The role of P-glycoprotein in intestinal tumorigenesis: disruption of mdr1a suppresses polyp formation in ApcMin/+ mice

Yasushi Mochida1,3, Ken-ichi Taguchi2, Shuichi Taniguchi1, Masazumi Tsuneyoshi2, Hiroyuki Kuwano3, Teruhisa Tsuzuki4, Michihiko Kuwano1,5 and Morimasa Wada1,6

1 Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
2 Department of Anatomical Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
3 Department of Surgery I, Faculty of Medicine, Gunma University, Maebashi, Japan
4 Medical Biophysics and Radiation Biology, Kyushu University, Fukuoka, Japan
5 Research Center for Innovative Cancer Therapy, Kurume University, Kurume, Japan

6 To whom correspondence should be addressed Email: wada{at}biochem1.med.kyushu-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
P-glycoprotein (P-gp) mediates the active transport of various substrates including xenobiotics, and it thus has a protective function in various cell types and tissues/organs including the intestinal epithelium. However, whether or not P-gp plays a positive role in the intestinal tumorigenesis is unclear. We have introduced disrupted alleles of the murine P-gp gene, mdr1a, into ApcMin/+ mice to evaluate whether P-gp plays any role in intestinal carcinogenesis. Spontaneously occurring DNA damage was significantly increased in both the small and large intestine of mdr1a-/-, ApcMin/+ mice compared with mdr1a+/+, ApcMin/+ mice. Furthermore, we observed active proliferation and rapid migration/disappearance of enterocytes in the intestine of the compound mice deficient in mdr1a. Finally, we found that the number of polyps and cancers was markedly decreased in mdr1a-/-, ApcMin/+ mice (P = 0.0016). P-gp thus appears to play a positive role during intestinal tumorigenesis.

Abbreviations: ABC, ATP-binding cassette; APC, adenomatous polyposis coli; BrdU, 5'-bromo-2'-deoxyuridine; 8-oxo-dG, 8-oxo-7,8-dihydro-2'-deoxyguanosine; P-gp, P-glycoprotein


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human genome is now known to contain 48 ATP-binding cassette (ABC) transporter genes. Dysfunction of several ABC transporter genes is closely linked with hereditary diseases (http://nutrigene.4t.com/humanabc.htm). Several ABC transporters, including P-glycoprotein (P-gp), not only confer multidrug resistance in cancer cells by enhancing drug efflux but also transport many different kinds of endogenous and exogenous toxic substrates (13). The expression level of P-gp/MDR1 is the highest among ABC transporter superfamily members in the intestine, and P-gp is oriented in the apical membrane of the mucosal epithelium. P-gp can thus pump xenobiotics from inside cells back into the intestinal lumen (4). On the other hand, over-expression of P-gp confers resistance to cell death induced by growth factor withdrawal, Fas, TNF-alpha or ultraviolet light irradiation (5). These results may support the possible role of P-gp in proliferation and survival of epithelial cells and malignant cells during tumorigenesis. Consistently, P-gp expression levels are enhanced in experimental systems following exposure to ultraviolet light irradiation and other environmental stimuli (6), reinforcing a major role of P-gp in protecting against cell apoptosis. Moreover, invasive colorectal carcinomas with P-gp expression showed a significantly greater incidence of vessel invasion and lymph node metastases compared with P-gp-negative invasive carcinomas (7).

