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 functional analysis of a novel cadmium-responsive proto-oncogene

The molecular mechanisms potentially responsible for cell transformation and tumorigenesis induced by cadmium, a human carcinogen, were investigated by differential gene expression analysis of BALB/c-3T3 cells transformed with cadmium chloride (CdCl(2)). Differential display analysis of gene expression revealed consistent overexpression of mouse translation initiation factor 3 (TIF3; GenBank accession number AF271072) in the cells transformed with CdCl(2) when compared with nontransformed cells. The predicted protein encoded by TIF3 cDNA exhibited 99% similarity to human eukaryotic initiation factor 3 p36 protein. A M(r) 36,000 protein was detected in cells transfected with an expression vector containing TIF3 cDNA. Transfection of NIH3T3 cells with an expression vector containing TIF3 cDNA resulted in overexpression of the encoded protein, and this was associated with cell transformation, as evidenced by the appearance of transformed foci exhibiting anchorage-independent growth on soft agar and tumorigenic potential in nude mice. Expression of the antisense RNA against TIF3 mRNA resulted in significant reversal of oncogenic potential of the CdCl(2)-transformed BALB/c-3T3 cells. Taken together, these findings demonstrate for the first time that the cell transformation and tumorigenesis induced by CdCl(2) are due, at least in part, to the overexpression of TIF3, a novel cadmium-responsive proto-oncogene.     

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.     

Molecular cloning and characterization of human WNT8A.

WNT - beta-catenin - TCF pathway is involved in carcinogenesis and fetal development. Xenopus wnt-8 is one of the most potent Wnts with the capacity to activate beta-catenin - TCF pathway in the Xenopus axis duplication assay. Here, we have cloned and characterized WNT8A, a novel human homologue of Xenopus wnt-8. The WNT8A gene, consisting of at least 6 exons, was found to encode a 351-amino-acid polypeptide with the N-terminal signal peptide, three N-linked glycosylation sites, and conserved amino-acid residues of the WNT family. WNT8A showed 63.2% total-amino-acid identity to WNT8B. C-terminal region of WNT8A, WNT8B, WNT2, WNT2B1 and WNT2B2 were longer than that of other WNTs. Among various normal human tissues and 34 human cancer cells lines, the 3.5-kb WNT8A mRNA was detected only in a human teratocarcinoma cell line NT2. These results strongly suggest that WNT8A might be implicated in development of early embryos as well as germ cell tumors through activation of the WNT - beta-catenin - TCF pathway.     

Molecular characterization of human trk proto-oncogene product monoclonal antibodies

Monoclonal antibodies specific for the human trk protooncogene product, a tyrosine kinase receptor, have been produced from mice in which tumors were generated by injection of stably transfected NIH3T3 cells expressing the human trk proto-oncogene product. The panel of eleven antibodies are reactive in ELISA, immunostaining and immunoprecipitation. These antibodies bind to the extracellular domain of the human trk proto-oncogene product and demonstrate no cross-reactivity to the trk oncogene or murine trkB gene products. The antibodies are equally effective in recognizing human proto-trk when expressed by transfected human or mouse cell lines. These antibodies either bind to the carbohydrate moieties or are dependent upon the conformational structure created by the extensive glycosylations. The localization and nature of the epitopes recognized by these monoclonal antibodies were defined by immunoprecipitation analyses using several different trk oncoproteins. Potential applications of these antibodies are discussed.     

