Enhancement of colon carcinogenesis by prostaglandin E2 administration

Toshihiko Kawamori1, Naoaki Uchiya, Takashi Sugimura and Keiji Wakabayashi2

Cancer Prevention Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan

2 To whom correspondence should be addressed Email: kwakabay{at}gan2.res.ncc.go.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although an accumulating body of evidence indicates that levels of prostaglandin E2 (PGE2) in human and rodent colon cancers are higher than those in surrounding normal tissues, the precise contribution of PGE2 to the process of colon cancer development has still been unclear. Therefore, we designed a study using a well-established azoxymethane (AOM)-induced colon carcinogenesis in male F344 rat model to investigate whether administration of exogenous PGE2 has a real impact on colon carcinogenesis. Intraperitoneal PGE2 injections (7.7 µg) once a week for 25 weeks significantly increased the AOM-induced colon tumor incidence (percent rats with tumors, 92 versus 53%, P < 0.05), especially adenocarcinomas (92 versus 47%, P < 0.05), and multiplicity (number of tumors per rat, 2.8 versus 1.0, P < 0.05). PGE2 treatment significantly increased 5-bromo-2'-deoxyuridine (BrdUrd) labeling index (11.8 versus 9.7%, P < 0.05) and reduced apoptotic index (0.34 versus 0.53%, P < 0.05) in colon cancers induced by AOM. PGE2 exhibits its physiological functions through binding to E-prostanoid (EP) membrane receptors EP1-4. All four types of EP receptors were detected in AOM-induced colon cancers using reverse transcription–polymerase chain reaction (RT–PCR). Our results provide evidence that PGE2 enhances colon carcinogenesis through induction of cell proliferation and reduction of apoptosis.

Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; BrdUrd, 5-bromo-2'-deoxyuridine; COX, cyclooxygenase; EP, E-prostanoid; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; RT–PCR, reverse transcription–polymerase chain reaction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Individuals taking non-steroidal anti-inflammatory drugs (NSAIDs) demonstrate a 40–50% reduction in the relative risk of colorectal cancer and colorectal cancer-associated mortality (1,2), and there is a large body of evidence that inhibition of cyclooxygenase (COX) is one of the underlying mechanisms (3,4). There are two isoforms of COX, referred to as COX-1 and COX-2. COX-1 is constitutively expressed in most tissues that generate prostaglandins (PGs) for normal physiological functions and does not fluctuate due to stimuli, whereas COX-2 expression is induced by various agents, such as cytokines, growth factors and tumor promoters (5,6). As COX-2 up-regulation is observed in colon tumors in humans and rodents, this enzyme may play a pivotal role in colon carcinogenesis (7,8). In fact, COX-2 gene deficiency reduces intestinal polyp formation in Apc{Delta}716 knockout mice (9) and overexpression in colorectal cancer cells increases their growth and invasiveness (10,11). Furthermore, selective COX-2 inhibitors cause significant reduction in adenoma burden in adenomatous polyposis coli patients (12) and azoxymethane (AOM)-induced colon cancer development in animals (13).

With regard to compounds produced by the actions of COX, such as PGs, data are relatively limited although elevation of serum and mucosal levels of PGE2 have been shown in colon cancers (14,15). Recently, two reports documented the effects of exogenous prostaglandin E2 (PGE2) on intestinal polyp formation in Min/+ mice, which possess a germ line mutation in the Apc gene and are employed as a model of familial adenomatous polyposis in humans (16,17). One report indicated that PGE2 exhibits inhibitory effects reflected reduced number and size of polyps although paradoxically PGE2 increased cell proliferation in polyps (16). The other report demonstrated that exogenous PGE2 administration increased the reduced number of polyp formation by NSAIDs through the elevation of the intracellular Ca2+ concentration (17). These results confuse our ideas for effects of PGE2 on colon carcinogenesis and exact underlying mechanism is still unclear. Some of the reasons may be due to using Min/+ mice model because Min/+ mice induce intestinal polyps mainly in small intestine and cover the early stages of intestinal tumorigenesis. On the other hand, another animal model for colon cancer using F344 rats and AOM as a carcinogen can develop colon cancers. The spectrum of colonic lesions in this model is similar to the various types of neoplastic diseases in the human colon including adenoma and adenocarcinoma sequence. Biological behaviors of AOM-induced rat colon tumors have close similarity to those of human colon tumors (18,19).

