Hemizygous mice for the angiotensin II type 2 receptor gene have attenuated susceptibility to azoxymethane-induced colon tumorigenesis

Tetsuo Takagi1, Yuichiro Nakano1, Susumu Takekoshi2, Tadashi Inagami1 and Masaaki Tamura1,3

1 Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, TN 37232, USA and
2 Department of Pathology, Tokai University, School of Medicine, Isehara, Kanagawa 259-1193, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Evidence suggests that the use of angiotensin-converting enzyme inhibitors potentially reduces the risk of cancer, though the mechanism is unclear. To clarify a potential involvement of angiotensin II (Ang II) signaling in cancer risk, we have examined the effect of Ang II receptor deficiency on azoxymethane (AOM)-induced colon tumorigenesis. Male Ang II type 2 receptor gene-disrupted (AT2-null) mice with a 129/Ola and C57BL/6J genetic background, AT2-null mice with an SWR/J genetic background, and their corresponding control wild type mice were treated once a week with AOM (10 mg/kg, i.p., 4 consecutive weeks) or saline vehicle. All mice were killed 23–26 weeks after the initial injection of AOM, and tumor burdens were examined. AOM treatment caused the development of colon tumors in all wild type control mice regardless of genetic background (100% tumor prevalence), but only one tumor was present in AT2-null mice with a 129/Ola and C57BL/6J genetic background (11.1% tumor prevalence). Although the introduction of the AOMsusceptible SWR/J genetic background induced AOM susceptibility in AT2 null mice, the tumor multiplicity (6.3) and tumor size (19.8 ± 3.0 mm3) were significantly smaller than those in wild type mice (multiplicity, 12.0 and size, 36.8 ± 3.2 mm3). AOM efficiently downregulated cytochrome P450 2E1 (CYP2E1) in the liver of wild type mice significantly more than in AT2-null mice. The levels of DNA methyl adducts formed in wild type mouse colon epithelium by AOM treatment were also significantly higher than in AT2-null mice. These results imply that the AT2 receptor functions to augment AOM-induced downregulation of CYP2E1 expression in the liver, and thus increases AOM-induced tumorigenesis in the colon. The AT2 receptor function in the liver may be a potential determinant of tumor susceptibility in chemical carcinogen-induced colon tumorigenesis.

Abbreviations: Ang II, angiotensin II; AT1, angiotensin II type 1 receptor; AT2, angiotensin II type 2 receptor; AOM, azoxymethane; CYP2E1, cytochrome P450 2E1; EDTA, ethylenediaminetetraacetic acid; ECL, enhanced chemiluminescence; H&E, hematoxylin and eosin; RT-PCR, reverse transcription-polymerase chain reaction; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been suggested that angiotensin-converting enzyme inhibitors which are commonly employed in the treatment of human clinical hypertension also attenuate tumor growth in experimental animals (1–4) and may reduce the risk of several human cancers (5). Angiotensin-converting enzyme inhibitors block the formation of angiotensin II (Ang II), an octapeptide which exerts the many diverse effects of the renin–angiotensin system through its receptors (6). This suggests that Ang II may have a modulating role in neoplasia.

There are two well-defined receptors of Ang II (7). The major isoform type 1 (AT1) receptor is expressed in a wide variety of tissues (7). The Ang II-AT1 receptor-mediated signal effects a variety of pathophysiological reactions, including constriction of blood vessels, secretion of mineralocorticoids, expression of protooncogenes such as c-fos, c-myc and c-jun, and promotion of cell proliferation (8,9). It also stimulates neovascularization, which is required for solid tumor growth (10,11). The second major isoform of the Ang II receptor, designated AT2, is expressed in a smaller quantity but is inducible and apparently functional under pathophysiological conditions (12,13). The Ang II-AT2 receptor frequently mediates signals that counteract the AT1 receptor-mediated biological actions (14,15). Although the roles of Ang II receptors in neoplasia have not been rigorously studied, Ang II receptors may be subtype-specifically involved in tumorigenesis. In order to evaluate this hypothesis, we have chosen to use male AT2 receptor-null (Agtr2–/y) mice, since AT2-null mutant mice fertilize and develop normally without apparent phenotype expression (16,17), whereas AT1 receptor-null mutant mice have obvious defects in body weight gain and morphological abnormalities in the kidney and heart (18,19). The study of tumorigenesis using AT2-null mice is feasible, and results should be comparable with effects observed in control wild type mice.