Despite previous speculation, it is still unclear whether P-gp plays a critical role in colorectal tumorigenesis either positively or negatively. To evaluate the effect of P-gp in intestinal tumorigenesis directly, we generated mdr1a-disrupted mice with an ApcMin/+ background and examined whether DNA damage and polyp formation were affected by the absence of functional P-gp. P-gp is encoded by the mdr1a, mdr1b and mdr2 genes in the mouse (8). Mdr1a is the murine ortholog of human MDR1 (8), and mdr1a mRNA levels are predominant in the mouse intestine (9). ApcMin/+ mice carry a heterozygous nonsense mutation at codon 850 of the mouse ortholog of human APC. Germ-line mutation of the adenomatous polyposis coli (APC/Apc) tumor suppressor gene predisposes both humans and mice to intestinal tumorigenesis. ApcMin/+ mice thus show multiple tumors in the small and large intestine (10), and the sporadic colorectal cancers in humans show the mutation in the APC gene in >85% of cases (11). In this study, using the compound mice deficient in mdr1a and Apc, we elucidate the possible role of P-gp in intestinal tumorigenesis and normal epithelium.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice
ApcMin/+ (C57Bl/6 J) (10) and mdr1a knockout mice (FVB/N) (9) were obtained from Jackson Laboratories (Bar Harbor, ME) and Taconic Farms (Germantown, NY), respectively. Mice were maintained under specific pathogen-free conditions at the Center of Biomedical Research, Kyushu University (Fukuoka, Japan). Mice were fed a standard diet and water. Male ApcMin/+ and mdr1a knockout mice were backcrossed to inbred female C57BL/6 J and FVB/N mice (both from CLEA Japan, Tokyo, Japan) for a generation before commencing experimental crosses, respectively. These backcrossed mice were mated to obtain mdr1a+/-, ApcMin/+ mice. These mdr1a+/-, ApcMin/+ mice were crossbred to obtain the mice for the experiments. For analysis, mice were killed at 12 months of age.

The protocol was reviewed by the Committee on the Ethics of Animal Experiments of the Faculty of Medical Sciences, Kyushu University, and carried out according to the Guidelines for Animal Experiments of the Faculty of Medical Sciences, Kyushu University, and Law No. 105 and Notification No. 6 of the Government of Japan.

Genotyping
DNA for genotyping was isolated from each mouse tail using the Genomic DNA Purification Kit (Gentra Systems, Minneapolis, MN), according to the manufacturer's protocol. The genotype at the Apc and mdr1a loci was determined by allele-specific PCR, respectively, as described previously (12,13). A 327 bp PCR product was generated specifically from ApcMin allele for Apc genotyping, and a 618 bp product surrounding the Min nonsense mutation was generated by either the wild-type or the Min allele of Apc (Figure 1A). The analysis for mdr1a genotyping requires two PCR reactions, because the molecular weights of the products from the knockout and wild-type alleles are similar (481 and 411 bp, respectively) and are not resolved when the heterozygotes are analyzed (Figure 1B).



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Fig. 1. Genotyping and immunohistochemical detection for P-gp and 8-oxo-dG in the intestinal tissues in mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice. (A) Allele-specific PCR for the wild-type and Min-type mutated allele. Arrows indicate the 618 bp control PCR product generated from wild or Min allele and the 327 bp band generated specifically from the Min allele, respectively. (B) Allele-specific PCR for the mdr1a allele. (CF) Immunohistochemical staining by anti-P-gp antibody (C219). Solid arrowheads indicate P-gp staining in large and small intestines of mdr1a+/+, ApcMin/+ mice (D and F, respectively). No P-gp staining is detected in either the large or small intestines of mdr1a-/-, ApcMin/+ mice (C and E, respectively). (G) Summary table of 8-oxo-dG staining. Black boxes represent samples showing strong 8-oxo-dG staining by specific antibody (N45.1). White boxes represent samples showing no staining. Strong staining for 8-oxo-dG was shown only in the samples of mdr1a-/-, ApcMin/+ mice, not those of mdr1a+/+, ApcMin/+ mice (small intestine, P = 0.038; large intestine, P = 0.0098 by {chi}2 test). (HK) Representative 8-oxo-dG staining of large (H and I) and small (J and K) intestines in mdr1a-/-, ApcMin/+ (H and J) and mdr1a+/+, ApcMin/+ (I and K) mice, respectively. Solid arrowheads indicate the 8-oxo-dG stained nuclei of epithelial cells. Stromal cells in intestines of mice with both genotypes show the same density of 8-oxo-dG staining and serve as internal controls (open arrowheads). Cell nuclei were not counterstained. Original magnifications were 400x and 200x in C–F and H–K, respectively.

