Prostaglandin E receptor subtype EP1 deficiency inhibits colon cancer development

Toshihiko Kawamori3, Tomohiro Kitamura, Kouji Watanabe, Naoaki Uchiya, Takayuki Maruyama1, Shuh Narumiya2, Takashi Sugimura and Keiji Wakabayashi*

Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan, 1 Minase Research Institute, Ono Pharmaceutical Co. Ltd, Osaka 618-8585, Japan and 2 Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606-8315, Japan

* To whom correspondence should be addressed Email: kawamori{at}musc.edu or kwakabay{at}gan2.ncc.go.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Prostaglandin E2 exerts its biological activity through binding to its membrane receptors, E-prostanoid (EP) receptors1–4. Our previous finding that lack of EP1 receptor inhibits the early stages of colon carcinogenesis led us to investigate whether EP1 receptor deficiency reduces colon cancer development induced by azoxymethane (AOM) using EP1 receptor knockout mice. At 6 weeks of age 33 homozygous EP1-deficient (EP1–/–) mice and 28 wild-type (EP1+/+) mice were given i.p. AOM (10 mg/kg body wt) once a week for 6 weeks. At 56 weeks of age all animals were killed and intestinal tumors were examined. The results clearly indicated that lack of EP1 receptor significantly reduced colon cancer incidence (27 versus 57%, P < 0.05) and multiplicity (0.30 versus 0.76, P < 0.05) as well as tumor volume (12.2 versus 75.6 mm3, P < 0.05). In EP1–/– mice, silver stained nucleolar organization region protein count as cell proliferation marker was significantly reduced (1.35 versus 2.17, P < 0.001) and apoptosis was significantly increased (0.685 versus 0.077, P < 0.001) in colon tumors induced by AOM compared with those in EP1+/+ mice. We confirmed that EP1 receptor mRNA was overexpressed in colon cancers of EP1+/+ mice using reverse transcription–polymerase chain reaction. These results provide strong evidence that the EP1 receptor is of major importance for colon cancer development and it could be a new target for a mechanism-based chemoprevention strategy against colon cancer development.

Abbreviations: AgNOR, silver stained nucleolar organizer region protein; AOM, azoxymethane; COX, cyclooxygenase; EP, E-prostanoid; NORs, nucleolar organizer regions; PGE2, prostaglandin E2; PKN, protein kinase N; RT-PCR, reverse transcription–polymerase chain reaction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Colon cancer development appears to be closely linked with alterations in the arachidonic acid cascade and non-steroidal anti-inflammatory drugs reduce the risk of colon cancer development in human (1) and the incidence of carcinogen-induced colon cancers in rodents (2) through inhibition of cyclooxygenase (COX) activity. There are two isozymes of COX, referred to as COX-1 and COX-2. COX-2, the inducible form, is known to be overexpressed in colon cancers in rodents (3) and humans (4,5), whereas COX-1 is constitutively expressed and may contribute to various physiological functions. Celecoxib, a selective COX-2 inhibitor, has shown strong chemopreventive effects against colon cancer development in animal models (6,7) and may significantly reduce the numbers of colorectal polyps in familial adenomatous polyposis patients (8). There is accumulating evidence that COX-2 plays a pivotal role in colon carcinogenesis from genetic and pharmacological studies (9,10). Recently, however, there was a report using knockout mice that COX-1 contributed to colon carcinogenesis as well as COX-2 (11). These results suggest that not only COX-2 but also COX-1 contribute to colon carcinogenesis. Several reports have documented increased levels of prostaglandin E2 (PGE2), one of the major prostanoids, in colon cancer tissues compared with surrounding normal appearing mucosa of rodents and humans (12,13). Taken together, both COX enzymes and the levels of their product PGE2 may be important factors and may play roles in colon carcinogenesis. In addition, recent studies provide evidence that PGE2 administration enhances AOM-induced colon carcinogenesis in male F344 rats (14). Thus, we hypothesized that decreased signaling via the PGE2 pathway may be associated with inhibition of colon cancer development. PGE2 exerts its biological activity through binding to the membrane receptors E-prostanoid (EP) receptors1–4. Therefore, it is likely that lack of specific receptors may contribute to the inhibitory effects on colon carcinogenesis. Recent development of mice lacking the genes encoding these receptors (1517) has allowed us to investigate which types of receptors are involved in colon carcinogenesis. In our previous studies, EP1 or EP4 receptor deficiency and specific antagonists against these receptors significantly reduced the number of azoxymethane (AOM)-induced aberrant crypt foci, which are thought to be preneoplastic lesions in the colon (18,19). The antagonists also inhibited intestinal polyp formation in Min mice, suggesting that receptors EP1 and EP4 play important roles in the early stages of colon carcinogenesis. However, the question remains as to whether EP1 receptor deficiency really has an impact on colon cancer development. Therefore, we designed the present study to analyze the effects of EP1 receptor deficiency on colon cancer development induced by AOM using knockout mice.

