Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin.

Several lines of evidence suggest that aberrant Notch signaling contributes to the development of several types of cancer. Activation of Notch receptor is executed through intramembrane proteolysis by γ-secretase, which is a multimeric membrane-embedded protease comprised of presenilin, nicastrin (NCT), anterior pharynx defective 1 and PEN-2. In this study, we report the neutralization of the γ-secretase activity by a novel monoclonal antibody A5226A against the extracellular domain of NCT, generated by using a recombinant budded baculovirus as an immunogen. This antibody recognized fully glycosylated mature NCT in the active γ-secretase complex on the cell surface, and inhibited the γ-secretase activity by competing with the substrate binding in vitro. Moreover, A5226A abolished the γ-secretase activity-dependent growth of cancer cells in a xenograft model. Our data provide compelling evidence that NCT is a molecular target for the mechanism-based inhibition of γ-secretase, and that targeting NCT might be a novel therapeutic strategy against cancer caused by aberrant γ-secretase activity and Notch signaling.

Expression of pentraxin 3 (PTX3) in human atherosclerotic lesions.

Pentraxin 3 (PTX3) and C-reactive protein (CRP) are members of the pentraxin superfamily. PTX3 expression is induced in response to inflammatory signals, and is produced at sites of inflammation by several types of cell, primarily monocytes/macrophages, dendritic cells (DCs), endothelial cells, smooth muscle cells (SMCs), and fibroblasts, but is not produced by hepatocytes, which are a major source of CRP. The aim of our study was to investigate the expression pattern of PTX3 in human atherosclerotic lesions using a novel monoclonal antibody against PTX3. We examined coronary arterial thrombi containing an atherosclerotic plaque component removed from patients with acute myocardial infarction and human aortic tissues with various degrees of atherosclerosis sampled from autopsy cases. Immunohistochemical study of paraffin and frozen sections indicated that macrophages, mainly foam cells, expressed PTX3 in advanced atherosclerotic lesions. Interestingly, we also clearly observed PTX3-positive neutrophils infiltrating into atherosclerotic plaques, suggesting that PTX3 derived from neutrophils as well as macrophages plays an important role in atherogenesis.

Aldo-keto reductase family 1, member B10 in uterine carcinomas: a potential risk factor of recurrence after surgical therapy in cervical cancer.

Aldo-keto reductase family 1, member B10 (AKR1B10), an enzyme that converts retinals into retinols is known to detect in non-small cell lung carcinoma (squamous cell- and adeno-carcinomas), but is barely expressed in normal tissues. Since these types of carcinoma occur frequently in the uterus (like in the lung), AKR1B10 may also be overexpressed in two major types of uterine cancer, cervical cancer (CC), and endometrial cancer (EMC). The objective of this study is to investigate AKR1B10 expression in uterine cancer and to analyze its clinical significance. In samples from uterine cancer patients, AKR1B10 was detected in 6 out of 30 (20.0%) CC cases and 6 out of 38 (15.8%) EMC cases. Statistical analysis indicated that AKR1B10 expression was associated with tumor recurrence after surgery and keratinization of squamous cell carcinoma only in CC. Although retinol (a metabolic product by AKR1B10) was observed in the normal epithelium, the molecule was not observed in cancer cells of AKR1B10-positive CC samples suggesting that the recurrence in CC may not depend on the convert of retinals into retinols via AKR1B10, a potential indicator in the management of patients with CC.

Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.

Hepatocyte nuclear factor-4alpha (HNF4alpha) exists in multiple isoforms that are generated by alternative promoter (P1 and P2) usage and splicing. Here we establish monoclonal antibodies (mAbs) for detecting P1 and P2 promoter-driven HNF4alpha, and evaluate their expression in normal adult human tissues and surgically resected carcinomas of different origins. Using immunohistochemical analysis, we demonstrate that, while P1 promoter-driven HNF4alpha is expressed in hepatocytes, small intestine, colon, kidney and epididymis, P2 promoter-driven HNF4alpha is expressed in bile duct, pancreas, stomach, small intestine, colon and epididymis. Altered expression patterns of P1 and P2 promoter-driven HNF4alpha were observed in gastric, hepatocellular and colorectal carcinomas. HNF4alpha was expressed in lung metastases from renal cell, hepatocellular and colorectal carcinoma but was not observed in lung tumours. The P1 and P2 promoter-driven HNF4alpha expression pattern of tumour metastases correlated with the primary site of origin. P1 promoter-driven HNF4alpha was also found in intestinal metaplasia of the stomach. These data provide evidence for the tissue distribution of P1 and P2 promoter-driven HNF4alpha at the protein level and suggest that HNF4alpha may be a novel diagnostic marker for metastases of unknown primary. We propose that the dysregulation of alternative promoter usage of HNF4alpha is associated with the pathogenesis of certain cancers.