In the present study, we examined the effect of the AT2 receptor deficiency on azoxymethane (AOM)-induced colon tumorigenesis in mice. In addition to the genetic manipulation of the receptor, we also studied the effect of pharmacological attenuation of the AT2 receptor function on the expression of hepatic cytochrome P4502E1, which mainly catalyzes the biotransformation of AOM and resultant DNA adduct formation in the target tissue (20).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Ang II was purchased from Peninsula Laboratories (Belmont, CA). Azoxymethane (AOM) and the AT2 receptor blocker PD123319 were from Sigma Chemical Co. (St Louis, MO). The protease inhibitor cocktail Complete was from Boehringer Mannheim (Mannheim, Germany). Anti-human cytochrome P4502E1 polyclonal antibodies were raised in Dr F.P.Guengerich's laboratory (Vanderbilt University) (21) and were presented as a gift. The anti-O6-methyldeoxyguanosine monoclonal antibody was a generous gift from Dr M.F.Rajewsky (Institute of Cell Biology, University of Essen Medical School, Germany). The enhanced chemiluminescence (ECL) Western blotting detection system was from Amersham Pharmacia Biotech (Piscataway, NJ). Primers for PCR were synthesized by the DNA Synthesis and Reagent Supply Core facility in the Vanderbilt University Diabetes Center. All other chemicals were of analytical grade.

Animals and genotyping
The original male hemizygote AT2-null mutant (Agtr2–/y) mice were the offspring of Agtr2 deletion mutants produced by homologous recombination in embryonic stem cells derived from strain 129/Ola(17). Chimeric males were mated with C57BL/6J females such that the genetic background of the mutants consisted of 129/Ola and C57BL/6J. Wild type male littermates served as controls. In order to introduce a tumor-susceptible genetic background, heterozygote female mice (Agtr2–/x) were mated with SWR/J male mice. F2 male hemizygote AT2-null mice were compared with wild type male littermates (Agtr2x/y) in the study. Southern blot analysis of tail DNA was used to screen for the Agtr2 genotype as previously described (17). All animals were maintained in a humidity- and temperature-controlled room on 12 h light/dark cycles. All procedures for handling animals were approved by the Institutional Committee for Animal Care and Use of Vanderbilt University.

Experimental protocol for azoxymethane administration in vivo
All AOM-handling procedures were approved by the office of Safety and Environmental Health of Vanderbilt University. Ten-week-old male wild type and AT2-null mice (minimum 5 mice/group) received regular mouse chow (#5015, Purina Mills, Inc., Indianapolis, IN). Mice were treated with four consecutive weekly administrations of AOM (10 mg/kg, i.p.) for tumorigenic study or with a bolus intraperitoneal administration of AOM (10 mg/kg) for short-term study. AT2 receptor blocker PD123319 treatment (15 mg/kg/12 h, gavage administration and 50 µg/ml in drinking tap water) was initiated 3 h prior to the AOM treatment. The control group for the AOM treatment received saline. Mice for the short-term study were killed 6 h or 24 h after AOM treatment. Mice for the tumorigenic study were killed 23–26 weeks after the first AOM treatment. The colons and livers were macroscopically examined. For quantitative estimation of tumor burdens, the colons were removed, longitudinally opened and placed flat on filter paper. Tumor size was measured with a caliper. The measurements were carried out blindly by a single observer throughout the study. Randomly selected tumors and adjacent normal tissues were fixed with 10% formalin, sectioned, and stained with H&E for histological examination. The remaining tissues were frozen in liquid nitrogen and stored at –80°C.

Preparation of microsomal fractions
Tissues were individually homogenized by a Polytron homogenizer in three volumes of 1 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA, 0.25 M sucrose and protease inhibitor cocktail. The supernatant from 10 000 g centrifugation of the homogenate for 15 min was further centrifuged at 100 000 g for 1 h. The microsomal membranes were suspended in 100 mM NaH2PO4, pH 7.4, 10 mM MgCl2, 20% glycerol, and protease inhibitor cocktail, and 25 µg of the membrane protein was subjected to western blot analysis.