The inhibitory mechanism was investigated by determining cell proliferation and apoptosis in colon tumors. An assessment of EP receptor expression in colonic normal mucosa and cancers of mice with or without EP1 receptors using the reverse transcription–polymerase chain reaction (RT-PCR) method was also examined. The mouse EP1 receptor gene, consisting of three exons, is predominantly expressed in the kidney and in the hypothalamus of the brain. Mouse PKN, a newly discovered gene encoding a protein kinase related to the protein kinase C family (20), overlaps the whole EP1 gene in a tail-to-tail manner (21), indicating that the whole EP1 gene is contained within the 3'-untranslated region of a long protein kinase N (PKN) gene transcript (Figure 1). Our EP1 knockout mice were made by inserting the neomycin-resistance gene into the FspI site of exon 2, which is located immediately before the sequence encoding the first transmembrane domain (15). Therefore, this EP1 receptor knockout strategy may influence a long PKN gene transcript. In this report, to confirm that the PKN gene is not a player in AOM-induced colon carcinogenesis, expression of the PKN gene in mice with or without EP1 receptor was also investigated using the RT-PCR method.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Gene organization at the mouse EP1/PKN gene locus and the transcripts produced. The center line represents the 7.2 kb of sequenced genomic DNA. Open boxes represent exons. The EP1 gene exons are numbered I–III and are shown above the line. Under the line are the seven last exons of the PKN gene. Solid bars illustrate mRNAs produced at this locus. a, b, c and d indicate primer positions within the locus.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Animals and chemicals
The mouse gene encoding the EP1 receptor was disrupted by gene knockout methods using homologous recombination, as reported previously (15). The chimeric mice generated were back-crossed with C57BL/6Cr mice and the resulting wild-type and homozygous mutant males of the F2 progeny were used at 6 weeks. The genotypes of the knockout mice were confirmed by PCR according to the method described previously (15). The animals were housed in plastic cages at 24 ± 2°C and 55% humidity with a 12 h light/dark cycle and maintained on powder diet (AIN-76A; Dyets Inc., Bethlehem, PA). AOM was purchased from Sigma Chemical Co. (St Louis, MO). The study was performed with the approval of the Institutional Animal Care and Use Committee.

Experimental procedure
At 6 weeks of age, 33 homozygous EP1-deficient (EP1–/–)and 28 wild-type (EP1+/+) mice were given i.p. injections of AOM (10 mg/kg body wt) once a week for 6 weeks. All mice were provided with food and tap water ad libitum, weighed weekly and killed with ether at 56 weeks of age. Complete autopsies were performed and, after laparotomy, the entire stomach and intestines were resected and opened longitudinally and the contents were flushed with normal saline. Using a dissection microscope, large intestinal tumors were noted grossly for their location, number and size. The length (L), width (W) and depth (D) of each tumor were measured with calipers and tumor volume was calculated using the formula V = L·W·D·{pi}/6 (6). Colon tumors and normal tissues were fixed in 10% buffered formalin and embedded in paraffin blocks for histological evaluation. Diagnosis of intestinal tumors using hematoxylin and eosin stained sections was performed according to the classification of Pozharisski (22). Half of the colon tumors sized >~20 mm3 and normal mucosa from each group were snap frozen with liquid nitrogen for analysis of RT-PCR.