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 of a 5' segment of the genomic phl gene defines a new breakpoint cluster region (bcr2) in Philadelphia-positive acute leukemias

Molecular rearrangements of Ph1 chromosome, the hallmark of CML, are clustered in a 5.8-kb DNA segment, the so-called breakpoint cluster region (bcr) of the phl gene that is localized to chromosome 22q11. In Ph1-positive (Ph1+) ALLs, the rearrangements have been shown to involve either the 5.8-kb bcr (called bcr+) or a region upstream of bcr in the 5' end of the phl gene (bcr-). To gain insight into the rearrangements occurring in Ph1+ acute leukemias, a 64-kb DNA fragment from the 5' end of phl was analyzed in order to generate molecular probes covering 40 kb of the phl gene first intron. A panel of seven cases of bcr-Ph1+ acute leukemia (three nonlymphocytic and four lymphocytic) was investigated with these intron 1-derived probes. Strikingly, in six of the seven leukemias, the breakpoints were located in a 10.8-kb DNA segment, defining a new bcr which appears to be specific for Ph1+ acute leukemias. By analogy with the CML bcr region located in the 3' part of the phl gene, we propose to designate this 10.8-kb fragment bcr2.     

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 analysis of 5' J region of immunoglobulin heavy chain gene in human acute leukemias

Ig heavy chain (IgH) J (JH) region organization in 83 human acute leukemias was studied. In 31 of the 35 precursor B acute lymphocytic leukemia (ALL) the rearrangements of the IgH gene involved sites between D regions 5' to DQ52 and JH. The study of the IgH gene organization in 43 T lineage ALL showed nine cases with the rearrangement at the IgJH region, one of which involved the D region 5' to DQ52. When analyzed with another restriction enzyme (BglII), three of the remaining eight showed rearrangement between DQ52 and JH. In the remaining five samples rearrangement of the IgJH region was not shown, but the restriction fragment length polymorphism (RFLP) between MspI sites in 5'DQ52 loci was observed. Thus the RFLP in 5'DQ52 locus was identified and was distinguished from the recombination between DQ52 and JH in non-B lineage leukemias.     

Molecular heterogeneity of splenic marginal zone lymphomas: analysis of mutations in the 5' non-coding region of the bcl-6 gene.

Splenic marginal zone lymphoma (SMZL) has been recognized as a distinctive type of small B cell lymphoma, and defined on the basis of its morphological, phenotypic, clinical and molecular characteristics. In spite of this, the borders of the entity, the homogeneity of the cases and the presumably cell origin of SMZL remain controversial issues. The frequency of mutation in the 5' non-coding region of the bcl-6 gene has been used as a marker of germinal center derivation, which may be used to establish the molecular heterogeneity of different non-Hodgkin lymphoma (NHL) types. This roughly parallels the characteristics and frequency of the somatic hypermutations found in the immunoglobulin heavy chain variable region (IgVH) genes. This study analyzed mutations of bcl-6 in the 5' non-coding region in 22 SMZL cases and, for the purpose of comparison with different B cell subsets, in microdissected germinal centers, mantle zones and marginal zone subpopulations from reactive splenic lymphoid follicles. A majority of the SMZL cases studied, 19/22 (87%), bear unmutated bcl-6 gene, while mutation was only observed in 3/22 (13%) cases. Analysis of normal B cell subpopulations showed bcl-6 hypermutation in 3/10 (30%) germinal center clones, 5/14 (35%) marginal zone clones; and unmutated sequences in all clones derived from mantle cells. The frequency of these mutations in normal spleen confirms previous findings on the hypermutation IgVH process in normal B cell populations. The data presented here support the existence of molecular heterogeneity in this entity, and give additional results in favor of the hypothesis that, in spite of initial morphological observations, a significant proportion of SMZL cases could derive from an unmutated naive precursor, different from the marginal zone, and possibly located in the mantle zone of splenic lymphoid follicles. Thus the marginal zone differentiation of these tumors could be related more with the splenic microenvironment than it is to the histogenetic characteristics of the tumor.     

