Up-regulation of Frizzled-10 (FZD10) by beta-estradiol in MCF-7 cells and by retinoic acid in NT2 cells.

Frizzled (FZD) genes encode receptors for WNTs, which play key roles in carcinogenesis and embryogenesis. We have previously cloned the FZD10 gene, and demonstrated up-regulation of FZD10 mRNA in the cervical cancer cell line HeLa S3, gastric cancer cell lines TMK1 and MKN74, and 4 cases out of 10 cases of primary gastric cancer. Here, effects of beta-estradiol, retinoic acid, and inflammatory cytokines on expression of FZD10 mRNA in human cancer cell lines were investigated. FZD10 mRNA was undetectable in MCF-7 cells derived from breast cancer, and was significantly up-regulated by beta-estradiol in MCF-7 cells with a peak at 24 h after treatment. FZD10 mRNA was expressed in NT2 cells, which are reported to differentiate into neuronal cells after exposure to retinoic acid. Although expression level of FZD10 mRNA was unchanged until 36 h after retinoic-acid treatment, FZD10 mRNA was up-regulated at 48 and 72 h after retinoic-acid treatment in NT2 cells. Effects of inflammatory cytokines on FZD10 mRNA expression in a gastric cancer cell line MKN45 was next investigated. FZD10 mRNA was undetectable in MKN45 cells, and was not up-regulated by IFNgamma and TNFalpha in MKN45 cells. Because we have previously demonstrated up-regulation of WNT2 mRNA by beta-estradiol in MCF-7 cells, FZD10 and WNT2 mRNAs were found to be up-regulated together by beta-estradiol in MCF-7 cells with the same time course. Synchronous up-regulation of FZD10 and WNT2 mRNAs might lead to activation of the WNT signaling pathway in human breast cancer.     

Expression and regulation of WNT1 in human cancer: up-regulation of WNT1 by beta-estradiol in MCF-7 cells

WNT family of secreted-type glycoproteins play key roles in carcinogenesis and embryogenesis. We have cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14 and WNT14B/WNT15 using bioinformatics and cDNA-PCR, and also reported frequent up-regulation of WNT2 in primary gastric cancer. Here, expression and regulation of WNT1 in human cancer were investigated using cDNA-PCR. WNT1 mRNA was relatively highly expressed in OKAJIMA cells (gastric cancer) and BxPC-3 cells (pancreatic cancer). Expression of WNT1 mRNA was up-regulated in 5 out of 10 cases of primary gastric cancer. Effects of beta-estradiol on expression of human WNT1 in MCF-7 cells (breast cancer) was next investigated, because mouse Wnt-1 induces mammary carcinogenesis even in estrogen receptor alpha (ERalpha) knockout mice. Expression of WNT1 mRNA was significantly up-regulated by beta-estradiol in MCF-7 cells. WNT1 was found to be one of estrogen target genes in human MCF-7 cells, which in part explains Wnt1-induced mammary carcinogenesis in ERalpha knockout mice.     

Regulation of WNT3 and WNT3A mRNAs in human cancer cell lines NT2, MCF-7, and MKN45.

We have previously cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT10A, WNT10B, WNT11, WNT14, and WNT14B/WNT15 by using bioinformatics, cDNA-library screening, cDNA-PCR, and RACE. WNT3 and WNT3A genes are two human paralogues of mouse proto-oncogene Wnt3, which induces carcinogenesis through activation of the beta-catenin - TCF signaling pathway. Here, regulation of WNT3 and WNT3A mRNAs in human cancer cell lines was investigated. WNT3 and WNT3A mRNAs were co-expressed in an embryonal carcinoma cell line NT2, which is reported to differentiate into postmitotic CNS neurons by treatment with retinoic acid for two weeks. Expression level of WNT3 mRNA in NT2 cells was not changed during 72 h after retinoic acid treatment, while expression of WNT3A mRNA was down-regulated in NT2 cells by retinoic acid. WNT3 and WNT3A mRNAs were also co-expressed in a breast cancer cell line MCF-7, and were down-regulated together by beta-estradiol in MCF-7 cells. Expression of WNT3 mRNA in a gastric cancer cell line MKN45 was not changed after treatment with tumor necrosis factor alpha (TNFalpha) or interferon gamma (IFNgamma), and that of WNT3A mRNA was undetectable before and after treatment with TNFalpha or IFNgamma. WNT3A, down-regulated by retinoic acid in NT2 cells, might play key roles in the maintenance of NT2 cells in the undifferentiated proliferation stage through activation of the beta-catenin - TCF signaling pathway.     

