Prostaglandin E receptor EP3 deficiency modifies tumor outcome in mouse two-stage skin carcinogenesis
Yutaka Shoji,
Mami Takahashi,
Nobuo Takasuka,
Naoko Niho,
Tomohiro Kitamura,
Hidetaka Sato 1,
Takayuki Maruyama 2,
Yukihiko Sugimoto 3,
Shuh Narumiya 4,
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 Department of Biological Safety Research, Japan Food Research Laboratories, Bunkyo 2-3, Chitose-shi, Hokkaido 066-0052, Japan, 2 Minase Research Institute, Ono Pharmaceutical Co. Ltd, 1-1, Sakurai 3-chome, Shimamoto-cho, Mishima-gunn, Osaka 606-8501, Japan, 3 Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto-shi, Kyoto 606-8501, Japan and 4 Department of Pharmacology, School of Medicine, Kyoto University, Kyoto-shi, Kyoto 606-8501, Japan
* To whom correspondence should be addressed. Tel: +81 3 3542 2511 ext. 4350; Fax: +81 3 3543 9305; E-mail: kwakabay{at}gan2.res.ncc.go.jp
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Abstract
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We have recently shown that the prostaglandin E2 (PGE2) receptor EP3 plays an important role in suppression of colon cancer cell proliferation and that its deficiency enhances late stage colon carcinogenesis. Here we examined the effects of EP3-deficiency on two-stage skin carcinogenesis. 7,12-Dimethylbenz[a]anthracene (50 µg/200 µl of acetone) was thus applied to the back skin of female EP3-knockout and wild-type mice at 8 weeks of age, followed by treatment with 12-O-tetradecanoylphorbol-13-acetate (5 µg/200 µl of acetone) twice a week for 25 weeks. First tumor appearance was observed in EP3-knockout mice at week 10, which was 3 weeks later than in EP3 wild-type mice, and multiplicity observed at week 11 was significantly lower in the EP3-knockout case. However, histological examination showed that the tumor incidence and multiplicity at week 25 were not significantly changed in knockout mice and wild-type mice (incidence, 19/19 versus 23/24; multiplicity, 3.58 ± 0.51 versus 3.17 ± 0.63, respectively). Interestingly, there were no squamous cell carcinomas (SCCs) in the EP3-knockout mice, while SCCs were observed in 3 out of 24 wild-type mice. Furthermore, benign keratoacanthomas only developed in EP3-knockout mice (6/19 versus 0/24, P < 0.01). The results suggest that PGE2 receptor EP3 signaling might contribute to development of SCCs in the skin.
Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; COX, cyclooxygenase; DMBA, 7,12-dimethylbenz[a]anthracene; PGE2, prostaglandin E2; SCC, squamous cell carcinoma; TPA, 12-O-tetradecanoylphorbol-13-acetate
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Introduction
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Prostaglandins are lipid autacoids synthesized by cyclooxygenase (COX) in response to numerous growth factors and environmental stimuli. Two isoforms of COXs have been described, the constitutively and ubiquitously expressed COX-1 and the inducible COX-2. Overproduction of prostaglandins attributable to overexpression of COX-2 in various tumors is critical for epithelial carcinogenesis and provides a target for cancer chemoprevention by non-steroidal antiinflammatory drugs (NSAIDs)(15). Furthermore, there is a large amount of evidence from epidemiological and pharmacological studies that COX inhibitors exhibit chemopreventive activities for various malignancies in humans, including skin cancer (6,7). Recent studies of chemical carcinogenesis in COX-2-overexpressing transgenic mice demonstrated a promotive role of COX-2 in tumorigenesis in the skin and breast epithelium (8,9). A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), was found to increase the epidermal prostaglandin E2 (PGE2) in mouse skin, and thus appears to be critical for TPA-induced hyperproliferation (10). PGE2 exerts its biological action through binding to four specific receptor subtypes, EP1, EP2, EP3 and EP4, with seven transmembrane domains. Activation of the EP1 receptor is associated with increases in intracellular Ca2+ and the EP2 and EP4 receptors are known to be coupled to Gs protein and stimulate cAMP production by activation of adenylate cyclase. In contrast, the major signaling pathway for the EP3 receptor is inhibition of adenylate cyclase via Gi. Several isoforms are also generated by alternative splicing from the single EP3 receptor gene, and these exhibit other functions through activation via G proteins other than Gi (11).