In addition, PGE2 exerts biological activity through binding to its receptors, E-prostanoid (EP)1-4. Our previous studies indicated that EP1 and EP4 receptors are involved in colon carcinogenesis using knockout mice and specific antagonists (20,21). Therefore, we hypothesized that PGE2 is strongly associated with colon carcinogenesis.

In order to clarify the exact role of PGE2 on colon carcinogenesis, we designed the present study using AOM-induced colon carcinogenesis in vivo rodent model. Cell proliferation and apoptosis in colon cancers and mucosa tissues to elucidate mechanism of PGE2 were examined. Also, EP receptor profile in colon carcinogenesis induced by AOM was investigated using reverse transcription–polymerase chain reaction (RT–PCR).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and chemicals
Thirty-nine male F344 rats, 4 weeks of age, purchased from Charles River Japan (Atsugi, Japan), were quarantined for 1 week and then randomly assigned to one of four treatment groups. The treatment groups were: (i) AOM alone; (ii) AOM + PGE2; (iii) PGE2 alone; and (iv) saline treatment (negative controls). All the animals were housed three to a plastic cage in a holding room controlled at 23 ± 2°C, 50 ± 10% humidity, with a 12-h light/dark cycle. Powdered AIN-76A (Dyets, Bethlehem, PA) was available, together with tap water, ad libitum during the experiment. AOM and 5-bromo-2'-deoxyuridine (BrdUrd) were purchased from Sigma Chemical (St Louis, MO). PGE2 ß-cyclodextrin clathrate including 7.7% of PGE2 was kindly provided from Ono Pharmaceutical (Osaka, Japan). As PGE2 is a very unstable form, PGE2 ß-cyclodextrin clathrate was synthesized by Ono Pharmaceutical as a stabilized form of PGE2 and confirmed by radioimmunoassay and radioactivity that pharmacologic behavior of PGE2 ß-cyclodextrin clathrate is the same as that of PGE2 (22). Enzyme Immunoassay (EIA) kit for detection of blood PGE2 level was purchased from Cayman Chemicals (Ann Arbor, MI). The study was performed with the approval of the Institutional Animal Care and Use Committee.

Experimental procedure
Starting at 5 weeks of age, all rats were given subcutaneous injections of AOM (15 mg/kg body wt) or saline vehicle once a week for 2 weeks (Figure 1). At 1 week after the last dosing of AOM or saline, they received intraperitoneal (i.p.) injections of PGE2 ß-cyclodextrin clathrate at a dose of 100 µg or saline vehicle once a week for 25 weeks. This dose was determined by being calculated from a previous study (23). All rats were monitored daily for their general health and weighed weekly. At 32 weeks of age, all animals were killed under ether euthanasia and blood was collected from rats in each group for PGE2 assay, 1 h after i.p. injection of BrdUrd in saline solution (50 mg/kg body wt) and complete autopsies were performed. After laparotomy, the entire stomach and intestines were resected and opened longitudinally, and the contents were flushed with saline. Using a dissection microscope, small and large intestinal tumors were noted grossly for their location, number, and size. The length, width and depth of each tumor were measured with calipers to allow calculation of tumor volume using the formula V = L · W · D · {pi}/6, where V is volume, L is length, W is width and D is depth (24). All other organs, with particular attention to the kidneys, lungs and liver, were also grossly examined under the dissection microscope for any abnormalities. The colon was laid flat on a glass plate, and half of each tumor and 1 in sample from each part of normal appearing colon (proximal, middle and distal) were removed and fixed in 10% buffered formalin and routinely processed for embedding in paraffin blocks for sectioning. Histological diagnosis of intestinal tumors using H&E stain sections were performed according to the classification of Pozharisski (25). The remaining normal mucosa was scraped with a stainless steel, disposable, microtome-bladed knife, S35 (Feather Safety Razor, Osaka, Japan). The other half of each colon tumor and scraped normal mucosa from each group were snap frozen in liquid nitrogen for analysis of EP receptors expression.