Up-regulation of WNT10A by tumor necrosis factor alpha and Helicobacter pylori in gastric cancer

WNT signaling pathway is implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized WNT10A and WNT6, which are clustered in human chromosome 2q35 region. In this study, we investigated expression of WNT10A and WNT6 in gastric cancer. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in gastric cancer cell lines MKN7, MKN45 and MKN74. The 2.0-kb WNT6 mRNA was expressed in gastric cancer cell lines MKN28 and MKN74. WNT10A was up-regulated in 3 out of 6 cases of primary gastric cancer, while WNT6 was not up-regulated in primary gastric cancer. Effects of inflammatory cytokines and Helicobacter pylori (H. pylori) on expression of WNT10A and WNT6 were next investigated. Interferon gamma (IFNgamma) failed to induce up-regulation of WNT10A and WNT6. Tumor necrosis factor alpha (TNFalpha) induced up-regulation of WNT10A in MKN45 cells. Up-regulation of WNT10A reached maximum at 6 h after TNFalpha treatment. H. pylori also induced up-regulation of WNT10A in MKN45 cells. These results strongly suggest that up-regulation of WNT10A induced by TNFalpha and H. pylori might play key roles in human gastric cancer through activation of WNT--beta-catenin--TCF signaling pathway.     

Proto-oncogene WNT10B is up-regulated by tumor necrosis factor alpha in human gastric cancer cell line MKN45.

WNT10A and WNT10B genes are human orthologues of mouse proto-oncogene Wnt-10b. We have previously cloned and characterized WNT10A, and demonstrated up-regulation of WNT10A by tumor necrosis factor alpha (TNFalpha) in gastric cancer. Here, we cloned and characterized human WNT10B, which showed Gly60Asp amino-acid substitution compared with human WNT10B previously reported by another group. Gly60 WNT10B allele was identified in 2 human genome draft sequences and 7 human ESTs, while Asp60 WNT10B allele was not identified in any human genome draft sequences or ESTs. The Gly60-type WNT10B cDNA isolated in this study might be derived from more common WNT10B allele. WNT10B was most homologous to WNT10A (64.5% total amino-acid identity) among human WNTs. Variable region in the WNT core domain of WNT10B and WNT10A were longer than that of other WNTs, such as WNT2B1, WNT2B2, WNT3, WNT3A, WNT5B, WNT7B, WNT8A, WNT11, WNT14, and WNT14B/WNT15. We next investigated expression of WNT10B in human gastric cancer. WNT10B was moderately expressed in MKN45 and MKN74 cells, and weakly expressed in Okajima, TMK1, MKN7, MKN28, and KATO-III cells. Because interferon gamma (IFNgamma) and TNFalpha were frequently elevated in gastric mucosa with Helicobacter pylori infection, effects of IFNgamma and TNFalpha on WNT10B expression in MKN45 cells were investigated. TNFalpha induced transient up-regulation of WNT10B mRNA in MKN45 cells. Up-regulation of WNT10B in human gastric mucosa might lead to gastric carcinogenesis through activation of the beta-catenin - TCF signaling pathway, just like up-regulation of Wnt-10b in mouse mammary gland leads to mammary carcinogenesis.     