Previously, we examined the role of PGE2 receptor subtypes in intestinal carcinogenesis using knockout mice, and the results showed that formation of aberrant crypt foci (ACF), putative preneoplastic lesions, induced by azoxymethane (AOM) was decreased in the EP1 and EP4 knockout cases, suggesting involvement of the receptors in ACF formation (12,13). Moreover, homozygous deletion of the gene encoding the EP2 receptor results in decrease of intestinal polyp formation in Apc gene-deficient mice (14). Recently, we also examined roles of the EP3 receptor in AOM-induced long-term colon carcinogenesis using EP3 receptor-knockout mice, and obtained evidence that it suppresses cell proliferation, downregulation enhancing late stage colon carcinogenesis (15).
In the present study, we examined two-step skin carcinogenesis with dimethylbenz[a
]anthracene (DMBA) and TPA (16) in EP3-knockout mice. These mice exhibited benign tumor formation, while wild-type mice developed malignant squamous cell carcinomas (SCCs). The results indicate that the EP3 receptor may play a critical role in the development of malignancies in skin carcinogenesis.
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Materials and methods
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Animals
Mice lacking EP3 receptor gene were generated as reported previously (17) and were backcrossed to the C57BL/6Cr strain for 10 generations. Female EP3 receptor-deficient mice were used at 6 weeks of age. Genotypes of the knockout mice were confirmed by PCR according to the method described previously (12). The animals were housed in plastic cages at 24 ± 2°C and 55% relative humidity with a 12/12 h light/dark cycle. Water and basal diet (AIN-76A; CLEA Japan, Tokyo) were given ad libitum. Body weights and food intake were measured weekly.
Two-stage skin carcinogenesis experiments
Initiation was achieved by a single application of 50 µg of DMBA dissolved in 200 µl of acetone to the skin of the backs of female EP3 receptor-deficient homozygous (EP3/) and wild-type mice at 8 weeks of age. From week 1, after the initiation, a 5 µg aliquot of TPA dissolved in 200 µl of acetone was applied to the initiated skin parts of the mice, twice a week for 25 weeks, as described previously (18). Skin tumors were noted grossly for their location, number and diameters, measured with calipers, and digital photographs of the backs of each animal were taken once a week. Mice were killed under ether euthanasia at the end of week 25 and complete autopsy was performed. All skin tumors were subjected to histological examination after routine processing and hematoxylin and eosin staining. The experimental protocol was according to the guidelines for Animal Experiments in the National Cancer Center.
Immunohistochemical staining
Immunohistochemical analyses of skin tumor samples from female EP3/ and wild-type mice were performed with the avidinbiotin complex immunoperoxidase technique, as previously reported (19). As primary antibodies, monoclonal mouse anti-ß-catenin and mouse anti-E-cadherin antibodies (Transduction Laboratories, Lexington, KY) were used at 100x dilution (20). As the secondary antibody, biotinylated anti-mouse IgG (H+L) raised in a horse, affinity purified, and absorbed with rat serum (Vector Laboratories, Burlingame, CA) was used at 200x dilution. Staining was performed using avidinbiotin reagents (Vectastain ABC reagents; Vector Laboratories), 3,3'-diaminobenzidine and hydrogen peroxide, and the sections were counterstained with hematoxylin to facilitate orientation. As a negative control, duplicate sections were immunostained without exposure to the primary antibody (20).
Analysis of EP receptor expression in skin tumors by RTPCR
Total RNA was extracted from papillomas (5 mm or more in diameter), keratoacanthoma, SCCs and normal skin tissues by direct homogenization in ISOGEN (Nippon Gene, Tokyo, Japan), and spectrophotometry was used for quantification. Three microgram aliquots of total RNA were subjected to the RT reaction with random 9mer primers using an Omniscript Reverse Transcriptase kit (Qiagen, Hilden, Germany). After reverse transcription, PCR was carried out with HotStartaq (Qiagen), according to the manufacturer's instructions. To test cDNA integrity, the cyclopilin (PPIA) gene was amplified for each sample (21). Primers were designed, based on published sequences from Genbank, using the computer program OLIGO 4.0-s (National Biosciences, MD). Primers were designed to cross an exonexon boundary or insert an intron to ensure that genomic DNA was not being amplified. BLAST searches confirmed that the primers were specific for the target gene. Primers for the cyclopilin and EP receptor genes are listed in Table I. PCR amplification was performed in a thermal cycler (Gene Amp PCR System 9600, PerkinElmer Applied Biosystems, Foster City, CA), with 1840 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 1 min, using the specific primer sets. The PCR products were then analyzed by electrophoresis on 2% agarose gels.