Phosphorylation of serine-rich protein encoded by open reading frame 3 of the TT virus genome.

TT virus (TTV) is a newly discovered human virus with a single-stranded, circular DNA genome. The TTV DNA sequence includes two major open reading frames (ORFs), ORF1 and ORF2. Recently, spliced TTV mRNAs were detected and revealed two additional coding regions, ORF3 and ORF4. We found sequence similarity between the TTV ORF3 protein and hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, which is a phosphoprotein and is thought to associate with various cellular proteins. To test whether the TTV ORF3 protein is phosphorylated, the state of phosphorylation was analyzed with a transient protein production system. The TTV ORF3 protein was phosphorylated at the serine residues in its C-terminal portion. Furthermore, the TTV ORF3 gene generated two forms of proteins with a different phosphorylation state, similar to the HCV NS5A region, suggesting that TTV ORF3 protein has function(s) similar to phosphorylated viral proteins such as the HCV NS5A protein.

Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product.

An ELISA was developed for serological determination of the six genotypes of hepatitis B virus (HBV) designated A, B, C, D, E, and F. Monoclonal antibodies were raised against genotype-specific epitopes in the preS2-region product, and labeled with horseradish peroxidase. Hepatitis B surface antigen (HBsAg) in sera was captured by immobilized antibodies against the common determinant, and evaluated for reactivity with genotype-specific monoclonal antibodies labeled with the enzyme. Serological genotyping was in complete accord with genotypes determined by S-gene sequences in a panel of 68 sera containing HBV/HBsAg of different genotypes. Of 514 sera with HBsAg from Japan, 507 (98.6%) were genotyped serologically: genotype A was identified in 24 (4.7%); B in 196 (38.1%); C in 282 (54.9%); D in 2 (0.4%); and F in 3 (0.6%). There were no sera containing HBV of genotype E. Likewise, 425 of 446 (95.3%) sera with HBsAg from Brazil, China, India, Indonesia, Kenya, Korea, Nepal, Papua New Guinea, the Philippines, and Thailand were classified into A (25.6%), B (24.2%), C (33.9%), and D (11.7%) genotypes; there were no sera with HBsAg of genotype E or F among them. Some sera unclassifiable by ELISA revealed mixed infection with HBV of distinct genotypes, or contained HBsAg deprived of genotype-specific epitopes by point mutations. The ELISA would be useful for large-scale surveys, because it allows serological detection of HBV genotypes without sequencing nucleotides.

Establishment of rat dermal papilla cell lines that sustain the potency to induce hair follicles from afollicular skin.

Dermal papilla cells in culture show a lower proliferative capacity compared with dermal fibroblasts, and lose their in situ potency to induce hair follicles in the epidermis at more than 10 passage numbers. This study overcomes these limitations of cultured papilla cells and for the first time demonstrates that papilla cells can be serially cultured for a long period without losing their hair-inductive potency. Outgrowth and the ensuing proliferation of papilla cells were markedly stimulated when explants of rat vibrissa papillae were cultured with rat sole-derived keratinocytes. Such feeder effects of the keratinocytes could be replaced to some extent with conditioned medium of the cells. Serial cultivation of papilla cells was established by maintaining them in the conditioned medium in which they were subcultured for more than 90 passages with an approximate population doubling time of 30 h, a value similar to that of rat dermal fibroblasts. During the subculture, they showed morphologic characteristics and phenotypic expressions of original papilla cells. Even after at least 70 passages, papilla cells sustained the innate hair follicle inductive ability at a level comparable with that of intact dermal papillae. The established cell lines did not show tumorigenicity when they were subcutaneously implanted into nude mice. The culture method developed in this study should facilitate the search for a biochemical entity of dermal papilla cells.

Significance of circulating hepatocyte growth factor level as a prognostic indicator in primary breast cancer.