Expression and regulation of WNT8A and WNT8B mRNAs in human tumor cell lines: up-regulation of WNT8B mRNA by beta-estradiol in MCF-7 cells, and down-regulation of WNT8A and WNT8B mRNAs by retinoic acid in NT2 cells

Xenopus wnt-8 (Xwnt-8) is one of the most potent Wnts to activate the WNT - beta-catenin - TCF signaling pathway. We have previously cloned and characterized WNT8A and WNT8B, two human homologues of Xwnt-8. Here, we investigated expression and regulation of WNT8A and WNT8B mRNAs in human tumor cell lines by using cDNA-PCR. WNT8A mRNA was undetectable in 7 pancreatic cancer cell lines, but WNT8B mRNA was detected in pancreatic cancer cell lines PSN-1, BxPC-3, MIA PaCa-2. Both WNT8A and WNT8B mRNAs were undetectable in 7 brain tumor cell lines. Although WNT8A mRNA was undetectable in 3 breast cancer cell lines, WNT8B mRNA was detected in the breast cancer cell line MCF-7. WNT8B mRNA, but not WNT8A mRNA, was significantly up-regulated by beta-estradiol in MCF-7 cells. WNT8A mRNA was detected in embryonal tumor cell lines NEC-14, NCC-IT, and NT2, while WNT8B mRNA was detected in embryonal tumor cell lines NEC-8, NEC-14, and NT2. Because NT2 cells differentiate into neuronal cells after all-trans retinoic-acid treatment, effects of all-trans retinoic acid on mRNA expression of WNT8A and WNT8B were next investigated. WNT8A and WNT8B mRNAs were down-regulated together in NT2 cells after all-trans retinoic-acid treatment. WNT8A and WNT8B might play key roles in embryonal tumors and embryonic stem cells through synergistic activation of the beta-catenin - TCF signaling pathway.     

Expression of WNT7A in human normal tissues and cancer,and regulation of WNT7A and WNT7B in human cancer

WNT signals are transduced through seven-transmembrane-type WNT receptors encoded by Frizzled (FZD) genes to the beta-catenin - TCF pathway, the JNK pathway or the Ca2+-releasing pathway. WNT signaling molecules are potent targets for diagnosis of cancer (susceptibility, metastasis, and prognosis), for prevention and treatment of cancer, and for regenerative medicine or tissue engineering. We have so far cloned and characterized human WNT signaling molecules WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, WNT14B/WNT15, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, FRAT1, FRAT2, NKD1, NKD2, VANGL1/STB2, ARHU/WRCH1, ARHV/WRCH2, GIPC2, GIPC3, betaTRCP2/FBXW1B, SOX17, and TCF-3 using bioinformatics, cDNA-library screening, and cDNA-PCR. Here, expression of WNT7A in human normal tissues and cancer, and regulation of WNT7A and WNT7B in human cancer were investigated. WNT7A was highly expressed in fetal lung, adult testis, lymph node, and peripheral blood leukocytes. WNT7A was relatively highly expressed in temporal lobe, occipital lobe, parietal lobe, paracentral gyrus of cerebral cortex, caudate nucleus, hippocampus, medulla oblongata and putamen within adult brain. WNT7A was highly expressed in SW480 (colorectal cancer), BxPC-3 and Hs766T (pancreatic cancer), and was also expressed in MKN7 and MKN45 (gastric cancer). WNT7B rather than WNT7A was expressed in MCF-7 (breast cancer) and NT2 (embryonal tumor). beta-estradiol did not affect expression levels of WNT7A and WNT7B in MCF-7 cells. WNT7B, but not WNT7A, was slightly up-regulated by all-trans retinoic acid in NT2 cells.     

Expression of WNT10A in human cancer

WNT signaling pathway is implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized WNT10A, and demonstrated up-regulation of WNT10A in gastric cancer. Here, we investigated expression of WNT10A mRNA in various types of human cancer. WNT10A mRNA was detected in 10 out of 12 esophageal cancer cell lines by cDNA-PCR, and was significantly up-regulated in esophageal cancer cell lines TE2, TE3, TE4, and a brain tumor cell line A-172. WNT10A mRNA was not up-regulated by retinoic acid in a teratocarcinoma cell line NT2. TFF1/pS2 mRNA, but not WNT10A mRNA, was up-regulated by beta-estradiol in a breast cancer cell line MCF-7. Expression of WNT10A mRNA in various types of primary cancers was next investigated by using Matched tumor/normal expression array filter. WNT10A mRNA was significantly up-regulated in 2 out of 8 cases of primary gastric cancer, and in 1 out of 7 cases of primary rectal cancer. Expression of WNT10A mRNA in esophageal cancer was not investigated, because such samples were not blotted on the expression array filter. Up-regulation of WNT10A mRNA might play key roles in some cases of esophageal, gastric, and colorectal cancer.     