Silver stained nucleolar organizer region protein (AgNOR) count and apoptotic index
Two serial sections (3 µm in thickness) of colon tumors were used for AgNOR staining and in situ end-labeling of fragmented DNA using Apo-BrdU-IHCTM (Chemicon International Inc., Temecula, CA). AgNOR staining was carried out according to the method described previously (23). For determination of AgNOR number in cell nuclei, AgNOR was counted on silver stained sections using a microscope at a magnification of x400. The other sections were stained with Apo-BrdU-IHC according to the manufacturer's instructions. For analysis of apoptosis, only well-defined and darkly stained cells were counted using a microscope. All tumor cells were counted for AgNOR number and apoptotic index in a section of colon tumor. Five tumors in each group provided data for AgNOR count and apoptotic index. The percentage of labeled cells (apoptotic index) was determined by calculating (labeled cell number:total cell number) x 100.

EP receptor and PKN mRNA expression
Using the RT-PCR method, EP receptor and PKN mRNA expression in colon tumors and normal mucosa was investigated. Total RNA was isolated from colon tumors and normal mucosa using Isogen (Nippon Gene Co. Inc., Tokyo, Japan) according to the manufacturer's instructions. Reverse transcription with random 9mers was used to generate cDNAs from 0.8 µg total RNA extract using reverse transcriptase (AMV Reverse Transcriptase XL) and a Takara RNA LA PCR kit (Takara Biomedical Co. Inc., Tokyo, Japan). The EP1 receptor (24) and PKN (20) primers were made for PCR amplification of the resulting cDNA (Figure 1): forward primer (a in Figure 1) for exon I, 5'-CCTGCATCCTGAGCAGCACT-3' (nt 33–52); reverse primer (b in Figure 1) for the middle of exon II, 5'-TGGCGACGAACAACAGGAAG-3' (nt 293–312); these being considered as reverse and forward primers for PKN (nt 4104–4123 and 2882–2901), respectively. The expected PCR products were 280 bp for the EP1 receptor and 1242 bp for PKN. The EP1 gene is located in the 3'-untranslated region of the PKN gene. To investigate the effects of the EP1 receptor knockout strategy on PKN gene expression, we used primers in the PKN encoding region as follows: forward primer (c in Figure 1), 5'-GAGAAGGCTACTGCGGAGGA-3' (nt 562–581); reverse primer (d in Figure 1); 5'-CGGCCACAAAGTCGAAATC-3' (nt 824–842). The expected PCR product was 281 bp. The following primers for the other EP receptors and ß-actin were used for PCR amplification of the resulting cDNA: EP2 (402 bp) forward primer, 5'-AGGACTTCGATGGCAGAGGAGAC-3' (nt 903–925); reverse primer 5'-CAGCCCCTTACACTTCTCCAATG-3' (nt 1282–1304) (25); EP3 (438 bp) forward primer, 5'-CCGGGCACGTGGTGCTTCAT-3' (nt 657–676); reverse primer, 5'-TAGCAGCAGATAAACCCAGG-3' (nt 1075–1094) (26); EP4 forward primer, 5'-GCCATAGAGAAGATCAAGTGCCT-3' (nt 1150–1172); reverse primer, 5'-CCCACTAACCTCATCCACCAA-3' (nt 1480–1500) (27); ß-actin (203 bp) forward primer, 5'-TCCTCCCTGGAGAAGAGCTA-3' (nt 763–782); reverse primer, 5'-CCAGACAGCACTGTGTTGGC-3' (nt 946–965). PCR conditions were 94°C for 120 s and then 30 cycles of 94°C for 30 s, 62–66°C for 60 s and 72°C for 60 s. ß-Actin was used as the internal control for normalization of sample amounts. Agarose gels (1.5%) were stained with ethidium bromide. All assays were performed in triplicate.

Statistical analysis
Body weights, tumor incidence, multiplicity, volume and AgNOR count, as well as apoptotic index, were compared between animals with and without EP1 receptors. Tumor incidence, expressed as the percentage of tumor-bearing animals, was analyzed using Armitage's {chi}2 method. Tumor multiplicity, expressed as the mean number of tumors per animal, tumor volume and body weights were analyzed by unpaired Welch's or Student's t-test. AgNOR count, expressed mean number of AgNORs per nucleus and apoptotic index, expressed as percentage of cells with positive staining of Apo-BrdU-IH in tumors, were analyzed by unpaired Student's t-test. Differences were considered statistically significant at P < 0.05.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Body weight changes for EP1+/+ and EP1–/– animals during the experiment are shown in Figure 2, values for the latter being significantly greater at 35 and 50 weeks after the first dosing of AOM (38.3 versus 42.0 g, P < 0.05 and 39.6 versus 44.6 g, P < 0.01, respectively). One EP1+/+ mouse at the age of 30 weeks was found to have three colon tumors diagnosed as adenocarcinomas. Therefore, mice alive on that day were counted as effective animals. At 56 weeks of age all survivors were killed and complete autopsies were performed. EP1–/– mice without AOM treatment had no tumors in their intestines and lived >1 year as previously described (15). No tumors other than colon tumors were observed in AOM-treated mice both with and without EP1 receptor.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Body weight changes of EP1+/+ and EP1–/– mice during the study.