 
Intestinal tumor scoring and size determination
Immediately after death, small intestines were removed from the mice and divided into nine equal segments; large intestines were cut at the junction between the cecum and the ascending colon. These segments were incised longitudinally, washed with phosphate-buffered saline, laid flat on filter paper and fixed for 24 h in 10% neutral-buffered formalin. The fixed intestinal segments were stained with 1% methylene blue and examined for tumors by gross inspection and light microscopy.

Histology and immunohistochemistry
Tissue fixing was performed as described above. The primary antibodies used were anti-P-gp (C219) (Signet Laboratories, Dedham, MA) and anti-8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxo-dG) (N45.1) (Nikken Foods Co., Shizuoka, Japan) at a working dilution of 1:20. Immunostaining of P-gp was done on deparaffinized sections using mouse to mouse IHC detection system (CHEMICON International, Temecula, CA) according to the manufacturer's protocol. Immunohistochemical detection of 8-oxo-dG was performed using the peroxidase-labeled streptavidin–biotin technique with the Histofine SAB-AP kit (Nichirei, Tokyo, Japan) for 8-oxo-dG according to the manufacturer's protocol. Antigen retrieval of 8-oxo-dG was done by autoclaving the sections at 120°C in citrate buffer (0.01 M citric acid, pH 6.0) for 5 min. The substrate for alkaline phosphatase was from Nichirei, Japan. Nuclei were not counterstained, and the intensity and localization of immunoreactivity was analyzed in each sample. The 8-oxo-dG staining was graded as strong or weak relative to the staining of stromal cells. All slides were interpreted pathologically by two of the investigators (K.Taguchi and M.Tsuneyoshi) who were blinded to the mice genotypes of samples.

Measurement of the proliferation and migration of enterocytes
Cells synthesizing DNA were labeled by injecting mice (three mice per genotype per time point) intraperitoneally with a mixture of 5'-bromo-2'-deoxyuridine (BrdU, 120 mg/kg) and 5'-fluoro-2'-deoxyuridine (12 mg/kg) (both from CN Biosciences, San Diego, CA) dissolved in normal saline solution containing 30% dimethyl sulfoxide. Mice were killed 2 h post-BrdU injection for analysis of cell proliferation, and 24 h later for cellular analysis. Formalin-fixed sections were prepared, and cells that had incorporated BrdU were visualized using a BrdU staining kit (CN Biosciences). The labeling index of the intestinal mucosa in mice killed after BrdU injection was calculated by counting the proportion of BrdU-labeled cells in 50 typical crypts and/or villi.

Statistical analysis
One-sided P values for significant difference in numbers and sizes of intestinal polyp were determined using the Mann–Whitney U test performed with STATVIEW software (SAS Institute, Cary, NC). All reported values are the mean ± SEM. We used a {chi}2 test to determine the significant difference of 8-oxo-dG staining or tumor incidence between two genotypes of mice.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Generation of compound mice deficient in P-gp and Apc
The mdr1a-/- mice and their counterparts showed no spontaneous intestinal tumors (n = 5 per each genotype), consistent with the findings of a previous report (9). We thus introduced an mdr1a-disrupted allele into mice carrying an ApcMin/+ background, an animal model for human familial adenomatous polyposis. Genotyping of the mice was performed by allele-specific PCR reactions (Figure 1A and B). We detected P-gp expression at the apical surface of the epithelium facing the intestinal lumen in small and large intestines of mdr1a+/+, ApcMin/+ mice (Figure 1D and F), as observed in previous reports (14), whereas no staining of P-gp was shown in mdr1a-/-, ApcMin/+ mice (Figure 1C and E). We also confirmed that there was no expression of mdr1a mRNA in the mdr1a-/- mice by real-time RT–PCR (data not shown). Moreover, compensatory induction of mdr1b or mrp2 expression in the small and large intestine of mdr1a-/-, ApcMin/+ mice compared with their mdr1a+/+, ApcMin/+ and mdr1a+/+, Apc+/+ littermates and mdr1a induction in mdr1a+/+, ApcMin/+ mice compared with their mdr1a+/+, Apc+/+, ApcMin/+ littermates were not observed (data not shown). These mRNA levels were all standardized against villin mRNA, which is expressed constantly at the apical surface (as is P-gp) of the intestines. The mdr1a-/-, ApcMin/+ mice gained weight at the same rate as did their mdr1a+/+, ApcMin/+ littermates up to 12 months of age.