Expression of WRCH1 in human cancer and down-regulation of WRCH1 by beta-estradiol in MCF-7 cells

Secreted-type glycoprotein WNTs bind to seven-transmembrane-type WNT receptors encoded by Frizzled genes (FZD1-FZD10) to transduce signals to the beta-catenin--TCF pathway, the JNK pathway, or the Ca(2+)-releasing pathway. Wrch1 gene is a down-stream target gene of Wnt1 in C57MG cells, and encodes a Cdc42-related GTPase with the potential to activate the JNK pathway. Here, we isolated human WRCH1 cDNAs (accession no. AB074878) from gastric cancer cell lines OKAJIMA, MKN7, MKN28, MKN45, MKN74, and KATO-III, all of which showed a nucleotide substitution (343 C-->T) without amino-acid substitution compared with WRCH1 cDNA isolated by another group. WRCH1 gene, consisting of at least 3 exons, was mapped to human chromosome 1q42.11-q42.3 by using bioinformatics. WRCH1 mRNA was more highly expressed in corpus callosum, hippocampus, cerebral cortex, and also in several parts within adult brain than in other normal tissues including stomach, pancreas, and placenta. Amounts of WRCH1 mRNA in 40 human cancer cell lines were lower than that in normal stomach, pancreas, or placenta. WRCH1 mRNA was significantly up-regulated in 4 cases of primary kidney tumors, 1 case each of primary colon, gastric, breast, ovarian, and uterus cancer. On the other hand, WRCH1 mRNA was significantly down-regulated in 6 cases of colon tumors, 2 cases of primary kidney cancer and breast cancer. Expression of WRCH1 mRNA was down-regulated by beta-estradiol in MCF-7 cells. This is the first report on comprehensive expression analyses on WRCH1 mRNA.     

Molecular cloning and characterization of WNT14B, a novel member of the WNT gene family

WNT14B was cloned and characterized in this study. WNT14B encoded 357-amino acid WNT family protein with the signal peptide and an N-linked glycosylation site. WNT14B was most homologous to WNT14 (61.4% total amino acid identity). WNT15 cDNA fragment previously isolated by another group corresponds to a part of ORF of the WNT14B cDNA (codon 216-335). Exon-intron boundaries were conserved between WNT14B and WNT14 genes. WNT14B and WNT3 genes were clustered in the human chromosome 17q21 region in head to head manner. Intergenic region between WNT14B and WNT3 genes was about 33 kb in size. The 6.6-kb WNT14B mRNA was moderately expressed in fetal kidney and adult kidney. Although WNT14B mRNA was not detected in fetal brain and adult brain by northern blot analyses, WNT14B mRNA was detected in brain, especially in occipital lobe, by RNA dot blot analysis. Among 48 human cancer cell lines derived from various tissues, WNT14B was expressed in a teratocarcinoma cell line NT2 with the potential to differentiate into neuronal cells. WNT14B mRNA was significantly up-regulated by all-trans retinoic acid in NT2 cells. These results strongly suggest that WNT14B might be implicated in the early process of neuronal differentiation of NT2 cells induced by retinoic acid.     

Molecular cloning and characterization of human WNT7B

WNT signaling molecules are implicated in carcinogenesis and embryogenesis. Only partial coding sequence of human WNT7B is reported so far, and human genome draft sequence corresponding to the WNT7B gene in human chromosome 22q13 region is not available at present. Here, we have cloned human WNT7B cDNAs, spanning the complete coding sequence, by using rapid amplification of cDNA ends (RACE) and cDNA-PCR. WNT7B encoded a 349-amino-acid polypeptide with three N-linked glycosylation sites and consensus amino-acid residues conserved among members of the WNT family. WNT7B showed 77.1% total-amino-acid identity with WNT7A. The 4.0-kb WNT7B was moderately expressed in fetal brain, weakly expressed in fetal lung and kidney, and faintly expressed in adult brain, lung and prostate. Expression levels of WNT7B mRNA in a lung cancer cell line A549, esophageal cancer cell lines TE2, TE3, TE4, TE5, TE6, TE7, TE10, TE12, a gastric cancer cell line TMK1, and pancreatic cancer cell lines BxPC-3, AsPC-1 and Hs766T were significantly higher than that in fetal kidney. In addition, WNT7B was up-regulated in 5 out of 10 cases of primary gastric cancer. These results strongly suggest that WNT7B might play important roles in various types of human cancer.     