 
Histopathological examination revealed colon tumors induced by AOM to be adenomas or adenocarcinomas. Table I summarizes the data for effects of EP1 receptor deficiency on colon tumor development. Colon tumors developed in 8 out of 26 EP1–/– mice (31%), including 1 mouse with an adenoma and 7 mice with adenocarcinomas. In the EP1+/+ case, 16 out of 28 mice (57%) had adenocarcinomas. In EP1–/– mice, 0.35 tumors/mouse were found, whereas the value was 0.79 tumors/mouse in EP1+/+ mice (P < 0.05). In addition, colon tumor volume was significantly decreased in EP1–/– mice compared with that in EP1+/+ mice (12.2 versus 75.6 mm3, P < 0.05). Thus, EP1 receptor deficiency significantly reduced colon cancer development as an end-point. These results strengthen our hypothesis that the EP1 receptor is involved in colon carcinogenesis and lack of the EP1 receptor significantly reduces colon cancer incidence and volume.


View this table:
[in this window]
[in a new window]
 
Table I. Effects of EP1 receptor deficiency on AOM-induced colon carcinogenesis

 
To elucidate the inhibitory mechanisms, we analyzed cell proliferation and apoptosis in colon tumors of mice with or without the EP1 receptor. The results are summarized in Table II. Nucleolar organizer regions (NORs) are loops of DNA which contain rRNA genes. They are transcribed by RNA polymerase I and are of vital importance for the ultimate synthesis of proteins (28). AgNORs are acidic proteins associated with the NORs which are selectively stained by a silver colloid technique. A series of studies indicates that the quantity of AgNOR protein is related to the rapidity of cell proliferation and there is evidence of a relationship between AgNOR counts and the prognosis of malignant tumors (29). Mean number of AgNORs per nucleus of tumors in EP1+/+ mice is significantly greater than that of EP1–/– mice (2.17 versus 1.35, P < 0.001). In addition to cell proliferation, we analyzed apoptosis of tumors induced by AOM in both mice. Apoptotic cell count (apoptotic index) in cancers of EP1–/– mice treated with AOM was significantly higher than that in EP1+/+ mice (0.685 versus 0.077, P < 0.001). The mechanisms by which activation of the EP1 receptor regulates cell proliferation and apoptosis in colon tumors are not known. However, the fact that colon cancers induced by AOM in EP1–/– mice showed lower cell proliferation rates and higher apoptotic indices than those in EP1+/+ mice suggests that lack of the EP1 receptor may down-regulate cell proliferation and up-regulate apoptosis, resulting in lower tumor incidence and volume. Further studies to investigate how the EP1 receptor regulates cell proliferation and apoptosis signaling in colon tumors are required.


View this table:
[in this window]
[in a new window]
 
Table II. Effects of EP1 receptor deficiency on cell proliferation and apoptosis in colon tumors induced by AOM