Quantitative real-time RTPCR analysis
Quantitative real-time RTPCR analysis was performed using the Smart Cycler system with an Ex Taq R-PCR kit and SYBR Green (Takara Shuzo, Shiga, Japan) according to the manufacturer's instructions. Primers for the cyclopilin and EP3 genes, and the cycle conditions for PCR are listed in Table I. To assess the specificity of each primer set, amplicons generated in PCR reactions were analyzed for their melting point curves and additionally run on 2% agarose gels to confirm the correct sizes of the PCR products. Each PCR product was subcloned into the TA cloning plasmid vector, pGEN-T easy vector (Promega, Madison, WI), and used as a positive control for real-time PCR analyses. The numbers of molecules of specific gene products in each sample were determined using a standard curve generated by amplification of 102108 copies of the control plasmid.
Statistical analysis
The significance of differences in the incidences of tumors was analyzed with the
2-test and other differences using the Student's t-test. Statistical significance was concluded at P < 0.05.
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Results
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Macroscopic findings for skin carcinogenesis
The mean food intake and body weights of EP3 receptor-knockout mice were comparable with those of wild-type mice. Data for macroscopic incidences (percentage of mice with tumors) and multiplicities (number of tumors per mouse) of skin tumors over time are summarized in Figure 1. Skin tumors began to appear at 7 weeks after DMBA treatment in wild-type mice, and at 10 weeks in EP3 receptor-knockout mice (Figure 1A). Multiplicities of skin tumors at week 11 were significantly different in EP3 receptor-knockout mice and wild-type mice (0.05 ± 0.05 versus 0.46 ± 0.18, P < 0.05) (Figure 1A and B), but no significant differences in incidences were observed (1/19 versus 8/24, P < 0.056). There were no significant differences in either incidences or multiplicities of macroscopic lesions at 25 weeks after TPA treatment (Figure 1A and B).

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Fig. 1. (A) Incidences (percentage of mice with tumors) and (B) multiplicities (number of tumors per mouse) of macroscopic skin tumors. Open squares, wild-type; closed circles, EP3/ mice. Note the significant differences in multiplicities at week 11 (0.05 ± 0.05 versus 0.46 ± 0.18) between EP3/ and wild-type mice. *Significantly different from the corresponding wild-type mice value at P < 0.05.
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Histological findings for skin carcinogenesis
Data for the histopathological diagnoses of skin tumors are summarized in Table II. Final incidences of skin tumors were 100% (19/19) and 95.8% (23/24), and multiplicities were 3.58 ± 0.51 and 3.17 ± 0.63 in EP3 receptor-knockout mice and wild-type mice, respectively. The incidences and multiplicities of papillomas also did not differ between the groups. However, SCCs occurred in three wild-type mice, one with two lesions, but were not observed in EP3 receptor-knockout mice. Instead of SCCs, keratoacanthomas were apparent in EP3 receptor-knockout mice, but were not apparent in their wild-type counterparts (6/19 versus 0/24, P < 0.01).
Microscopical features of skin tumors observed in hematoxylin and eosin stained sections are illustrated in Figure 2. One of the SCCs was moderately-differentiated and this lesion appeared as a solid mass forming shallow encrusted ulcers, with squamous epithelial cells proliferating downward and invading the adjacent subcutis and muscle (Figure 2A). This SCC was composed of irregular masses of squamous epithelial cells and some spindle cells (Figure 2B). The other three SCCs were well-differentiated and featured irregular structures of squamous cells with well defined basal cell layers (Figure 2C), and a number of epithelial pearls in the basal cell layers (Figure 2D). All keratoacanthomas exhibited a characteristic bowl shape filled with mature keratin (Figure 2E), and the walls had a buttress-like appearance. They were composed of thick, folded layers of well-differentiated stratified squamous epithelium enclosed by a prominent basal layer, and the structure of stratified squamous epithelium was similar to normal epidermis (Figure 2F).