Molecular cloning and characterization of human WNT3

Mouse Wnt-3 is a proto-oncogene, which is activated by mouse mammary tumor virus (MMTV). Human WNT3 cDNA fragment, previously isolated by another group, corresponds to a partial coding sequence. WNT3 cDNA, spanning the complete coding sequence, was isolated in this study. WNT3 encoded 355-amino-acid polypeptide with the N-terminal signal peptide and two N-linked glycosylation sites. WNT3 was most homologous to WNT3A (84.2% total amino-acid identity) among human WNTs. The WNT3 gene on the human chromosome 17q21 region consisted of five exons. WNT3 mRNAs were detected in fetal brain, adult brain, and testis by Northern blot analyses. WNT3 mRNA was relatively highly expressed in A549 cells (lung cancer) and MKN45 cells (gastric cancer) among 37 human cancer cell lines. WNT3 was significantly up-regulated in a case of primary breast cancer and in a case of primary rectal cancer among various types of human primary cancers. These results strongly suggest that WNT3 might play a key role in some cases of human breast, rectal, lung, and gastric cancer through activation of the WNT - beta-catenin - TCF signaling pathway, similar to mouse Wnt-3.     

Molecular cloning and characterization of human WNT5B on chromosome 12p13.3 region.

WNT signaling pathway plays a key role in carcinogenesis and embryogenesis. We have cloned and characterized the human WNT5B. Overlapping WNT5B cDNAs, containing 1080-bp ORF, were isolated. WNT5B encoded a 359-amino-acid polypeptide with the N-terminal signal peptide, four N-linked glycosylation sites, and consensus amino-acid residues conserved among the WNT family. WNT5B showed 80.5% total-amino-acid identity with WNT5A. Comparison between nucleotide sequence of WNT5B cDNA and human genome draft sequences revealed that the WNT5B gene, consisting of 4 exons, was located on human chromosome 12p13.3 region. Northern blot analyses with W5B2 probe detected the 2.8- and 2.4-kb WNT5B mRNAs. WNT5B was moderately expressed in adult prostate and fetal brain, and weakly expressed in fetal lung, kidney, adult liver, ovary, and small intestine. Among human cancer cell lines, WNT5B was expressed in gastric cancer cell lines MKN7, MKN45, KATO-III, and a teratocarcinoma cell line NT2. WNT5B might be implicated in human carcinogenesis through activation of the WNT-beta-catenin-TCF signaling pathway, just like Wnt5a.     

Molecular cloning and expression of proto-oncogene FRAT1 in human cancer

FRAT1 and FRAT2 genes, clustered in human chromosome 10q24, are human homologues to mouse proto-oncogene Frat1, which promotes carcinogenesis through activation of the WNT - beta-catenin - TCF signaling pathway. FRAT1 and FRAT2 mRNAs are up-regulated together in a gastric cancer cell line TMK1, and also in 2 out of 10 cases of primary gastric cancer. Here, we isolated FRAT1 cDNA (AB074890), which showed two amino-acid substitutions (Gln57X and His58Asp) compared with human FRAT1 cDNA previously reported by another group (U58975). The Gln57-His58 FRAT1 allele isolated in this study was also identified in human genome draft sequences. FRAT1 mRNA was almost ubiquitously expressed in human pancreatic cancer cell lines. Expression level of FRAT1 mRNA was relatively higher in esophageal cancer cell lines TE2, TE3, TE4, a cervical cancer cell line SKG-IIIa, and breast cancer cell lines MCF-7 and T-47D. Expression level of FRAT1 mRNA was not significantly changed after all-trans retinoic-acid treatment in NT2 cells with the potential of neuronal differentiation. Expression of FRAT1 mRNA in MCF-7 cells derived from breast cancer was down-regulated by beta-estradiol. This is the first report on isolation of FRAT1 cDNA derived from the more common FRAT1 allele, and also on regulation of FRAT1 mRNA in human cancer cells.     

