoptotic nuclei (arrows) are detected in the intestinal crypts. Apoptosis in intestinal crypts was determined by using previously described histological criteria and confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay (brown).

 
AOM-mediated reduction in mitoses is COX-1 mediated. In addition to inducing apoptosis, AOM and other DNA-damaging agents reduce the number of mitotic figures observed in colonic crypt epithelial cells (11). To determine whether AOM mediated a similar reduction in epithelial mitosis in the small intestine and whether the reduction is COX-1 dependent, we quantified crypt epithelial mitotic figures in WT and COX-1–/– mice. The appearance of crypt epithelial mitotic figures is shown in Fig. 6A. The baseline number of mitotic figures per crypt was the same in WT and COX-1–/– mice (0.547 vs. 0.540, P = 0.91; Fig. 6B). At 8 h after AOM administration there was a 60% reduction in the number of crypt epithelial mitotic figures in WT mice (Fig. 6B). In COX-1–/– mice, AOM had no effect on the number of mitotic figures observed; however, the mitotic figures were frequently TUNEL positive, indicating that these cells were undergoing apoptosis or mitotic catastrophe (Fig. 6, C and D). In WT mice, crypt epithelial mitotic figures, although reduced in number, rarely stained TUNEL positive.


Figure 6
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  		Fig. 6. A: histochemical identification of mitotic figures in WT mice. Representative jejunal section from an untreated adult WT mouse at baseline is shown. Note the mitotic figures in crypt epithelial cells (arrows). B: histochemical identification of mitotic figures in WT and COX-1–/– mice. Identification of mitotic figures (arrowheads) in crypt epithelial cells of WT (a) and COX-1–/– mice (b) mice at 8 h after AOM (10 mg/kg) is shown. Note that apoptotic bodies are also present (arrows). C: effect of AOM on mitosis in mouse intestine. The baseline mitotic index in WT and COX-1–/– mice was the same. AOM (10 mg/kg) treatment at 8 h before death caused a 60% decrease in the occurrence of mitotic figures in WT mice but had no effect in COX–/– mice. *P < 0.002, WT AOM vs. COX-1–/– AOM; n = 3 mice/group. D: histochemical localization of apoptotic and mitotic figures in a COX-1–/– mouse. The morphology of apoptotic and mitotic figures (M) in a representative intestinal crypt cross section 8 h after AOM is shown. Apoptotic cells (arrows) are easily identified via their morphological appearance as well by TUNEL staining (brown). Several cells appear to be undergoing mitosis and apoptosis (MC). This likely represents programmed cell death in cells attempting mitosis.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have examined the early molecular response of intestinal stem cells to genotoxic insult by AOM. The results demonstrate that AOM elicits in the first few hours after treatment an increased prosurvival response specific to the functional stem cells. The survival response is mediated through a COX-1-dependent increase in PGE2. COX-2 had no role in AOM-induced PGE2 synthesis or crypt regeneration. COX-2 had no role in AOM-induced PGE2 synthesis or crypt regeneration.

AOM, a chemical and metabolic derivative of 1,2-dimethylhydrazine, is a powerful and specific colon carcinogen in rodents (10). AOM-induced aberrant crypt foci have been used as putative preneoplastic markers to evaluate colonic carcinogenesis (1, 41). AOM is metabolized to methylazoxymethanol by cytochrome P-450 (CYP)2E1 in the liver, which can methylate DNA (10, 39). DNA methylation results in a complex multistep sequence of tumorigenesis. AOM-treated rodents exhibit light microscopic and histological features similar to those observed in human disease (36, 37). Moreover, AOM induces both adenomas and carcinomas in some models.

Measuring crypt stem cell survival by use of the microcolony formation assay provides a surrogate means of assessing the cellular response to AOM. Scored by the microscopic appearance of regenerating crypts 3.5 days after severe radiation injury, this assay measures the clonogenic capacity of the remaining stem cells (44). If a crypt contains a surviving stem cell, it will proliferate to form a regenerative crypt and these regenerative crypts will ultimately repopulate the entire intestinal epithelium (28). Agents given before irradiation can modulate the cellular response to radiation injury in the intestine. IL-1, IL-11, PGE2, transforming growth factor-beta, and FGF-2 are radioprotective when given before radiation in that they increase the number of surviving crypts after radiation (2, 12, 27). Radioprotection initiated by agents described above potentially act through several different mechanisms. They may protect against DNA damage, cause cell cycle arrest, promote DNA repair, or diminish stem cell apoptosis.

In this study, WT, COX-1–/–, and COX-2–/– mice on the C57BL/6 background were used to explore the roles of COX-1- and COX-2-derived PGE2 in AOM-induced changes in the gut that increase stem cell survival. AOM treatment led to radioprotection in both WT and COX-2–/– mice but not in COX-1–/– mice. This finding was reproduced when COX activity was inhibited pharmacologically before administration of AOM. The events initiated by AOM administration leading to protection from radiation injury and increased stem cell survival appear to be the induction of COX-1 and subsequent synthesis of COX-1-derived PGE2. AOM induced only COX-1 and not COX-2 mRNA and protein expression in the intestine. This is an important observation because COX-1 has been considered to be a strictly constitutively expressed enzyme and COX-2 is considered to be the inducible form. An expanding body of evidence demonstrates that COX-1 can also be induced by cytokines under appropriate circumstances. For example, c-kit ligand, either alone or in combination with IL-3, IL-9, or IL-10, was able to induce expression of COX-1 in mouse mast cells (23). Similarly, stem cell factor, especially when combined with dexamethasone, can induce COX-1 expression (35). The increased synthesis of PGE2 at 8 h after AOM is COX-1 derived. AOM did not increase PGE2 production in mice that lack functional COX-1, whereas mice that lack functional COX-2 exhibited markedly increased PGE2 production. In WT mice treated with the nonspecific COX inhibitor indomethacin, the AOM-induced increase in PGE2 production was completely abolished. Conversely, the COX-2-specific inhibitor NS-398 failed to inhibit the AOM-induced PGE2 production. These data taken together demonstrate that COX-1 is required for the AOM-mediated increase in PGE2 production in the intestine and that COX-1-derived PGE2 is likely responsible for the AOM-induced increased stem cell survival in the mouse intestine.