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Fig. 2. Microscopical features of skin tumors observed in hematoxylin and eosin stained sections. (AD), SCCs in wild-type mice. (E and F), keratoacanthomas in EP3/ mice. Moderately-differentiated SCC appearing as a solid mass forming shallow encrusted ulcers (A), and irregular masses of squamous epithelial cells and some spindle cells (B). A well-differentiated SCC demonstrates loss of keratin (C), and a number of epithelial pearls in the basal cell layer (D). Keratoacanthomas exhibits a bowl shape filled with mature keratin (E), and well-differentiated stratified squamous epithelium (F). Original magnification: 40x for A, C and E; 200x for B, D and F.
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The four SCCs and six keratoacanthomas developed at 10, 11, 17, 20 and 12, 12, 14, 17, 17, 22 weeks after the beginning of TPA treatment, respectively. SCCs were originally observed as wart-like tumors in the shape of a dome without a stem, and then grew inside mouse skin. On the other hand, keratoacanthomas arose from papilloma-like tumors in the shape of a pole or a mushroom, and then grew on the surface of mouse skin into node-like or crater-like tumors.
The size distribution of papillomas (excluding SCCs and keratoacanthomas) is provided in Figure 3. The number of papillomas 3 to <5 mm in diameter was significantly increased in EP3 receptor-knockout compared with wild-type mice (0.68 ± 0.19 versus 0.22 ± 0.09, P < 0.05).

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Fig. 3. Size distribution of papillomas in wild-type (open columns) and EP3/ mice (closed columns). The mean number of papillomas/mouse in each size class is given; bars, SE. *Significantly different from the corresponding wild-type value at P< 0.05.
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Localization of E-cadherin and ß-catenin in skin tumors
Immunohistochemical analysis of paraffin-embedded specimens of skin SCCs in wild-type mice (Figure 4AC) and keratoacanthomas in EP3 receptor-knockout mice (Figure 4DF) was performed to examine the localization of cellcell adhesion molecules E-cadherin and ß-catenin, using specific antibodies. Sections without primary antibody treatment were also stained as negative controls (Figure 4A and D). Reduced expression of E-cadherin in cellular membranes and abnormal localization of ß-catenin in the cytoplasm were found in basal cells of SCCs (Figure 4B and C). In contrast, normal localization of E-cadherin and ß-catenin were observed in basal cells of keratoacanthomas (Figure 4E and F).

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Fig. 4. Immunohistochemical staining of E-cadherin and ß-catenin in a SCC in a wild-type mouse (AC) and a keratoacanthoma in an EP3/ mouse (DF). (A and D), negative controls without primary antibody; (B and E), E-cadherin; (C and F), ß-catenin.
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Expression of PGE2 receptors in skin tumors
Expression of PGE2 receptors in four samples each of DMBA/TPA-treated mouse skin lesions and adjacent normal skin tissues were examined by RTPCR. Expression of EP1, EP2 and EP4 receptor mRNAs in SCCs and keratoacanthomas was very similar to that in adjacent normal skin samples. EP3 expression in keratoacanthomas and adjacent normal skin samples from EP3 receptor-knockout mice could not be detected, in clear contrast to the positive results obtained for all SCC and adjacent normal skin samples in wild-type mice. However, expression of EP3 receptor mRNA in skin SCCs of wild-type mice was markedly lower than that in normal skin tissue (Figure 5A). Quantitative real-time RTPCR analysis suggested the EP3 receptor to be downregulated in papillomas and SCCs to 5.7 and 17.1%, respectively, of the average value of normal skin tissues (Figure 5B).

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Fig. 5. Expression of PGE2 receptors in DMBA/TPA-treated mouse skin lesions and adjacent normal skin tissues. (A) Expression of EP14 receptors examined by RTPCR. PPIA was used as an internal control. N indicates adjacent normal skin tissue. SC and K indicate SCCs in wild-type mice and keratoacanthomas in EP3/ mice. (B) Expression of the EP3 receptor examined by real-time RTPCR. The PCR primers of mouse and rat EP3 receptors were designed to target a sequence common to all EP3 receptor variants expressed in the mouse. N indicates adjacent normal skin tissue (n = 6). Pa and SC indicate papillomas (n = 3) and SCCs (n = 4), respectively, in wild-type mice. Columns, mean; bars, ± SE; **P < 0.01.