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Fig. 1. Experimental protocol.

 
BrdUrd labeling and apoptotic indices
Two serial sections (3 µm in thickness) of colon tumors and normal mucosa were used for immunohistochemical examination. One section was used for immunohistochemical detection of BrdUrd incorporation with avidin–biotin complex method (Vectastain ABC kit, Vector Laboratories, Burlingame, CA). The other section was stained with in situ end-labeling of fragmented DNA using ApopTag in situ detection peroxidase kit (Intergen Co., Purchase, NY) according to the manufacturer's instructions. For detection of BrdUrd labeling and apoptotic indices, in normal colon samples, at least 20 well-oriented crypts, in which the base, lumen and top of each could be seen by a microscope, were selected in each section of three parts of the colon. All tumor cells were counted in each colon tumor. For analyses of BrdUrd and apoptosis, only well-defined and darkly stained cells were counted using a microscope. The percentage of labeled cells (labeling index) was determined by calculating the labeled cell number: total cell number x 100.

Blood levels of PGE2
At death, blood was collected from rats in each group. PGE2 levels were analyzed using an EIA kit according to the manufacturer's instructions. Briefly, whole blood samples were purified through a C-18 reverse phase cartridge (Bond Elut, Varian, CA) to remove organic solvents. Blood samples were applied to a plate pre-coated with mouse monoclonal antibody and incubated with PGE2 acetylcholinesterase tracer and PGE2 antiserum for 18 h at room temperature. Then, all the wells were emptied and rinsed five times, and incubated with Ellman's reagent for 60 min to produce 5-thio-2-nitrobenzoic acid, which has a strong absorbance at 412 nm; the plate was read at 412 nm. We calculated the results with negative and positive controls using the standard curve. The results were expressed as picogram per millilitre. All assays were performed in triplicate.

RT–PCR for EP receptors expressions
We analyzed EP receptors expressions using RT–PCR method in normal mucosa and tumors of the colon in each group. Total RNA was isolated from half of each tumor and normal mucosa of the colon using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Reverse transcription with random 9mers was used to generate cDNAs from 0.8 µg of total RNA extracts using reverse transcriptase (AMV Reverse Transcriptase XL) and a Takara RNA LA PCR kit (Takara Biomedical, Japan). The following primers were used for PCR amplification of the resulting cDNA: EP1 (182 bp) forward primer 5'-TCGTGCATCTGCTGGAGCCC-3' (nucleotides 1510–1529), and reverse primer 5'-AGGAGGCGAAGAAGTTGGCG-3' (nucleotides 1672–1691); EP2 (233 bp) forward primer 5'-GTGCTGGCTTCTTATTCGAG-3' (nucleotides 377–396), and reverse primer 5'-AGCAAGGAGACCCCATAGAT-3' (nucleotides 590–619); EP3 (218 bp) forward primer 5'-ATCCTCGTGTACCTGTCGCA-3' (nucleotides 348–367), and reverse primer 5'-AGCCACACACCCAGCAGTA-3' (nucleotides 547–565); EP4 (220 bp) forward primer 5'-CTACTTCTACAGCCACTACG-3' (nucleotides 372–391), and reverse primer 5'-GAG-GATGAGGAAGGAACTGA-3' (nucleotides 572–591); ß-actin (203 bp) forward primer 5'-TCCTCCCTGGAGAAGAGCTA-3' (nucleotides 741–760), and reverse primer 5'-CCAGACAGCACTGTGTTGGC-3' (nucleotides 924–943). PCR conditions were 94°C for 120 s and then 25–35 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. ß-Actin was used for the internal control to normalize the sample amounts. Agarose gels (1.5%) were stained with ethidium bromide and the band intensity was quantified using an NIH image program. All assays were performed in triplicate.