The circulating hepatocyte growth factor (HGF)/scatter factor level is frequently increased in advanced cancer patients. In this study, we have assessed the prognostic value of the circulating HGF level determined by enzymatic immunoassay in primary breast cancer patients. Of 200 primary breast cancer patients, 54 (27.0%) showed the increase of serum HGF level according to the age-matched cutoff values. The prognosis of the patients with the increased HGF level was statistically worse than that of the patients with normal HGF level (P = 0.0001, log-rank test). Multivariate analysis confirmed that the increase in HGF level was an independent prognostic indicator in primary breast cancer patients. In the background analysis, the increase in serum HGF level was significantly associated with tumor size, nodal status, and histological evidence of venous invasion. The data indicate that up-regulation of the circulating HGF level may predict systemic tumor spread and early relapse in primary breast cancer patients.

Domain structures and immunogenic regions of the 90-kDa heat-shock protein (HSP90). Probing with a library of anti-HSP90 monoclonal antibodies and limited proteolysis.

Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library of anti-HSP90 monoclonal antibodies (mAbs) and by limited proteolysis with trypsin and chymotrypsin. Thirty-three mAbs were obtained by immunization with bacterially expressed human HSP90alpha and HSP90beta isoforms. Among them, ten and three mAbs reacted specifically with HSP90alpha and HSP90beta, respectively. Immunoblotting and enzyme-linked immunosorbent analyses revealed that major immunogenic domains were located at two restricted regions of HSP90alpha, i.e. amino acids 227-310 (designated Region I) and 702-716 (Region II), corresponding to a highly charged region and a region near the C terminus, respectively. Taken together with the characteristics of the amino acid sequences, these two immunogenic regions appeared to be exposed at the outer surface of HSP90. We further investigated the domain structures of HSP90 by limited proteolysis in combination with N-terminal sequencing and immunoblotting analyses. Tryptic cleavages of HSP90alpha at low concentrations revealed the existence of major susceptible sites at Arg400-Glu401, Lys615-Ala616, and Arg620-Asp621. Proteolysis at higher trypsin concentrations caused successive cleavages only toward the N-terminal direction from these sites, and Region I was included in the region selectively deleted during this process, thereby further suggesting its surface location. From these results, we propose three domain structures of HSP90 consisting of amino acids 1-400, 401-615, and 621-732. Differences in the protease sensitivity and immunogenicity further suggest that every domain is composed of two subdomains. This is the first study describing the domain structures and the immunogenic regions of HSP90.

Isoform-specific monoclonal antibodies against HSP90.

The purpose of this study was to develop monoclonal antibodies (mAbs) that distinguish between the two isoforms of human 90-kDa heat shock protein (HSP90), i.e., HSP90 alpha and beta. Human HSP90 alpha and beta isoforms expressed in Escherichia coli were separately used as antigens for developing the mAbs. Twenty-three and ten mAbs were obtained by immunization of mice with HSP90 alpha and beta, respectively. Among them, ten and three mAbs specifically recognized HSP90 alpha and beta isoforms, respectively, on the criteria of both enzyme-linked immunosorbent and immunoblotting analyses. Immunochemical analysis by use of the mAbs revealed that both of the HSP90 isoforms were present in human cells even under unstressed conditions and that the expression of HSP90 alpha was more strongly induced when the cells were exposed to arsenate. This is the first report of the development of the mAbs discriminating between the two isoforms of HSP90. The mAbs specific for HSP90 isoforms should be useful for the regulational and functional analyses of HSP90 isoforms.

Increased concentration of hepatocyte growth factor in the sera of patients with gastrointestinal cancer.

We measured the concentration of hepatocyte growth factor (HGF) in the sera of 138 patients with gastrointestinal cancer. The mean value and positive rate for this protein (assuming 0.4 ng/ml as the cut-off value) were 0.29 ng/ml (11.4%) in healthy donors (n=44), 0.90 ng/ml(70.8%) in esophageal cancer (n=24), 0.39 ng/ml (33.3%) in gastric cancer (n=39), and 0.40 ng/ml (42.7%) in colorectal cancer (n=75). Thus, the mean values of the esophageal and colorectal cancer group were significantly higher than that of healthy donors. There was a tendency for elevated mean value of serum HGF in the sera of patients with distant metastases, compared to those with no such evidence, i.e., 1.36:0.77 ng/ml in esophageal cancer, 0.69:0.30 ng/ml in gastric cancer, and 0.66:0.37 ng/ml in colorectal cancer. This difference was statistically significant in gastric and colorectal cancer. These findings suggest that serum HGF is useful as a tumor marker with a close correlation to the metastatic state of gastrointestinal cancer.