Western blot analysis.
The liver microsomal membranes were lysed with 0.5 ml lysis buffer (10 mM Tris-HCl (pH 7.4), 1% SDS). After sonication, the lysate was boiled for 5 min. The boiled lysate was subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to a nitrocellulose membrane by electroblotting. The membrane was blocked for 16 h at 4°C with non-fat milk in Tris-buffered saline (TBS, 10 mM Tris (pH 7.5), 100 mM NaCl) containing 0.1% Tween-20, and then incubated with anti-rabbit anti-human CYP2E1 antibodies for 1 h. After washing, the membrane was incubated for 1 h with goat anti-rabbit IgG conjugated with horseradish peroxidase; peroxidase activity was visualized with an ECL Western blotting detection system.

DNA isolation
Immediately after the mice were killed by cervical dislocation, the colon was excised, washed with phosphate-buffered saline, divided into proximal and distal segments, and snap-frozen in liquid N2. Small portions of each lobe of the liver were also removed and frozen in the same manner. Tissue was digested in a lysis buffer consisting of 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% SDS, 5X SSC and 0.2 mg/ml Proteinase K. DNA was extracted by phenol/chloroform/isoamyl alcohol and precipitated with ice-cold ethanol by standard protocols. Samples were then treated with RNase A (50 mg/ml) and DNA was fragmented by sonication. The concentration of DNA was determined by the absorbance at 260 nm.

Immuno-slot-blot assay
The immuno-slot-blot method described earlier (22) was used with the following modifications. DNA samples were heat-denatured for 10 min, immediately chilled on ice, and mixed with an equal volume of 2 M ammonium acetate. The resulting single-stranded DNA was then immobilized on a nitrocellulose membrane and fixed to the membrane by UV crosslinking. The membrane was then treated with TBS containing 5% skimmed milk for 2 h. The membrane was first incubated overnight at 4°C with a monoclonal antibody raised against O6-methyldeoxyguanosine. After washing, the membrane was incubated for 1 h with goat anti-rabbit IgG conjugated with horseradish peroxidase (Amersham Life Sciences). DNA-methyl adducts were visualized using the ECL Western blotting detection system. Relative blot intensities were measured by densitometry using a Fluor-S image analyzer (Bio-Rad Laboratories, Hercules, CA).

Isolation of total RNA and reverse transcription-polymerase chain reaction (RT-PCR) of AT2 receptor mRNA
Liver and colonic tissues were ground in liquid nitrogen and total RNA was extracted by TRI reagent (Sigma, St Louis, MO) according to the manufacturer's protocol. RT-PCR was carried out using the same conditions and primers for the AT2 receptor as described previously (13).

Statistical analysis
Data obtained from the western blot analysis and immuno-slot-blot assay were averaged and are presented as means ± SE. Significant differences between groups were evaluated by one-way analysis of variance with the Student–Newman–Keuls test. A value of P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Identification of genotype and AT2 receptor mRNA expression
The genotype and expression of the mRNA were confirmed by Southern blot analysis and RT-PCR, respectively. Genomic DNA of male wild type and hemizygous mice exhibited single bands of 9.5 kb or 6.5 kb, respectively, in agarose gel electrophoresis (Figure 1AGo). Results from RT-PCR indicated that AT2-null mutant mice did not express AT2 receptor mRNA in any tissue examined (figure 1BGo), whereas expression of the AT1 receptor and angiotensinogen were unaffected (data not shown). Thus, the targeted disruption of the AT2 receptor gene was effective and specific.



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Fig. 1. Verification of the targeted disruption of the murine Agtr 2 gene. (A) Identification of genomic DNA of male wild type (Agtr 2x/y) and hemizygous (Agtr 2–/y) mice by Southern blot analysis. DNA isolated from the tails of mice was electrophoresed as described in Materials and methods. The higher 9.5 kb band indicates the wild type allele and the lower 6.5 kb band corresponds to the mutant allele (Agtr –/y). (B) Demonstration of tissue expression pattern of the AT2 receptor mRNA. Total RNA was isolated from various mouse tissues, and expression of the AT2 receptor mRNA was examined by RT-PCR. The odd-numbered lanes represent PCR products derived from the wild type mice, and the even-numbered lanes show results from the AT2-null mice.