 
To investigate which EP receptors are involved in colon cancer tissues, we analyzed expression of EP receptor genes using the RT-PCR method. We first examined the EP1 receptor mRNA levels in normal mucosa and cancer tissues in the mice. Representative results on EP receptor expression in normal mucosa and cancer tissues of the colon from EP1+/+ and EP1–/– mice are shown in Figure 3. Using primers a and b (Figure 1) we found that the EP1 receptor was evident in colon cancer, whereas it was not detectable in normal mucosa of EP1+/+ mice (Figure 3, upper panel). As expected, EP1 receptor mRNA was not detected either in normal colon mucosa or cancer tissues of EP1–/– mice. The whole mouse EP1 receptor gene is contained within the 3'-untranslated region of a long PKN gene transcript. Primers a and b (Figure 1) were designed to distinguish EP1 receptor from PKN by product length. Although PKN was found to be constitutively expressed in both normal mucosa and cancer of the colon in EP1+/+ mice, interestingly, PKN appeared not to be expressed in EP1–/– mice. These results suggest that the EP1 gene knockout strategy may influence the stability of the long form of the PKN gene. To clarify the contribution of PKN gene instability in EP1 knockout mice during colon carcinogenesis, we examined whether the PKN encoding region was influenced in these mice. Using primers c and d (Figure 1) we found no differences in mRNA levels of the PKN encoding region between normal mucosa and cancer of the colon in both EP1+/+ and EP1–/– mice (Figure 3, second panel). In addition, expression of mRNA for the PKN encoding region was at the same level in the kidneys of all EP1+/+ and EP1–/– mice (data not shown). These results suggest that the EP1 knockout strategy and AOM-induced colon carcinogenesis do not affect PKN gene stability. Thus, we conclude that colon cancer development was reduced by lack of the EP1 receptor, not PKN gene instability. Next, we examined expression of other EP receptor mRNAs in normal mucosa and cancers of the colon in EP1+/+ and EP1–/– mice (Figure 3, lower panels). The data clearly indicate the EP2 receptor to be up-regulated and the EP3 receptor down-regulated in colon cancers. The EP4 receptor was detected in both normal mucosa and cancers of the colon. There were no differences in the results on EP receptors expression except for EP1 between EP1–/– and EP1+/+ mice. Recently it was reported that the EP2 receptor is important for intestinal polyp formation in Apc{Delta}716 heterozygote mice (30). The authors found analogous results for EP receptor mRNA expression using RT-PCR, as in the present study. They, however, indicated no differences in EP1 receptor expression between polyps and normal mucosa in both small and large intestine. The difference between our and their results may be due to the use of different samples, such as colon adenocarcinomas and intestinal polyps, respectively.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 3. EP receptors and PKN gene expression using RT-PCR. Lanes M, N and C indicate molecular weight control, normal mucosa and cancer of the colon, respectively. The left two lanes represent samples from EP1+/+ mice and the right two lanes represent samples from EP1–/– mice. (Upper panel) Agarose gel indicating results for EP1 receptor and PKN gene expression using primers a and b. (Second panel) Agarose gel indicating results for PKN encoding region expression using primers c and d. (Third, fourth, fifth and sixth panels) Agarose gels indicating results for EP2, EP3, EP4 and ß-actin gene expression, respectively.

 
The results of this study clearly show that EP1 receptor deficiency decreases colon cancer incidence induced by AOM from 57 to 27%. However, 27% of EP1–/– mice still developed colon cancers. Interestingly, the EP2 receptor was up-regulated and the EP3 receptor was down-regulated in colon cancers in EP1–/– mice. The EP1 receptor mediates a PGE2-induced elevation in free Ca2+ concentration in Chinese hamster ovary cells and is able to regulate Ca2+ channel gating via an as yet unidentified G protein (24). The EP2 and EP4 receptors are coupled to Gs and mediate increases in cAMP levels (25). Although alternative splicing of the EP3 receptor gene results in several isoforms in animal species, including the mouse, rat and human (3133), the major signaling pathway of the EP3 receptor is inhibition of adenylate cyclase via Gi. Therefore, there may be cross-talk between second messengers after ligand binding to EP receptors and all four types of EP receptor may interact with each other. Further investigations to identify down stream coordination of these receptors are required.

In conclusion, EP1 receptor knockout mice demonstrate significantly reduced AOM-induced colon cancer development compared with wild-type mice due to down-regulation of cell proliferation and up-regulation of apoptosis in cancer cells. Also, EP1 receptor mRNA was found to be up-regulated in colon cancers. These results suggest that the EP1 receptor plays a pivotal role in colon carcinogenesis and support the hypothesis that lack of the EP1 receptor has the potential for a mechanism-based chemoprevention strategy against colon cancer development. Therefore, EP1 receptor antagonists may be a promising chemopreventive agent against colon carcinogenesis. Further studies on the chemopreventive potential of EP1 receptor antagonists for colon cancer in long-term in vivo animal models are ongoing in our laboratory.