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Discussion
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In the present study, examination of the significance of PGE2 receptor EP3 for two-stage DMBA/TPA skin carcinogenesis in EP3 receptor-knockout mice showed no overall difference in incidences and multiplicities of papillomas at week 25, but a shift from SCC to keratoacanthoma development was evident, indicating that the EP3 receptor may promote malignant change in skin carcinogenesis.
The EP3 receptor is widely distributed throughout the body, and its mRNA has been identified in almost all tissues in mice and rats, as well as humans (11,22,23). In situ hybridization analysis has revealed high level expression of EP3 and EP4 receptor mRNA in the dermal papilla cells of hair follicles (24). We confirmed the prostaglandin E2 receptors EP14 to be expressed in wild-type mouse normal skin tissues by RTPCR (Figure 5A), consistent with a previous report (25).
TPA treatment is known to increase epidermal PGE2 production in mice skin (10), and PGE2 promotes mast cell activation and IL-6 production through the EP3 receptor (2628). Furthermore, acute cutaneous inflammation induced by arachidonic acid is markedly attenuated in EP3 receptor-knockout mice (29). Thus, PGE2/EP3 signaling is considered to be a major pathway of acute inflammation in mouse skin. In the present study, macroscopic observation demonstrated that formation of tumors was delayed for 3 weeks in EP3 receptor-knockout mice, and multiplicity was significantly lower at week 11 than in wild-type mice. DMBA/TPA two-stage skin tumorigenesis using IL-6-knockout C57BL/6 mice also features a delay in tumor formation compared with wild-type mice (30) so that the delay of 3 weeks in formation of tumors in EP3 receptor-knockout mice may have been caused by attenuation of acute inflammation due to deficiency of the EP3 receptor. Therefore, it is important to determine whether the temporal delay in tumor formation in EP3 receptor-knockout mice consequently influenced formation of keratoacanthomas. Examination of the time points of occurrence of the individual keratoacanthomas and SCCs, however, suggested no clear difference in the developing period between two types of tumor. Therefore, it is considered that the lack of SCCs in EP3-knockout mice was not simply due to a delay, but rather to a block in malignant development because of the EP3 deficiency. Indeed, characteristic differences in generation of keratoacanthomas and SCCs were observed as macroscopic findings, described in the Results. These might reflect variation, especially regarding cell invasiveness, in the early promotion stage occurring in wild-type but not EP3 receptor-knockout mice. Clearly, a number of molecules downstream of the EP3 receptor could play important roles in early stages of skin carcinogenesis; however, which are actually critical for SCC formation have yet to be clarified.
The present histological assessment suggested that tumor development in wild-type and EP3 receptor-knockout mice distinctly differs (Figure 2). The structural differences apparent between SCCs and keratoacanthomas would be expected to correlate with several factors including cellcell adhesion, cell invasiveness and cell polarity, and this was confirmed by our immunohistochemical findings for adhesion molecules, E-cadherin and ß-catenin. Chu et al. (31,32) earlier reported E-cadherin and catenin (
-, ß- and
-) to be normally localized in cell membranes of keratoacanthomas, whereas abnormal cytoplasmic localization or loss of expression are characteristic of SCCs and our results are in agreement.
There was no significant difference in incidences and multiplicities of tumor formation at the end point of the experiment as shown in Figure 1 and Table II. Although formation of tumors was delayed for 3 weeks in EP3 receptor-knockout mice (Figure 1), papillomas of 3 to <5 mm in diameter were increased (Figure 3), suggesting elevated proliferation of tumor cells. Indeed, our previous study has shown that downregulation of EP3 receptor expression in colon cancer might be associated with increased multiplicity of lesions 25 mm in diameter. Furthermore, the EP3 receptor-selective agonist, ONO-AE-248 decreases cell proliferation in the HCA-7 human colon adenocarcinoma cell line (15). Konger et al. (33) reported that growth stimulation of human kerationocyte cells occurs via an EP2 and/or EP4 receptor-adenylate cyclase coupled response, while cell growth was inhibited by EP3 receptor agonist sulprostone. Our studies, together with recent reports, suggest that the EP3 receptor plays a role in suppression of skin epithelial cell proliferation and inhibits tumor growth.