Up-regulation of GIPC2 in human gastric cancer

GIPC1/GIPC, GIPC2, and GIPC3 are a family of central PDZ-domain proteins. GIPC1/GIPC interacts with TGFbeta type III receptor, receptor tyrosine kinase TrkA, integrin alpha6A subunit, and GTPase-activating protein RGS-GAIP, while Xenopus homologue of human GIPCs interacts with Frizzled-3 (FZD3) class of WNT receptor. Here, we investigated expression of GIPC2 mRNA in human gastric, pancreatic, and breast cancer cell lines. GIPC2 mRNA was relatively highly expressed in OKAJIMA, TMK1, MKN45, and KATO-III cells derived from diffuse type of gastric cancer, but was almost undetectable in MKN7, MKN28, and MKN74 cells derived from intestinal type of gastric cancer as well as in other cell lines derived from pancreatic and breast cancer. Tumor necrosis factor alpha and interferon gamma, which are elevated in gastric mucosa with Helicobacter pylori infection, did not affect the expression level of GIPC2 mRNA in MKN45 cells. Up-regulation of GIPC2 mRNA was detected in 7 out of 10 cases of primary gastric cancer by using cDNA-PCR, and in 4 out of another 8 cases of primary gastric cancer by using expression array filter hybridization. GIPC2 might play important roles in human gastric cancer through modulation of growth factor signaling or cell adhesion.     

Up-regulation of WNT8B mRNA in human gastric cancer.

WNT - beta-catenin - TCF signaling pathway is activated by Xenopus wnt-8 (Xwnt-8) during Xenopus early development, and dysregulated activation of beta-catenin - TCF signaling pathway in mammalian cells leads to carcinogenesis. We have previously cloned and characterized human WNT8A, one of human orthologues of Xwnt-8. Here, we cloned and characterized human WNT8B by using bioinformatics, cDNA-PCR, and RACE. WNT8B gene of about 23-kb in size consisted of six exons, and encoded a 351-amino-acid polypeptide with the N-terminal signal peptide and two N-linked glycosylation sites. C-terminal region of WNT8B, WNT8A, WNT2, and WNT2B were longer than that of other human WNTs. Thirty-five nucleotide changes between WNT8B isolated by us and WNT8B isolated by another group resulted in Gly230Ala and Arg284Leu amino-acid substitutions. Gly230 and Arg284 of WNT8B were conserved in WNT8A. Gly230-Arg284 WNT8B allele was also identified in human genome draft sequences AL133352.10, AL359759.18, and human EST BF732616. These results indicate that the Gly230-Arg284 WNT8B cDNA isolated in this study is derived from the more common WNT8B allele. WNT8B mRNAs of 4.4- and 3.5-kb in size were weakly detected in a colorectal cancer cell line SW480, but were undetectable in any normal human tissues by using Northern blot analyses. WNT8B was significantly up-regulated in gastric cancer cell lines KATO-III (signet-ring cell carcinoma) and MKN45 (poorly differentiated adenocarcinoma), and also in 5 out of 10 cases of primary gastric cancer. WNT8B might play key roles in gastric cancer through activation of the beta-catenin - TCF signaling pathway.     

Expression of TFF1, TFF2 and TFF3 in gastric cancer

Expression of some members of the trefoil factor (TFF) and the WNT gene families is regulated together by estrogen. We have cloned and characterized human WNT signaling molecules using bioinformatics, cDNA-library screening and cDNA-PCR to investigate expression profile of WNT signaling molecules in human gastric cancer. Here, we investigated expression profile of TFF1/pS2, TFF2/SP and TFF3/ITF in human gastric cancer. Among 7 gastric cancer cell lines, TFF1 was expressed in OKAJIMA, TMK1, MKN45, and KATO-III, TFF2 in KATO-III, and TFF3 in MKN45 and KATO-III. TFFs were preferentially expressed in diffuse-type gastric cancer cell lines. Expression of TFFs in primary gastric cancer was next investigated. TFF1 was down-regulated in 7 cases out of 12 cases (58.3%) of primary gastric cancer. TFF2 was down-regulated in 10 out of 12 cases (83.3%) of primary gastric cancer. TFF3 was down-regulated in 2 out of 12 cases (16.7%) of primary gastric cancer, and was up-regulated in 5 out of 12 cases (41.7%). TFF1 and TFF2 were frequently down-regulated in primary gastric cancer, while TFF3 was up-regulated in some cases of primary gastric cancer. This is the first report on comprehensive expression analyses on TFFs in gastric cancer.     