 
AT2 receptor deficiency inhibited AOM-induced colon tumorigenesis
To evaluate if the AT2 receptor function is associated with chemical carcinogen-induced tumorigenesis in the colon, AT2-null mutant and wild type control mouse groups were treated with either AOM (10 mg/kg, i.p., four consecutive weekly administrations) or the saline vehicle. AOM caused the development of multiple colon tumors in all wild type mice, but only one AT2-null mouse developed a single macroscopic tumor 23 weeks after the first AOM injection (table IGo). The macroscopic tumors were predominantly observed in the lower half of the colon. The size of the tumors was relatively large (55% tumors >=22 mm3). The majority of the tumors were histologically observed to be moderately to well-differentiated adenocarcinoma. These results suggest that the AT2 receptor-mediated signal(s) is essential in AOM-induced colon tumorigenesis in this mouse strain.


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Table I. Summary of tumor results in wild type and AT2-null mice with a 129/Ola and C57BL/6J genetic background. Male mice in both wild type and AT2-null groups, 10 weeks old, were treated with AOM (10 mg/kg, i.p., 4 consecutive weeks) or the saline vehicle. Mice were killed 23 weeks after the initial injection of AOM
 
Although we obtained clear-cut results indicating that the AT2 receptor is essential in the formation of AOM-induced colon tumors, this may be true only in this mouse strain crossbred from 129/Ola and C57BL/6J mouse strains. In order to resolve this issue, the heterozygote female AT2-null mice in this crossbred strain were further crossed with male SWR/J mice, whose susceptibility to AOM-induced colon tumorigenesis has been established (23). We conducted identical experiments as above, using SWR/J crossbred F2 male wild and AT2-null mice. We again obtained similar results, indicating that the disruption of the AT2 receptor markedly attenuates AOM-induced tumorigenesis in the colon (table IIGo). Although AT2-null mice in this F2 strain developed tumors, presumably due to the strong tumor susceptibility characteristic of the SWR/J genetic background, the tumor multiplicity and volume in the null mice were significantly smaller than in the wild type mice. Histological analysis revealed that tumor types in the wild type and the AT2-null mice were identical. The majority of the tumors were microscopically observed to be moderately to well-differentiated adenocarcinoma. This additional experiment further strengthened the significance of the role of the AT2 receptor in colon tumorigenesis induced by AOM. Metastasis of the primary colon tumors to other tissues such as liver and lung was not detected by macrosopic and microscopic observations in these two series of in vivo studies.


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Table II. Summary of tumor results in wild type and AT2-null mutant mice crossbred from the original AT2-null strain and SWR/J. F2 male mice, both wild type and AT2-null offspring of F1 female AT2-null mice (AT2–/+, crossbreed of 129/Ola and C57BL/6J) and male SWR/J mice, were treated with AOM (10 mg/kg, I.P., 4 consecutive weeks) or the saline vehicle. Mice were killed 26 weeks after the initial injection of AOM
 
AT2 receptor deficiency attenuated AOM-induced CYP2E1 downregulation
Biotransformation enzymes often play a critical role in chemical carcinogen-induced tumorigenesis (24–26), and they are potential candidates in the determination of susceptibility to certain carcinogens. Since AOM is mainly metabolized and bioactivated by CYP2E1 in the liver(20), the effect of AT2 receptor expression on CYP2E1 expression in the liver was examined. Although basal expression levels of the hepatic CYP2E1 proteins in the wild type and the AT2-null mice were identical, their responses to AOM injection were significantly different (figure 2Go). AOM (10 mg/kg, bolus i.p. injection) significantly downregulated CYP2E1 protein expression in both wild type and AT2-null mice livers. However, the expression levels were higher in the AT2-null mouse liver than in the wild type mouse liver at 24 h after AOM injection. This observation coincided with results obtained following pharmacological attenuation of the AT2 receptor function with the subtype-specific receptor antagonist PD123319. Pretreatment of wild type animals with PD123319 (15 mg/kg/12 h by gavage administration and 0.35 mg/kg/h in drinking water) significantly attenuated AOM-induced CYP2E1 downregulation in the liver (figure 2Go). The extent of the attenuation was similar to that observed in the AT2-null mice. Agreement between these two methods of attenuating the AT2 receptor function indicates that the differing responses of CYP2E1 expression to AOM in the two mouse strains are due to a disruption of the AT2 receptor function. These results indicate that wild type mice lose hepatic CYP2E1 enzyme activity more quickly and significantly than AT2-null mice after AOM administration.