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


    Acknowledgments
 
We thank Ms Yukari Teramoto for her excellent technical support. This work was supported in part by Grants-in-Aid for Cancer Research and the Second-Term Comprehensive 10-Year Strategy for Cancer Research from the Ministry of Health, Labour and Welfare, Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

  1. Thun,M.J., Namboodiri,M.M. and Heath,C.W.,Jr (1991) Aspirin use and reduced risk of fatal colon cancer. N. Engl. J. Med., 325, 1593–1596.[Abstract]
  2. Reddy,B.S., Tokumo,K., Kulkarni,N., Aligia,C. and Kelloff,G. (1992) Inhibition of colon carcinogenesis by prostaglandin synthesis inhibitors and related compounds. Carcinogenesis, 13, 1019–1023.[Abstract]
  3. Singh,J., Hamid,R. and Reddy,B.S. (1997) Dietary fat and colon cancer: modulation of cyclooxygenase-2 by types and amount of dietary fat during the postinitiation stage of colon carcinogenesis. Cancer Res., 57, 3465–3470.[Abstract]
  4. Kargman,S.L., O'Neill,G.P., Vickers,P.J., Evans,J.F., Mancini,J.A. and Jothy,S. (1995) Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res., 55, 2556–2559.[Abstract]
  5. Sano,H., Kawahito,Y., Wilder,R.L. et al. (1995) Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res., 55, 3785–3789.[Abstract]
  6. Kawamori,T., Rao,C.V., Seibert,K. and Reddy,B.S. (1998) Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res., 58, 409–412.[Abstract]
  7. Jacoby,R.F., Seibert,K., Cole,C.E., Kelloff,G. and Lubet,R.A. (2000) The cyclooxygenase-2 inhibitor celecoxib is a potent preventive and therapeutic agent in the min mouse model of adenomatous polyposis. Cancer Res., 60, 5040–5044.[Abstract/Free Full Text]
  8. Steinbach,G., Lynch,P.M., Phillips,R.K. et al. (2000) The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N. Engl. J. Med., 342, 1946–1952.[Abstract/Free Full Text]
  9. Tsujii,M. and DuBois,R.N. (1995) Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell, 83, 493–501.[ISI][Medline]
  10. Oshima,M., Dinchuk,J.E., Kargman,S.L., Oshima,H., Hancock,B., Kwong,E., Trzaskos,J.M., Evans,J.F. and Taketo,M.M. (1996) Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell, 87, 803–809.[ISI][Medline]
  11. Chulada,P.C., Thompson,M.B., Mahler,J.F. et al. (2000) Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min mice. Cancer Res., 60, 4705–4708.[Abstract/Free Full Text]
  12. Yamaguchi,A., Ishida,T., Nishimura,G., Katoh,M. and Miyazaki,I. (1991) Investigation of colonic prostaglandins in carcinogenesis in the rat colon. Dis. Colon Rectum, 34, 572–576.[ISI][Medline]
  13. Rigas,B., Goldman,I.S. and Levine,L. (1993) Altered eicosanoid levels in human colon cancer. J. Lab. Clin. Med., 122, 518–523.[ISI][Medline]
  14. Kawamori,T., Uchiya,N., Sugimura,T. and Wakabayashi,K. (2003) Enhancement of colon carcinogenesis by prostaglandin E2 administration. Carcinogenesis, 24, 985–990.[Abstract/Free Full Text]
  15. Ushikubi,F., Segi,E., Sugimoto,Y. et al. (1998) Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP3. Nature, 395, 281–284.[CrossRef][ISI][Medline]
  16. Segi,E., Sugimoto,Y., Yamasaki,A. et al. (1998) Patent ductus arteriosus and neonatal death in prostaglandin receptor EP4-deficient mice. Biochem. Biophys. Res. Commun., 246, 7–12.[CrossRef][ISI][Medline]
  17. Hizaki,H., Segi,E., Sugimoto,Y. et al. (1999) Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP(2). Proc. Natl Acad. Sci. USA, 96, 10501–10506.[Abstract/Free Full Text]