Expression of ST7R (ST7-like,ST7L) in normal tissues and cancer

We have recently cloned and characterized ST7R (ST7-like, ST7L), WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, WNT14B/WNT15, NKD1, NKD2, ARHU/WRCH1, ARHV/WRCH2, and VANGL1/STB2 using bioinformatics, cDNA-PCR and RACE. ST7R is a paralog of tumor suppressor gene ST7 in the human genome. ST7R gene is clustered with WNT2B gene in human chromo-some 1p13 region, while ST7 gene is clustered with WNT2 gene in human chromosome 7q31 region. Multiple ST7R mRNAs (ST7R1-ST7R4) are transcribed due to alternative splicing. ST7R4 is divergent from ST7R1-ST7R3 in the C-terminal region. Here, we investigated expression of ST7R mRNAs in normal human tissues and human cancer. Northern blot analysis with S7S1 probe corresponding to ST7R1, ST7R2 and ST7R3 isoforms detected 4.2 kb ST7R mRNA in various normal tissues, and also large amounts of 2.2-2.4 kb ST7R mRNAs in testis. Northern blot analysis with S7S4 probe corresponding to ST7R4 isoform detected 2.0 kb ST7R mRNA in testis. Expression of ST7R mRNAs in human cancer was next investigated using cDNA-PCR. Although ST7R mRNAs were almost ubiquitously expressed in 7 gastric cancer cell lines, expression levels of ST7R mRNAs were relatively lower in TMK1 cells. ST7R mRNAs were expressed in most cases of primary gastric cancer, and were up-regulated in 2 out of 10 cases of primary gastric cancer. This is the first report on expression analyses on ST7R.     

Expression profiles of 10 members of Frizzled gene family in human gastric cancer

Frizzled (FZD) genes encode seven-transmembrane type WNT receptors, which are implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, and FZD10. Here, we investigated expression profiles of all members of the FZD gene family in human gastric cancer. FZD mRNAs were detected by one-step cDNA-PCR. Specificity of cDNA-PCR was confirmed by nucleotide sequence analyses of cDNA-PCR products. Among seven gastric cancer cell lines, FZD7 was up-regulated in MKN7, which was consistent with a previous report. FZD5 was up-regulated in MKN45. FZD9 and FZD10 were up-regulated together in TMK1 and MKN74. FZD2 was up-regulated in TMK1, MKN7, MKN28, MKN45, MKN74 and KATO-III. Among 10 cases of primary gastric cancer, FZD9 was up-regulated in 2 cases, FZD2 and FZD8 were up-regulated in 4 cases. Effects of Helicobacter pylori (H. pylori) on expression of FZDs were further investigated, and it was revealed that FZDs were not up-regulated by H. pylori in MKN45 cells. These results indicate that FZD2, FZD8, and FZD9 might play key roles in human gastric cancer.     

The anti-progestin RU-486 inhibits viability of MCF-7 breast cancer cells by suppressing WNT1

The Wnt signaling pathway is activated in over 50% of women with breast cancer and contributes to tumor progression. Here, we investigated the effects of RU-486 on Wnt signaling in breast cancer cell lines. RU-486 reduced viability of the progesterone receptor-positive MCF-7 and T-47D cells, but had no effect on the triple-negative MDA-MB-231 cells. Furthermore, RU-486 suppressed WNT1 expression of MCF-7 cells by 99%. The addition of recombinant WNT1 partially reversed the RU-486-dependent inhibition of viability in MCF-7, but not in T-47D cells. In conclusion, we identified WNT1 as a novel mediator of the anti-tumor effects of RU-486 in MCF-7 cells.