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Fig. 2. Effect of AOM on the expression of hepatic CYP2E1 in wild type and AT2-null mice. Mice (5 mice/group) were treated with AOM (10 mg/kg, i.p., bolus injection) and were killed 0 h ({blacksquare}), 6 h ({square}) or 24 h ({square}) later. AT2 receptor antagonist PD123319 treatment (15 mg/kg/12 h, gavage administration, and 50 µg/ml in drinking tap water) was initiated 3 h prior to the AOM treatment. The liver was dissected out and the microsomal fractions were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-human CYP2E1 antibodies. Representative western blots of the hepatic CYP2E1 are displayed in the upper panel. Averages of the expression levels of CYP2E1 (n = 5) are displayed in the histogram. (a) P <= 0.05 compared with the corresponding control levels in the wild type or AT2-null mouse livers. (b) P <= 0.05 compared with the level in the wild type mice treated with AOM for 24 h.

 
AT2 receptor deficiency decreased AOM-induced DNA adduct formation
DNA adduct formation is the earliest step in chemical carcinogen-induced tumorigenesis. AOM increases O6-methylguanine adduct levels in the liver and colon, and this increase in the colon epithelium is apparently associated with AOM-induced colon tumorigenesis (27,28). To evaluate if disruption of the Agtr2 gene attenuates DNA methyl adduct formation in the colon epithelium, the levels of O6-methylguanine adduct in the colon were determined by the immuno-slot-blot method with an anti-O6-methyldeoxyguanosine antibody (22). Bolus intraperitoneal administration of AOM (10 mg/kg) time-dependently increased colonic O6-methylguanine levels in both wild type and AT2-null mice (figure 3Go). However, the adduct level in the wild type mice was significantly higher than that in the AT2-null mice 24 h after the AOM administration. Pharmacological attenuation of the AT2 receptor function in wild type mice with PD123319 (15 mg/kg/12 h by gavage administration and 0.35 mg/kg/h in drinking water) markedly attenuated the AOM-induced increase in the colonic O6-methylguanine level 24 h after the AOM administration, although this attenuation was significantly larger than that observed in the AT2-null mice. These results strongly suggest that the disruption of the AT2 receptor gene attenuates DNA methyl adduct formation by AOM.



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Fig. 3. Effect of AOM on the expression of colonic O6-methylguanine levels in wild type and AT2-null mice. Mice (5 mice/group) were treated with the identical procedure as described in the legend to Figure 2Go. The colon was dissected out and whole DNA was extracted as described in Materials and methods. O6-methylguanine adduct levels were determined by immuno-slot-blot analysis with a monoclonal anti-O6-methyldeoxyguanosine antibody. *P <= 0.05 compared with the level in the wild type mice treated with AOM for 24 h. **P <= 0.01 compared with the level in the wild type mice treated with AOM alone for 24 h.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Increasing evidence suggests that Ang II signaling may play important roles in tumorigenesis in several tissues (1–4). However, the specific role of the Ang II receptors in tumorigenesis has not been elucidated. We used the well-characterized model of AOM-induced rodent colon tumorigenesis to examine the effect of AT2 receptor gene disruption on AOM-induced tumorigenesis. Although AOM may not be a practical carcinogen for human colorectal cancer, AOM-induced colon cancer in rodents is similar to human colorectal cancer in its morphology, proliferation characteristics and gene mutation involvement (29–31). Therefore, the outcome from this study with the rodent colon cancer model should be valuable.