Molecular cloning and characterization of Strabismus 2 (STB2)

WNT signaling pathway is implicated in carcinogenesis and embryogenesis. WNT signal is transduced to the beta-catenin - TCF pathway, the JNK pathway, or the Ca2+-releasing pathway through seven-transmembrane-type WNT receptors encoded by Frizzled (FZD) genes. Xenopus Strabismus (Stbm) is a tetra-spanning transmembrane protein interacting with Dishevelled, and is a negative regulator of the WNT - beta-catenin - TCF signaling pathway. STB1/KIAA1215/VANGL2 is a human orthologue of Xenopus Stbm (90.6% total-amino-acid identity). Here, STB2/VANGL1 gene fragments were identified in human genome draft sequences by using bioinformatics, and STB2 cDNAs were isolated by using cDNA-PCR. STB2 gene consisted of at lest 7 exons, and encoded a 524-amino-acid protein with 4 transmembrane domains and the C-terminal Ser/Thr-X-Val motif. Human STB2 was homologous to human STB1 (73.1% total-amino-acid identity) and Xenopus Stbm (72.7% total-amino-acid identity). STB2 gene was clustered with Calsequestrin 2 (CASQ2) gene in tail-to-tail manner (interval less than 5.0 kb), and CASQ2 gene is mapped to human chromosome 1p11-p13.3 or linked to human chromosome 1p13-p21. STB2 mRNAs of 4.8- and 6.8-kb in size were expressed almost ubiquitously in various normal tissues. STB2 mRNA was significantly up-regulated in gastric cancer cell lines MKN28, MKN74, pancreatic cancer cell lines BxPC-3, PSN-1 and Hs766T. On the other hand, STB2 mRNA was significantly down-regulated in a pancreatic cancer cell line AsPC-1. This is the first report on molecular cloning and characterization of STB2.     

Molecular cloning and characterization of a novel esophageal cancer related gene

We previously identified four novel cDNA fragments related to human esophageal cancer. One of the fragments was named esophageal cancer related gene 2 (ECRG2). We report here the molecular cloning, sequencing, and expression of the ECRG2 gene. The ECRG2 cDNA comprises a 258 bp nucleotide sequence which encodes for 85 amino acids with a predicted molecular weight of 9.2 kDa. Analysis of the protein sequence reveals the presence at the N terminus of a signal peptide followed by 56 amino acids with a significant degree of sequence similarity with the conserved Kazal domain which characterizes the serine protease inhibitor family. Pulse-chase experiments showed that ECRG2 protein was detected in both cell lysates and culture medium, indicating that the ECRG2 protein was extracellularly secreted after the post-translational cleavage. In vitro uPA/plasmin activity analysis showed the secreted ECRG2 protein inhibited the uPA/plasmin activity, indicating that ECRG2 may be a novel serine protease inhibitor. Northern blot analysis revealed the presence of the major band corresponding to a size of 569 kb throughout the fetal skin, thymus, esophagus, brain, lung, heart, stomach, liver, spleen, colon, kidney, testis, muscle, cholecyst tissues and adult esophageal mucosa, brain, thyroid tissue and mouth epithelia. However, ECRG2 gene was significantly down-regulated in primary esophageal cancer tissues. Taken together, these results indicate that ECRG2 is a novel member of the Kazal-type serine protease inhibitor family and may function as a tumor suppressor gene regulating the protease cascades during carcinogenesis and migration/invasion of esophageal cancer.     

Molecular cloning and functional analysis of the mouse homologue of the KILLER/DR5 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors are members of the tumor necrosis factor superfamily. TRAIL selectively kills cancer cells but not normal cells. We report here the cloning of the mouse homologue of the TRAIL receptor KILLER/DR5 (MK). The cDNA of MK is 1146 bp in length and encodes a protein of 381 amino acids. MK contains an extracellular cysteine-rich domain, a transmembrane domain, and a cytoplasmic death-domain characteristic of Fas, tumor necrosis factor, and human TRAIL receptors. MK is highly homologous and binds TRAIL with similar affinity as human DR4 and KILLER/DR5. MK induces apoptosis in mouse and human cells and inhibits colony growth of NIH3T3 cells. Expression of MK is p53-dependent and up-regulated by tumor suppressor p53 and by DNA damaging agents in mouse cells undergoing apoptosis. This is the first report describing a mouse TRAIL receptor gene and also demonstrating that the p53-dependent regulation of KILLER/DR5-mediated apoptosis is conserved between human and mouse.