Statistical analysis
Body weights, blood PGE2 levels and BrdUrd labeling and apoptotic indices were analyzed among all groups. Tumor incidence, multiplicity and volume as well as apoptotic indices were compared between the animals treated with AOM alone and AOM plus PGE2. Tumor incidence, expressed as the percentage of tumor-bearing animals, was analyzed by Fisher's exact probability test. Tumor multiplicity, expressed as the mean number of tumors per animal, tumor volume, blood PGE2 levels and BrdUrd labeling and apoptotic indices were analyzed by the unpaired Welch's or Student's t-test. Differences were considered statistically significant at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General observations
The body weights of animals treated with saline or AOM, with or without PGE2, were comparable throughout the study, weight loss being consistently observed with PGE2 (Figure 2). All animals were sacrificed at 26 weeks after the last AOM dosing and autopsy revealed no gross or histological changes in the colon, liver, kidneys, lungs and stomach of rats treated with saline, with or without PGE2 treatment. No neoplastic lesions other than colon tumors were found in rats treated with AOM.



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Fig. 2. Body weight changes during the study.

 
Colon tumor data
Table I summarizes data for effects of PGE2 on development of colon tumors induced by AOM. All tumors were classified as adenomas and adenocarcinomas through histopathological examination. There were no tumors in saline-treated animals injected with PGE2 or saline. Ninety-two percent of rats (11/12) treated with AOM and PGE2 developed colon tumors including 33% (4/12) with adenomas and 92% (11/12) with adenocarcinomas, whereas 53% (8/15) of rats treated with AOM alone had colon tumors including 7% (1/15) with adenomas and 47% (7/15) with adenocarcinomas. Regarding tumor multiplicity (number of tumors per animal), the figures were 2.8 for the AOM and PGE2 treatment group and 1.0 with AOM alone (P < 0.05). Also, colon tumor volume in rats treated with AOM and PGE2 was 2-fold greater than that in rats treated with AOM (23 versus 11 mm3), although the difference was not significant.


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Table I. Effects of PGE2 on the incidence, multiplicity and volume of AOM-induced colon tumors in male F344 rats

 
Blood PGE2 levels
To confirm the effects of PGE2, we examined blood levels of PGE2 in each group (Table II). The mean blood PGE2 level in rats treated with AOM and PGE2 was greater than those of the other groups. As expected, PGE2 blood levels in rats treated with PGE2 were significantly higher than those of rats without PGE2 treatment with/without AOM (P < 0.01). In groups without PGE2 treatment, blood PGE2 level in rats treated with AOM alone was significantly higher than that of rats treated with saline (13.6 versus 5.7 pg/ml, P < 0.05).


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Table II. Blood levels of PGE2 in each group

 
Cell proliferation and apoptosis
To elucidate mechanism of PGE2 on AOM-induced colon carcinogenesis, BrdUrd labeling and apoptotic indices were examined. The results are shown in Table III. Representative figures of BrdUrd and ApopTag staining are shown in Figure 3. PGE2 treatment significantly increased BrdUrd labeling indices not only in colon cancers (11.8 versus 9.7%, P < 0.05) but also in normal mucosa (8.5 versus 5.7%, P < 0.05) of rats receiving AOM. Interestingly, PGE2 treatment alone did not show any effects on BrdUrd labeling index in normal mucosa of rats receiving saline (3.3 versus 3.1%), while AOM treatment significantly increased cell proliferation than saline treatment in normal mucosa (5.7 versus 3.1%, P < 0.05). Apoptotic cells in cancers of rats treated with AOM and PGE2 were significantly lower than those in rats treated with AOM alone (0.34 versus 0.53, P < 0.05). In normal colon mucosa, only very few apoptotic cells in rats treated with/without AOM and PGE2 or saline injections were observed. Since their values were <0.1%, the results did not allow us to analyze effects of PGE2 on apoptosis in colon normal mucosa.