The mouse EP3 receptor has three isoforms, EP3
, EP3ß and EP3
generated by alternative splicing from the single EP3 receptor gene. The major signaling pathway for the EP3
and EP3ß receptors is inhibition of adenylate cyclase via Gi (34,35). In contrast, the EP2 and EP4 receptors are coupled to Gs and stimulate cAMP production by adenylate cyclase (11). Konger et al. (36) reported that inhibition of EP2 receptor expression by its anti-sense construct in a HaCat immortalized human keratinocyte cell line, which expresses the EP2 receptor predominantly and trace amounts of EP3 and EP4 receptors, is associated with decreased expression of paxillin, a critical component for focal adhesion assembly. Inhibition of EP2 receptor expression decreased EP2 agonist-induced cAMP production in HaCat cells, and endowed extensive deep invasion capacity in a 3D organ culture model of normal skin. Our findings provide indirect evidence that attenuation of cAMP production by PGE2 via EP3 receptor in keratinocyte cells might enhance neoplastic progression. It has been reported that the EP3 selective agonist ONO-AE-248 blocks the rise in intracellular cAMP induced by forskolin, an activator of adenylate cyclase, in CHO cells transfected with EP3
receptor (37). Additional studies are now needed to investigate interactions between the EP3 and other EP receptors in skin carcinogenesis.
In conclusion, our present data suggest that the PGE2 receptor EP3 may play a role in neoplastic progression in skin carcinogenesis.
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Acknowledgments
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We thank Professor Hiroyuki Tsuda (Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences) for his comments on histology of skin tumors. We are also grateful to Mr Naoaki Uchiya and Miss Yurika Teramoto for excellent technical assistance. This work was supported in part by Grants-in-Aid for Cancer Research, for the Third-Term Comprehensive 10-Year Strategy for Cancer Control and for Research on Advanced Medical Technology, from the Ministry of Health, Labor and Welfare of Japan. Y.S. was the recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research during performance of this work.
Conflict of Interest Statement: None declared.
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References
|
---|
- Asano,T., Shoda,J., Ueda,T., Kawamoto,T., Todoroki,T., Shimonishi,M., Tanabe,T., Sugimoto,Y., Ichikawa,A., Mutoh,M., Tanaka,N. and Miwa,M. (2002) Expressions of cyclooxygenase-2 and prostaglandin E-receptors in carcinoma of the gallbladder: crucial role of arachidonate metabolism in tumor growth and progression. Clin. Cancer Res., 8, 11571167.[Abstract/Free Full Text]
- Fujita,T., Matsui,M., Takaku,K., Uetake,H., Ichikawa,W., Taketo,M.M. and Sugihara,K. (1998) Size- and invasion-dependent increase in cyclooxygenase 2 levels in human colorectal carcinomas. Cancer Res., 58, 48234826.[Abstract]
- Murata,H., Kawano,S., Tsuji,S., Tsuji,M., Sawaoka,H., Kimura,Y., Shiozaki,H. and Hori,M. (1999) Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma. Am. J. Gastroenterol., 94, 451455.[CrossRef][ISI][Medline]
- Denkert,C., Kobel,M., Pest,S., Koch,I., Berger,S., Schwabe,M., Siegert,A., Reles,A., Klosterhalfen,B. and Hauptmann,S. (2002) Expression of cyclooxygenase 2 is an independent prognostic factor in human ovarian carcinoma. Am. J. Pathol., 160, 893903.[Abstract/Free Full Text]
- Ristimaki,A., Sivula,A., Lundin,J., Lundin,M., Salminen,T., Haglund,C., Joensuu,H. and Isola,J. (2002) Prognostic significance of elevated cyclooxygenase-2 expression in breast cancer. Cancer Res., 62, 632635.[Abstract/Free Full Text]