Although these studies define one of the molecular intermediates involved in AOM-induced increased stem cell survival in the mouse intestine, they do not absolutely define the cell types involved. The immunohistochemical data suggest that COX-1-expressing crypt epithelial cells in the stem cell zone are the source of the AOM-induced radioprotective prostaglandin. We have not excluded other potential cell types including endothelial cells, fibroblasts, or other inflammatory cell types. However, prostaglandins are rapidly metabolized, and, as a consequence, they usually act on the cells in which they are produced or on adjacent cells. Villus epithelial cells are a considerable distance from crypt epithelial stem cells, and it is unlikely that prostaglandins made by villus cells would act on crypt stem cells. COX-2 is expressed predominantly on villi, which may provide another explanation as to why COX-2 does not appear to play a role in protecting stem cells in this experimental model. Subepithelial myofibroblasts are immediately adjacent to stem cell zone, and prostaglandins produced by these COX-2-expressing cells could affect the stem cell response to radiation injury (33). In this study there was no AOM-induced COX-2 staining in subepithelial myofibroblasts, nor was there any upregulation of COX-2 mRNA or protein following AOM. Thus COX-1 expression in the cytoplasm of crypt epithelial cells in the putative stem cell zone as well as in columnar epithelial cells interspersed between the Paneth cells could potentially act as the PGE2 source required for the AOM-induced increased stem cell survival. It has been proposed that these cells may act as stem cells and express the putative stem cell marker protein Musashi-1 (15, 31).

Treatment of mice with AOM has been shown to induce intestinal epithelial cell apoptosis and to reduce the appearance of mitotic figures (11, 47). In the present study, AOM induces apoptosis equally in the intestine of both WT and COX-1–/– mice (Fig. 6A). These data suggest that COX-1-derived endogenous PGE2 plays little or no role in protection of the non-stem-cell epithelium from AOM-induced apoptosis in the intestine. Although AOM elicits an equal degree of apoptosis in WT and COX-1–/– mice, there is a greater selective degree of protection of the functional stem cells in WT mice compared with COX-1–/– mice as measured by the surrogate measure of crypt survival. AOM reduces intestinal mitotic figures in the stem cell zone of the crypt in WT mice but not in COX-1–/– mice. The mitotic figures observed in COX-1–/– mice are often TUNEL positive. In the absence of PGE2, damaged cells progress through the cell cycle to mitosis where they accumulate as a result of mitotic catastrophe (Fig. 6C). These data suggest that, following AOM-induced DNA damage, PGE2 is an important participant regulating cell cycle delay and/or DNA repair. This enables the DNA repair process to proceed, particularly in the stem cell population or in cells that have clonogenic capacity.

The microcolony assay in the small intestine has allowed us to ask how the immediate effects of AOM alter the stem cell population. We have presented evidence that changes in COX-1-derived prostaglandin levels in the appropriate region of the crypt are related to the increase in stem cell survival elicited by AOM. In the colon, where AOM initiates tumor formation, AOM also induces COX-1 expression and increases COX-1-derived PGE2 within 8 h of exposure (unpublished observations). Although we are unable to determine, for lack of an assay comparable to the microcolony assay in the small intestine, whether AOM induces an early COX-1-dependent increase in stem cell survival in the colon, Kawamori et al. have shown that PGE2 enhances AOM-dependent tumor formation and that the administration of a specific COX-1 inhibitor caused a decrease in tumor formation in rats treated with AOM (17). An increase in COX-1 derived PGE2 and the potential increase in stem cell survival may contribute to AOM tumor initiation in the colon by subverting the surveillance function served by mitotic catastrophe. Cells that fail to adequately repair but that escape mitotic failure are likely to divide asymmetrically in the next round of cell division, with the consequent generation of aneuploid cells.

This study suggests that specific inhibition of COX-1 in the early stages of AOM-induced DNA damage may lead to reduced stem cell survival and thus attenuate the initiation phase of tumorigenesis and neoplasia in the AOM tumor model. These findings suggest that COX-1 inhibition may be an excellent target for chemoprevention strategies. Furthermore, evaluation of the effects of chemoprevention and chemotherapeutic agents on mitotic catastrophe should be explored.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-066161 and DK-002822 and by Washington University Digestive Disease Research Cores Center Grant P30-DK-52574 (to C. W. Houchen) and by DK-62265 (to S. Anant).


   ACKNOWLEDGMENTS
 
We thank Dr. William F. Stenson for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. W. Houchen, Div. of Digestive Diseases and Nutrition, University of Oklahoma Health Sciences Center, PO Box 26901, WP 1360, Oklahoma City, OK 73190 (e-mail: courtney-houchen{at}oumsc.edu)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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