Increased DNA damage in mdr1a-/-, ApcMin/+ mice
To determine whether P-gp serves a protective function against xenobiotics in the intestine, we first examined spontaneous DNA damage generated by intestinal contents by immunohistochemical staining for 8-oxo-dG, one of the most frequently detected markers of such damage in genomic DNA, in the intestines of mdr1a-/-, ApcMin/+ mice and their mdr1a+/+, ApcMin/+ littermates. Strong staining of 8-oxo-dG in epithelial cells of small and large intestines was observed in three and four out of 5 mdr1a-/-, ApcMin/+ mice, respectively, whereas none of their five mdr1a+/+, ApcMin/+ littermates showed 8-oxo-dG staining in either the small or large intestines (Figure 1G). Differential staining density is apparent between 8-oxo-dG positive (Figure 1H and J) and negative (Figure 1I and K) specimens. In both the small and large intestines of mdr1a-/-, ApcMin/+ mice, the epithelial cells facing the intestinal lumen showed strong immunostaining for 8-oxo-dG but the crypts did not (Figure 1H and J). As described above, P-gp is also expressed at the apical surface of the enterocytes facing the intestinal lumen but not in crypts.

Proliferation and migration of the intestinal mucosa in mdr1a-/-, ApcMin/+ mice
To further clarify the biological role of P-gp in the intestine, we next performed analyses of the proliferation and turnover of intestinal epithelial cells in mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice by immunohistochemical detection after administration of BrdU. The proportion of BrdU-labeled cells in crypts of mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice at 2 h after BrdU injection was 13.2 ± 1.2 and 5.4 ± 1.2% in the large intestine (P = 0.0495) and 50.4 ± 5.3 and 37.3 ± 5.4% in the small intestine (P = 0.127), respectively. This suggests that the BrdU-labeled cells in the crypts of large and small intestines tend to be increased in mdr1a-/-, ApcMin/+ mice compared with their mdr1a+/+, ApcMin/+ littermates (Figure 2A–D).



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Fig. 2. Proliferation and migration of enterocytes in mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice. (A–D) Proliferation was detected by immunohistochemical detection of BrdU-labeled cells in the small (A and B) and large (C and D) intestines of mdr1a-/-, ApcMin/+ (A and C) and mdr1a+/+, ApcMin/+ (B and D) mice, respectively. Mice were killed 2 h after BrdU injection and the number of cells in S phase was determined by immunohistochemistry. (E–H) Migration of labeled cells in small (E and F) and large (G and H) intestine at 24 h after BrdU injection of mdr1a-/-, ApcMin/+ (E and G) and mdr1a+/+, ApcMin/+ (F and H) mice, respectively. Original magnifications were 200x and 400x in A–D and E–H, respectively.

 
We next examined the migration and turnover of epithelial cells by following BrdU-labeled cells at 24 h after BrdU injection. The BrdU-labeled cells in the small and large intestine of mdr1a-/-, ApcMin/+ mice had migrated further toward the intestinal lumen and the top of the villi than had the epithelial cells in mdr1a+/+, ApcMin/+ mice (Figure 2E–H). All small and large intestines in mdr1a-/-, ApcMin/+ mice showed faster migration of enterocytes than did intestines in their mdr1a+/+, ApcMin/+ littermates. The labeling indices of cells in the small intestine at 24 h after BrdU injection were similar in mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice (32.8 ± 6.8 and 35.1 ± 1.8%, respectively). Given that BrdU incorporation at 2 h after injection was 50.4 ± 5.3 and 37.3 ± 5.4% of the labeling index, respectively, in enterocytes of mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice, 35% of the labeled cells of small intestine were expected to be lost in mdr1a-/-, ApcMin/+ mice, compared with 6% loss of the labeled cells in mdr1a+/+, ApcMin/+ littermates. We confirmed no significant differences in the lengths or cell numbers of intestinal crypts and/or villis between the two genotypes.