- Fischer,S.M., Lo,H.H., Gordon,G.B., Seibert,K., Kelloff,G., Lubet,R.A. and Conti,C.J. (1999) Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis. Mol. Carcinog., 25, 231240.[CrossRef][ISI][Medline]
- Prescott,S.M. and Fitzpatrick,F.A. (2000) Cyclooxygenase-2 and carcinogenesis. Biochim. Biophys. Acta, 1470, M69M78.[CrossRef][ISI][Medline]
- Müller-Decker,K., Neufang,G., Berger,I., Neumann,M., Marks,F., and Fürstenberger,G. (2002) Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis. Proc. Natl Acad. Sci. USA, 99, 1248312488.[Abstract/Free Full Text]
- Chang,S.H., Liu,C.H., Conway,R., Han,D.K., Nithipatikom,K., Trifan,O.C., Lane,T.F. and Hla,T. (2004) Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression. Proc. Natl Acad. Sci. USA, 101, 591596.[Abstract/Free Full Text]
- Fürstenberger,G., Gross,M. and Marks,F. (1989) Eicosanoids and multistage carcinogenesis in NMRI mouse skin: role of prostaglandins E and F in conversion (first stage of tumor promotion) and promotion (second stage of tumor promotion). Carcinogenesis, 10, 9196.[Abstract]
- Narumiya,S., Sugimoto,Y. and Ushikubi,F. (1999) Prostanoid receptors: structures, properties, and functions. Physiol. Rev., 79, 11931226.[Abstract/Free Full Text]
- Watanabe,K., Kawamori,T., Nakatsugi,S. et al. (1999) Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis. Cancer Res., 59, 50935096.[Abstract/Free Full Text]
- Mutoh,M., Watanabe,K., Kitamura,T. et al. (2002) Involvement of prostaglandin E receptor subtype EP4 in colon carcinogenesis. Cancer Res., 62, 2832.[Abstract/Free Full Text]
- 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 ApcDelta 716 knockout mice. Nat. Med., 7, 10481051.[CrossRef][ISI][Medline]
- Shoji,Y., Takahashi,M., Kitamura,T. et al. (2004) Downregulation of prostaglandin E receptor subtype EP3 during colon cancer development. Gut, 53, 11511158.[Abstract/Free Full Text]
- Armuth,V. and Berenblum,I. (1976) Phorbol as a possible systemic promoting agent for skin carcinogenesis. Z. Krebsforsch Klin. Onkol. Cancer Res. Clin. Oncol., 85, 7982.[Medline]
- Ushikubi,F., Segi,E., Sugimoto,Y. et al. (1998) Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP3. Nature, 395, 281284.[CrossRef][ISI][Medline]
- Morikawa,T., Wanibuchi,H., Morimura,K., Ogawa,M. and Fukushima,S. (2000) Promotion of skin carcinogenesis by dimethylarsinic acid in Keratin (K6)/ODC transgenic mice. Jpn. J. Cancer Res., 91, 579581.[ISI][Medline]
- Takahashi,M., Fukuda,K., Ohata,T., Sugimura,T. and Wakabayashi,K. (1997) Increased expression of inducible and endothelial constitutive nitric oxide synthases in rat colon tumors induced by azoxymethane. Cancer Res., 57, 12331237.[Abstract]
- Takahashi,M., Fukuda,K., Sugimura,T. and Wakabayashi,K. (1998) Beta-catenin is frequently mutated and demonstrates altered cellular location in azoxymethane-induced rat colon tumors. Cancer Res., 58, 4246.[Abstract]
- Weisinger,G., Gavish,M., Mazurika,C. and Zinder,O. (1999) Transcription of actin, cyclophilin and glyceraldehyde phosphate dehydrogenase genes: tissue- and treatment-specificity. Biochim. Biophys. Acta, 1446, 225232.[ISI][Medline]
- Sugimoto,Y., Namba,T., Honda,A., Hayashi,Y., Negishi,M., Ichikawa,A. and Narumiya,S. (1992) Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. J. Biol. Chem., 267, 64636466.[Abstract/Free Full Text]
- Kotani,M., Tanaka,I., Ogawa,Y., Usui,T., Mori,K., Ichikawa,A., Narumiya,S., Yoshimi,T. and Nakao,K. (1995) Molecular cloning and expression of multiple isoforms of human prostaglandin E receptor EP3 subtype generated by alternative messenger RNA splicing: multiple second messenger systems and tissue-specific distributions. Mol. Pharmacol., 48, 869879.[Abstract/Free Full Text]