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Table III. Effects of PGE2 on cell proliferation and apoptosis in the colon

 


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Fig. 3. Immunohistochemical stainings. Figures in left column indicate samples from rats treated with AOM alone and those in right column indicate samples from rats treated with AOM and PGE2. Immunohistochemical stainings with BrdUrd are shown in figures in the upper two rows (A) and staining with ApopTag are shown in figures in the lower row (B). Figures in the upper row indicate normal colon mucosa with BrdUrd staining. Figures in the middle row indicate colon cancers with BrdUrd staining. Figures in the lower row indicate colon cancers with ApopTag staining.

 
EP receptor expressions in colon carcinogenesis induced by AOM
PGE2 activates seven transmembrane receptors EP1-4 as a ligand. Using RT–PCR, EP receptor expressions were investigated in colon normal mucosa and cancers in each group (Figure 4). PCR products were confirmed by sequencing analyses (data not shown). All four types of EP receptor were expressed in AOM-induced colon cancers. EP1 and EP2 receptors mRNA were overexpressed in colon cancers as compared to the levels of normal mucosa. On the other hand, EP3 and EP4 receptors mRNA were found to be similar expression levels in both colonic normal mucosa and cancers.



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Fig. 4. RT–PCR results of EP receptor expression in colonic normal mucosa and cancers induced by AOM in male F344 rats. Lanes 1–4, normal mucosa samples from rats treated with saline, PGE2, AOM and AOM + PGE2, respectively; lanes 5–7, adenocarcinomas from rats treated with AOM; lanes 8 and 9, adenocarcinomas from rats treated with AOM + PGE2.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study provided evidence that exogenous PGE2 administration enhances AOM-induced colon cancer development. All four types of EP receptor were expressed in AOM-induced colon cancers. Its enhancing effects are associated with induction of cell proliferation and reduction of apoptosis.

It is clear that COX-2 plays a role in the promotion of colorectal cancer (26). However, a recent report demonstrated that deficiency of either COX-1 or COX-2 causes a decrease in intestinal tumorigenesis in the Min/+ mouse with both COX-1 and COX-2 contributing to PGE2 production in polyps (27). It is therefore probable that the PGE2 level is important for tumor development, and that decreased production attributable to the loss of either isoform significantly reduces development of the lesions. DuBois and his group indicated that decreased cell death caused by PGE2 through up-regulated Bcl-2 expression would enhance the tumorigenic potential in human colon cancer cells (28) and PGE2 can induce phenotypic alterations including increased motility, changes in cell shape and stimulation of cell growth in colon cancer cells through the phosphatidylinositol 3-kinase/Akt signaling pathway (29). In the present study, to evaluate the effects of PGE2 on the behavior of in vivo colon carcinogenesis, we employed an AOM-induced colon cancer development model. Because it is a well-established rodent model, and it takes around 16 weeks and 50 weeks to develop adenomas and adenocarcinomas in the colon, respectively. This model allows us to investigate sequential analysis of colon carcinogenesis, such as ACF, adenomas and adenocarcinomas. Short life span of Min/+ mouse may make it very difficult to identify effects of PGE2 on intestinal tumorigenesis. Because the number of intestinal polyps may reach the maximum and plateau in Min/+ mouse at 16 weeks of age. In addition, neoplasms arise when there is abnormal accumulation of altered cells, characterized by excessive proliferation and decreased cell death. The results in this study clearly indicated that exogenous administration of PGE2 significantly enhances AOM-induced colon cancer development through induction of cell proliferation and reduction of apoptosis.