We detected apoptotic cells at very low frequency in crypts of small and large intestines of both mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice (data not shown), when we examined apoptosis in intestinal epithelium by Terminal Deoxynucleotide Transferase dUTP Nick End Labeling assay. The apoptotic enterocytes were rarely if ever detected in the crypts of mouse intestines under spontaneous conditions, which is consistent with previous reports (15,16).

Decreased polyp formation in mdr1a-/-, ApcMin/+ mice
We next determined the number and size of polyps found in mdr1a-/-, ApcMin/+ mice compared with their mdr1a+/+, ApcMin/+ mice littermates. The number of large and/or small intestinal polyps in mdr1a-/-, ApcMin/+ mice was significantly smaller than the number in mdr1a+/+, ApcMin/+ mice (Table I). In particular, in the distal small intestine, the mean number of tumors in mdr1a-/-, ApcMin/+ mice was approximately four times smaller than the number in mdr1a+/+, ApcMin/+ mice (P = 0.0008) (Table I). Polyps are known to appear most often in the distal small intestine/ileum of the digestive tract of ApcMin/+ mice (17). The mean size of tumors in the distal small intestines of the mdr1a-/-, ApcMin/+ mice was also significantly smaller than the mean size of tumors in mdr1a+/+, ApcMin/+ mice (P = 0.0103) (Table I). The number of mice carrying large intestinal tumors (>3 mm in diameter) was also decreased in mdr1a-/-, ApcMin/+ mice compared with mdr1a+/+, ApcMin/+ mice, especially in the distal small intestine (total intestine, P = 0.0099; small intestine, P = 0.051; distal small intestine, P = 0.0006) (Table II). The histopathological character of the polyps found in the small and large intestines was similar in mice carrying mdr1a-disrupted alleles and mice with a wild-type mdr1a genotype (data not shown).


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Table I. Number and size of intestinal tumors in mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice

 

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Table II. Proportion of mice with tumors >3 mm in diameter

 
To evaluate the genetic features of the intestinal polyps in mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice, we used PCR as described previously to examine the loss of heterozygosity status of Apc (18). The loss of heterozygosity at the Apc locus was observed in polyps dissected from both mdr1a-/-, ApcMin/+ mice and mdr1a+/+, ApcMin/+ mice (data not shown), suggesting that intestinal tumors in mice with both genotypes might be formed through the tumorigenesis pathway where the wild-type Apc allele is lost. P-gp thus might be involved in polyp formation at both an earlier stage and subsequent progression during intestinal tumorigenesis.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we demonstrate for the first time the novel role of P-gp in tumorigenesis. We found that DNA damage detected by 8-oxo-dG staining was significantly increased at the apical surface in mdr1a-/-, ApcMin/+ mice compared with their mdr1a+/+, ApcMin/+ littermates, and both the labels of 8-oxo-dG and P-gp are in the apical surface of the enterocytes. Shih et al. (19) have reported that genetically predisposed cells in the superficial portions of the mucosa spread laterally and downward to form new crypts in the human colon, and in this way, enterocytes at the apical surface in the intestine might be very important for intestinal tumorigenesis. We also found decreased proliferation activity and slower migration of enterocytes in mdr1a+/+, ApcMin/+ mice compared with mdr1a-/-, ApcMin/+ mice.