  18. Watanabe,K., Kawamori,T., Nakatsugi,S. et al. (1999) Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis. Cancer Res., 59, 5093–5096.[Abstract/Free Full Text]
  19. Mutoh,M., Watanabe,K., Kitamura,T. et al. (2002) Involvement of prostaglandin E receptor subtype EP(4) in colon carcinogenesis. Cancer Res., 62, 28–32.[Abstract/Free Full Text]
  20. Mukai,H. and Ono,Y. (1994) A novel protein kinase with leucine zipper-like sequences: its catalytic domain is highly homologous to that of protein kinase C. Biochem. Biophys. Res. Commun., 199, 897–904.[CrossRef][ISI][Medline]
  21. Batshake,B. and Sundelin,J. (1996) The mouse genes for the EP1 prostanoid receptor and the PKN protein kinase overlap. Biochem. Biophys. Res. Commun., 227, 70–76.[CrossRef][ISI][Medline]
  22. Pozharisski,K.M. (1990) Tumours of the intestines. In Turusov,V. and Mohr,U. (eds) Pathology of Tumours in Laboratory Animals, IARC Scientific Publications no. 1. IARC, Lyon, pp. 159–180.
  23. Tanaka,T., Takeuchi,T., Nishikawa,A., Takami,T. and Mori,H. (1989) Nucleolar organizer regions in hepatocarcinogenesis induced by N-2-fluorenylacetamide in rats: comparison with bromodeoxyuridine immunohistochemistry. Jpn. J. Cancer Res., 80, 1047–1051.[ISI][Medline]
  24. Watabe,A., Sugimoto,Y., Honda,A., Irie,A., Namba,T., Negishi,M., Ito,S., Narumiya,S. and Ichikawa,A. (1993) Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor. J. Biol. Chem., 268, 20175–20178.[Abstract/Free Full Text]
  25. Katsuyama,M., Nishigaki,N., Sugimoto,Y., Morimoto,K., Negishi,M., Narumiya,S. and Ichikawa,A. (1995) The mouse prostaglandin E receptor EP2 subtype: cloning, expression and northern blot analysis. FEBS Lett., 372, 151–156.[CrossRef][ISI][Medline]
  26. Sugimoto,Y., Negishi,M., Hayashi,Y., Namba,T., Honda,A., Watabe,A., Hirata,M., Narumiya,S. and Ichikawa,A. (1993) Two isoforms of the EP3 receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with Gi proteins. J. Biol. Chem., 268, 2712–2718.[Abstract/Free Full Text]
  27. Honda,A., Sugimoto,Y., Namba,T., Watabe,A., Irie,A., Negishi,M., Narumiya,S. and Ichikawa,A. (1993) Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype. J. Biol. Chem., 268, 7759–7762.[Abstract/Free Full Text]
  28. Egan,M.J. and Crocker,J. (1992) Nucleolar organiser regions in pathology. Br. J. Cancer, 65, 1–7.[ISI][Medline]
  29. Ruschoff,J., Bittinger,A., Neumann,K. and Schmitz-Moormann,P. (1990) Prognostic significance of nucleolar organizing regions (NORs) in carcinomas of the sigmoid colon and rectum. Pathol. Res. Pract., 186, 85–91.[ISI][Medline]
  30. Sonoshita,M., Takaku,K., Sasaki,N., Sugimoto,Y., Ushikubi,F., Narumiya,S., Oshima,M. and Taketo,M.M. (2001) Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc (Delta 716) knockout mice. Nature Med., 7, 1048–1051.[CrossRef][ISI][Medline]
  31. Kotani,M., Tanaka,I., Ogawa,Y., Usui,T., Tamura,N., Mori,K., Narumiya,S., Yoshimi,T. and Nakao,K. (1997) Structural organization of the human prostaglandin EP3 receptor subtype gene (PTGER3). Genomics, 40, 425–434.[CrossRef][ISI][Medline]
  32. Namba,T., Sugimoto,Y., Negishi,M., Irie,A., Ushikubi,F., Kakizuka,A., Ito,S., Ichikawa,A. and Narumiya,S. (1993) Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity. Nature, 365, 166–170.[CrossRef][ISI][Medline]
  33. Negishi,M., Sugimoto,Y., Irie,A., Narumiya,S. and Ichikawa,A. (1993) Two isoforms of prostaglandin E receptor EP3 subtype. Different COOH-terminal domains determine sensitivity to agonist-induced desensitization. J. Biol. Chem., 268, 9517–9521.[Abstract/Free Full Text]
Received May 26, 2004; revised October 11, 2004; accepted October 19, 2004.