PGE2 activates seven transmembrane receptors EP1-4 as a ligand. Our results that all four types of EP receptor were detected by RT–PCR in AOM-induced colon cancers in male F344 rats confirm that PGE2 signaling pathways exist in this model. We found that mRNA of EP1 and EP2 receptors are up-regulated in colon carcinogenesis-induced by AOM in male F344 rats. These results support our previous conclusion that EP1 and EP4 receptors are involved in the early event of AOM-induced colon carcinogenesis (20,21,30) and the report from Taketo and his group that EP2 is the major receptor mediating the PGE2 signaling pathway in intestinal polyp formation in Apc{Delta}716 mice (31). According to binding assays, PGE2 has affinity for all four types of EP receptor in the rat (32) as well as the human (33), while 16,16-dimethyl PGE2 demonstrates binding to EP2, EP3 and EP4, but not EP1 and 17-phenyl-trinor-PGE2 plays as an agonist for EP1 and EP3 (34). In the present study, we employed PGE2 ß-cyclodextrin clathrate for PGE2 because of its stability and solubility in saline. PGE2 ß-cyclodextrin clathrate acts as PGE2 after solution in saline, in fact, we confirmed high blood levels of PGE2 in rats treated with PGE2. There were two contrary results using Min/+ mouse intestinal polyp formation model. A report from Wilson and Potten indicated that 16,16-dimethyl PGE2 reduces both number and size of intestinal polyps in Min/+ mice (16). One of the reasons for inhibitory effect of 16,16-dimethyl PGE2 on intestinal polyp formation may be related to its binding affinity to EP receptors. Because 16,16-dimethyl PGE2 cannot activate EP1 receptor which plays an important role in intestinal polyp formation in Min/+ mice since a selective EP1 antagonist significantly inhibits intestinal polyp formation in this model (20). Another study using the Min/+ mouse model by Hansen-Petrik et al. indicated that administration of a mixture of 16,16-dimethyl PGE2 and 17-phenyl-trinor PGE2 attenuates the reduced intestinal polyp formation by NSAIDs treatment (17). Based on binding assays, a mixture of 16,16-dimethyl PGE2 and 17-phenyl-trinor PGE2 can activate all four types of EP receptor. In addition, they found that NSAIDs decrease intracellular Ca2+ concentration in tumors of Min/+ mice and the effects are attenuated by treatment of 17-phenyl-trinor PGE2, which can activate EP1 and EP3 receptors. As stimulation of EP1 receptor results in elevation of intracellular Ca2+ concentration, they suggested that intracellular Ca2+ plays a role to maintain colon tumor integrity. Taken together, it is possible that PGE2 plays a role in colon carcinogenesis and its effects may be dependent on EP receptors, especially EP1 receptor. In down-stream of EP receptors signaling pathways, activation of EP3 receptor is coupled with Gi protein, leading to an inhibition of adenylate cyclase activity and resulting in a decrease of cAMP concentration. EP2 and EP4 receptors, in contrast, are coupled with Gs protein, increasing intracellular cAMP concentration through activation of adenylate cyclase. Protein kinase A activity appears to alter proliferation and differentiation in several cancer cell lines (35). Therefore, there may be crosstalk of second messengers after ligand binding to EP receptors and all four types of EP receptor may be involved in colon carcinogenesis.

In conclusion, the present study provides additional evidence that exogenous administration of PGE2 indeed enhanced development of AOM-induced colon cancers in in vivo rodent model through up-regulation of cell proliferation and down-regulation of apoptosis. All four types of EP receptors express in AOM-induced colon cancers. Additional studies to investigate the effects of EP receptor deficiency in mice on colon carcinogenesis using cancer as an end point are now ongoing in our laboratory.


    Notes
 
1Present address: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Avenue, Suite 309, Charleston, SC 29425, USA Back


    Acknowledgments
 
We thank Ono Pharmaceutical Co. Ltd. for generously supplying PGE2 ß-cyclodextrin clathrate. We thank Dr Masahiko Watanabe for his helpful discussion on RT–PCR method and Ms Yurika Teramoto for her excellent technical assistance. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare, Japan, and for Scientific Research (C) from the Japan Society for the Promotion of Science, the Ministry of Education, Culture, Sports, Science and Technology, Japan (12670226 to T.K.). This study was also supported by grants from the Second Term Comprehensive 10-year Strategy for Cancer Control and a Research Grant of the Princess Takamatsu Cancer Research Fund (99-23104 to T.K.).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received January 17, 2003; accepted February 21, 2003.