- Torii,E., Segi,E., Sugimoto,Y., Takahashi,K., Kabashima,K., Ikai,K. and Ichikawa,A. (2002) Expression of prostaglandin E2 receptor subtypes in mouse hair follicles. Biochem. Biophys. Res. Commun., 290, 696700.[CrossRef][ISI][Medline]
- Muller-Decker,K., Leder,C., Neumann,M., Neufang,G., Bayerl,C., Schweizer,J., Marks,F. and Furstenberger,G. (2003) Expression of cyclooxygenase isozymes during morphogenesis and cycling of pelage hair follicles in mouse skin: precocious onset of the first catagen phase and alopecia upon cyclooxygenase-2 overexpression. J. Invest. Dermatol., 121, 661668.[CrossRef][ISI][Medline]
- Rao,T.S., Currie,J.L., Shaffer,A.F. and Isakson,P.C. (1993) Comparative evaluation of arachidonic acid (AA)- and tetradecanoylphorbol acetate (TPA)-induced dermal inflammation. Inflammation, 17, 723741.[CrossRef][ISI][Medline]
- Beetz,A., Messer,G., Oppel,T., van Beuningen,D., Peter,R.U. and Kind,P. (1997) Induction of interleukin 6 by ionizing radiation in a human epithelial cell line: control by corticosteroids. Int. J. Radiat. Biol., 72, 3343.[CrossRef][ISI][Medline]
- Nguyen,M., Solle,M., Audoly,L.P., Tilley,S.L., Stock,J.L., McNeish,J.D., Coffman,T.M., Dombrowicz,D. and Koller,B.H. (2002) Receptors and signaling mechanisms required for prostaglandin E2-mediated regulation of mast cell degranulation and IL-6 production. J. Immunol., 169, 45864593.[Abstract/Free Full Text]
- Goulet,J.L., Pace,A.J., Key,M.L. et al. (2004) E-prostanoid-3 receptors mediate the proinflammatory actions of prostaglandin E2 in acute cutaneous inflammation. J. Immunol., 173, 13211326.[Abstract/Free Full Text]
- Suganuma,M., Okabe,S., Kurusu,M., Iida,N., Ohshima,S., Saeki,Y., Kishimoto,T. and Fujiki,H. (2002) Discrete roles of cytokines, TNF-alpha, IL-1, IL-6 in tumor promotion and cell transformation. Int. J. Oncol., 20, 131136.[ISI][Medline]
- Papadavid,E., Pignatelli,M., Zakynthinos,S., Krausz,T. and Chu,A.C. (2001) The potential role of abnormal E-cadherin and alpha-, beta- and gamma-catenin immunoreactivity in the determination of the biological behaviour of keratoacanthoma. Br. J. Dermatol., 145, 582589.[CrossRef][ISI][Medline]
- Papadavid,E., Pignatelli,M., Zakynthinos,S., Krausz,T. and Chu,A.C. (2002) Abnormal immunoreactivity of the E-cadherin/catenin (alpha-, beta-, and gamma-) complex in premalignant and malignant non-melanocytic skin tumours. J. Pathol., 196, 154162.[CrossRef][ISI][Medline]
- Konger,R.L., Malaviya,R. and Pentland,A.P., (1998) Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes. Biochim. Biophys. Acta, 1401, 221234.[CrossRef][ISI][Medline]
- 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, 27122718.[Abstract/Free Full Text]
- Irie,A., Sugimoto,Y., Namba,T., Harazono,A., Honda,A., Watabe,A., Negishi,M., Narumiya,S. and Ichikawa,A. (1993) Third isoform of the prostaglandin-E-receptor EP3 subtype with different C-terminal tail coupling to both stimulation and inhibition of adenylate cyclase. Eur. J. Biochem., 217, 313318.[CrossRef][ISI][Medline]
- Konger,R.L., Scott,G.A., Landt,Y., Ladenson,J.H. and Pentland,A.P. (2002) Loss of the EP2 prostaglandin E2 receptor in immortalized human keratinocytes results in increased invasiveness and decreased paxillin expression. Am. J. Pathol., 161, 20652078.[Abstract/Free Full Text]
- Zacharowski,K., Olbrich,A., Piper,J., Hafner,G., Kondo,K. and Thiemermann,C. (1999) Selective activation of the prostanoid EP3 receptor reduces myocardial infarct size in rodents. Arterioscler. Thromb. Vasc. Biol., 19, 21412147.[Abstract/Free Full Text]
Received February 16, 2005;
revised July 16, 2005;
accepted July 22, 2005.