First, we examined the role of the AT2 receptor in AOM-induced tumorigenesis using our original AT2 receptor-deficient mice with a C57BL/6J and 129/Ola genetic background. The results clearly demonstrated that the presence of the AT2 receptor is very important in AOM-induced colon tumorigenesis, since AOM treatment induced multiple tumors in all of the wild type mice but only one in one of the AT2-null mice (table IGo). In the second in vivo study, with crossbred SWR/J mice possessing the AT2-null genotype, the importance of the AT2 receptor in AOM-induced tumorigenesis was confirmed again (table IIGo). Although the AT2-null mice in this strain developed tumors, the tumor multiplicity and volume in the null mice were significantly smaller than in the control wild type mice. The reason that the AT2-null mice developed tumors in the second study appears to be due to the strong tumor susceptibility characteristic of the SWR/J genetic background (23). These series of in vivo studies demonstrate that the AT2 receptor functions to favor AOM-induced colon tumorigenesis. In addition, the development of significantly smaller sized tumors in the null mice, despite their SWR/J genetic background, may suggest that the AT2 receptor may also be associated with the growth of AOM-induced colon tumors. The clarification of this possibility, however, awaits further study.

The development of chemical carcinogen-induced colon cancer can be divided into four chronological stages: initiation, promotion, malignant conversion and progression (32). Although the involvement of multiple gene mutations or alterations is postulated at each stage of tumorigenesis, cytochrome P450-dependent biotransformation of carcinogenic xenobiotics is the first step of the initiation. Among many cytochrome P450 enzymes, CYP2E1 has been identified as the major enzyme to catalyze the biotransformation of AOM (20). In fact, the manipulation of CYP2E1 enzyme expression by dietary ethanol (28) or benzylselenocyanate (33), which increases CYP2E1 levels, has been shown to attenuate the tumorigenicity of AOM. In the present study, AOM significantly downregulated CYP2E1 protein expression in both wild type and AT2-null mouse livers. However, the CYP2E1 protein expression levels were significantly higher in the AT2-null mouse liver than in the wild type mouse liver at 24 h after AOM administration (figure 2Go). An identical pattern was also observed in wild type mice following pharmacological attenuation of the AT2 receptor function with a receptor antagonist (figure 2Go). These results suggest that AT2 receptor signaling enhances AOM-induced CYP2E1 downregulation. The results further suggest that AT2-null mice maintain relatively higher CYP2E1 enzyme activity in the liver after AOM administration and metabolize AOM more effectively. Wild type mice, however, may lose CYP2E1 enzyme activity quickly after AOM administration, and therefore a relatively larger quantity of unmetabolized AOM and/or the intermediate metabolite methylazoxymethanol (MAM) may be delivered to the colon target tissue where they are bioactivated and cause tumorigenesis. A natural implication of the present study is that bioactivation of AOM and/or MAM in colonic tissue may play a critical role in AOM-induced colon tumorigenesis. This interpretation appears to be supported by a series of studies by Fiala (28,33) in which hepatic CYP2E1 enzyme activity was found to be negatively associated with AOM-induced colon tumorigenesis in rats. A very recent report by the same group (34), describing how MAM, but not AOM, significantly increases DNA methyl adduct levels in CYP2E1-deficient mouse colon, may also support the importance of bioactivation of the carcinogen at the colon target tissue. Thus, it is concluded that the oncogenic function of AT2 receptor signaling is, at least in part, associated with CYP2E1-dependent biotransformation of the colon carcinogen AOM.