Finally, we observed fewer and smaller intestinal tumors in P-gp-deficient mice compared with wild-type mice. Just before the publication of this report, Yamada et al. (20) have also reported the suppression of polyp number in ApcMin/+ mice deficient in mdr1a/1b genes. Although the report was not accompanied by analyses of DNA damage and proliferation/migration in intestinal epithelium, it further supports our result. The underlying mechanism of the effect is a subject of future studies. Anti-apoptotic activity of P-gp (5) is one possible explanation for the difference. Actually, the migrated cells were lost seven times more frequently in mdr1a-disrupted mice than in their wild-type littermates, as we learned by tracing BrdU-labeled cells, in spite of the cells being of identical length and number in intestinal crypts and/or villis between the two genotypes. It is known that senescent enterocytes are lost from the surface epithelium by a type of programmed cell death, sometimes called ‘anoikis’, in which exofoliation is thought to precede apoptosis (16) (see also Figure 3). This may be why apoptotic enterocytes were detected rarely on the apical surface of epithelium in the previous and the present study, which highlights the importance of further analysis of ‘anoikis’.



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Fig. 3. A hypothetical model of the role of P-gp in normal intestinal mucosa and intestinal tumorigenesis. (A) Intestine of mdr1a knockout mouse. The absence of functional P-gp at the apical surface leads to an increase in spontaneously occurring DNA damage, and plausibly to excess cell death. Furthermore, active proliferation in the bottom of the crypts and rapid migration of enterocytes also contribute to their shorten life span. (B) Intestine of mdr1a wild-type mouse. P-gp expressed at the apical surface of enterocytes avoid the DNA damage generated by xenobiotics, and plausibly excess cell death in normal mucosa. This effect of P-gp may lead to a prolonged life span of the enterocytes, resulting in increasing chance for cells to transform. In our present study, however, we did not determine anoikis in vivo (see Discussion for details).

 
The decreased proliferation activity and slower migration of enterocytes in mdr1a+/+, ApcMin/+ mice, compared with mdr1a-/-, ApcMin/+ mice, might lead to prolonged life span of enterocytes in the former mice (Figure 3). This idea is supported by the observation that the intestinal crypt and villi of mdr1a-/-, ApcMin/+ and mdr1a+/+, ApcMin/+ mice are similar in length and in cell number. Once enterocytes with a longer life span are transformed, they may avoid cell death or apoptosis during tumorigenesis, plausibly by the presence of P-gp, and begin to proliferate. In this way, one can expect dual roles of P-gp in cellular defence mechanisms; one is a protective role by exporting xenobiotics from cells, and the other is an anti-apoptotic role. Both functions may be a double-edged sword as they may enable dysplastic cells to survive as do normal epithelial cells exposed to cell- and DNA-damaging agents (Figure 3).

Our results raise the possibility that expression of P-gp plays a role in human colorectal carcinogenesis. Large inter-individual variation of P-gp/MDR1 expression level and the existence of functional single nucleotide polymorphisms in the MDR1 gene exon (21) and in its promoter region (S.Taniguchi, M.Wada, Y.Mochida and M.Kuwano, unpublished data) have been observed in humans. Strong staining by anti-8-oxo-dG antibody in the mucosa of mdr1a-/- mice compared with that of their wild-type mdr1a littermates is consistent with our observation that human colorectal samples with low P-gp/MDR1 expression show more DNA damage than do those with high P-gp/MDR1 expression (Y.Mochida, M.Wada and M.Kuwano, unpublished observation). The reduced number and size of tumors in the mdr1a-disrupted mice suggests that suppression of P-gp function might prevent tumor formation. Based on our present finding that the number of mice tumor-free in the small intestine was increased up to 4-fold by disruption of mdr1a, P-gp inhibitors may be considered as candidate chemopreventive agents for colorectal cancer.


    Acknowledgments
 
We thank Susan P.C.Cole (Queen's University, Ontario, Canada) for valuable discussion and proofreading. This work was supported by a Grant-in-Aid for Cancer Research and Scientific Research on Priority Areas (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 24, 2003; revised April 16, 2003; accepted April 22, 2003.