In chemical carcinogen-induced tumorigenesis, DNA adduct formation follows the bioactivation of carcinogens. AOM generates O6-methylguanine adducts in the colon mucosa (28,35). This methyl adduct formation is postulated to occur in the DNA of target genes such as the ß-catenin gene and/or the K-ras oncogene and causes irreversible DNA mutation, thereby inducing tumors in the colon (31,36). The significance of this pathway is strongly supported by a recent report by Wali et al. (37) in which the inhibition of O6-methylguanine-DNA methyltransferase increased AOM-induced colon tumorigenesis. In the present study, the AOM-dependent increase in colonic O6-methylguanine levels was significantly higher in wild type mice than in the AT2-null mice (figure 3Go). Pharmacological attenuation of the AT2 receptor function with a receptor antagonist also significantly attenuated this increase in O6-methylguanine levels (figure 3Go). These results derived from both genetic and pharmacological studies imply that the AT2 receptor favors AOM-induced DNA adduct formation. Since the AOM-induced colonic DNA adduct level was inversely correlated with the hepatic CYP2E1 expression level, AT2 receptor-associated regulation of the hepatic CYP2E1 expression appears to play an important role in the reduced DNA adduct formation and significantly decreased tumorigenesis in the AT2-null mice. However, these studies do not rule out the potential involvement of additional mechanisms, such as DNA repair, detoxification of the AOM and its metabolites and/or tumor suppression mechanisms in the decrease in AOM-induced DNA adduct formation and resultant colon tumorigenesis in AT2-null mice.

There has been debate about the effect of angiotensin-converting enzyme inhibitors on human cancers (5,38–40). Several recent population studies suggest that the use of ACE inhibitors, which are primarily utilized to control peripheral blood pressure, do not change overall cancer risk (38–40). These results are contradictory to the retrospective study reported by Lever et al. (5) and a number of reports with experimental animals (1–4). Although an ACE inhibitor was not employed in the present study, our results suggest that an attenuation of the AT2 receptor-specific signaling cascade within the entire Ang II signaling system may reduce the risk of colon cancer. These seemingly conflicting results may be explained by the following. (i) The dose of ACE inhibitor used in human studies is an antihypertensive dose, whereas in experimental animal studies the dose is designed to control tumor growth and is perhaps relatively higher than that used in human studies. (ii) An ACE inhibitor attenuates the entire Ang II generation, thus suppressing all downstream signaling of Ang II. (iii) The consequences of attenuating the entire Ang II system are most likely different than attenuating the pathway of one receptor type alone. Therefore, the negligible effect of ACE inhibitors on human cancer development reported epidemiologically does not necessarily contradict the results of the present study.

Nevertheless, the present study demonstrates an oncogenic function of the AT2 receptor in AOM-induced tumorigenesis in mouse colon. It also suggests that the AT2 receptor function in the liver may be a determinant of tumor susceptibility in AOM-induced tumorigenesis. Since pharmacological control of the AT2 receptor function in vivo is a viable therapeutic technique (41), it is feasible to determine whether AT2 receptor blockers attenuate AOM-induced colon tumorigenesis. This may lead to the development of a potential chemoprevention procedure for human colorectal cancer. However, the importance of the AT2 receptor function in other types of chemical carcinogen-induced colon cancer and in human colorectal cancer awaits further study. To the best of our knowledge, the present study is the first to demonstrate that the Ang II AT2 receptor possesses an oncogenic function in AOM-induced colon tumorigenesis in mice.


    Notes
 
3 To whom correspondence should be addressed at: 622G Light Hall, Department of Biochemistry, Vanderbilt University Medical School, Nashville, TN 37232, USA Email: masaaki.tamura{at}vanderbilt.edu Back


    Acknowledgments
 
The authors thank Dr Fred P.Guengerich (Department of Biochemistry, Vanderbilt University) and Dr Manfred F.Rajewsky (Institute of Cell Biology (Cancer Research), University of Essen Medical School, Essen, Germany) for sharing anti-human CYP2E1 antibodies and anti-O6-methyldeoxyguanosine monoclonal antibody, respectively. We thank Dr Tatsuo Tani (Department of Biochemistry, Vanderbilt University) for his assistance in histological tumor typing. We are grateful to Ms Pamela J.Tamura (Department of Chemistry, Vanderbilt University) for critical reading and constructive comments during the preparation of the manuscript. We thank Mr Eric F.Howard (Department of Biochemistry, Vanderbilt University) for his assistance in the preparation of the manuscript. This work was supported in part by a Grant-in-Aid from the American Heart Association (9750624N) and a National Cancer Institute grant (CA091428).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 5, 2001; revised March 11, 2002; accepted February 22, 2002.