The interaction between Ku antigen and REF1 protein mediates negative gene regulation by extracellular calcium.

Through the specific binding of a negative calcium-responsive element to its binding protein in response to extracellular Ca (Ca2+e), negative calcium-responsive element-bearing genes, such as the human parathyroid hormone gene, are negatively regulated by Ca2+e. The Ku antigen mediated negative gene regulation by Ca2+e by interacting with a redox factor protein, REF1. Although sequence-nonspecific DNA binding activity of the Ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure. Here, we report that the specific binding of the Ku antigen to another protein, REF1, leads to DNA-protein complex formation with a novel sequence specificity and thereby regulates gene expression.

Loss of immunostaining of the RBP-J kappa transcription factor upon F9 cell differentiation induced by retinoic acid.

RBP-Jkappa is a novel type of transcriptional regulatory protein that does not contain any known DNA-binding motif. We raised anti-RBP-Jkappa monoclonal antibodies (K0043 and T6709) to investigate the roles of RBP-Jkappa in cell differentiation. These antibodies stained nuclei of undifferentiated embryonic stem cells and F9 cells but not those of the other differentiated cell lines tested so far although the RBP-Jkappa protein is expressed at similar levels. Interestingly, differentiated F9 cells lost the immunostaining reaction with the anti-RBP-Jkappa monoclonal antibodies. Biochemical subcellular fractionation study showed that the majority of RBP-Jkappa was localized in nuclei of F9 cells and that there are at least two forms of the RBP-Jkapppa protein in the nuclei of undifferentiated F9 cells, a free form and a chromatin-bound form. Upon induction of F9 cell differentiation, free nuclear RBP-Jkappa disappeared concomitantly with the loss of immunostaining, suggesting that the anti-RBP-Jkapppa antibodies cannot recognize chromatin-bound RBP-Jkappa. Since there is no evidence to indicate covalent modification of RBP-Jkappa, we assume that chromatin-bound RBP-Jkappa interacts with a large number of proteins which block the exposure of RBP-Jkappa epitopes to the monoclonal antibodies.

Biochemical and immunological characterization of the DNA binding protein (RBP-J kappa) to mouse J kappa recombination signal sequence.

We have investigated whether J kappa recombination signal sequence (RS) binding protein (RBP-J kappa) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-J kappa protein purified by J kappa-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-J kappa protein, recombinant RBP-J kappa was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-J kappa produced in silkworm cells could bind J kappa-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-J kappa has the ligase activity, the RBP-J kappa protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-J kappa fusion protein which was synthesized in E. coli and unable to bind J kappa-RS. Using the anti-RBP-J kappa monoclonal antibody we have shown that the RBP-J kappa protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-J kappa protein is consistent with the hypothesis that the RBP-J kappa protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].

Hyoscyamine 6 beta-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root.

Hyoscyamine 6 beta-hydroxylase (H6H; EC 1.14.11.11) catalyzes the first reaction in the biosynthetic pathway from hyoscyamine to scopolamine in several solanaceous plants. Four monoclonal antibodies were raised against H6H purified from cultured roots of Hyoscyamus niger. The IgG1 antibody mAb5 inhibited H6H activities present in cell-free extracts of H. niger roots and specifically recognized 38-40-kDa proteins from six different scopolamine-producing plant species in Western blot analysis after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The other three monoclonal antibodies all recognized SDS-denatured H6H protein from Hyoscyamus species, but did not bind to native H6H. Western blot analysis of protein extracts from various tissues of H. niger using these antibodies showed that H6H is abundant in cultured roots, present in plant roots, but absent in leaf, stem, calyx, cultured cells, and cultured shoots. Immunohistochemical studies using monoclonal antibody and immunogold-silver enhancement detected H6H only in the pericycle cells of the young root in several scopolamine-producing plants. Mature roots that underwent secondary growth and lacked the pericycle did not react with the antibody. This pericycle-specific localization of scopolamine biosynthesis provides an anatomical explanation for the tissue-specific biosynthesis of tropane alkaloids and may be important for translocation of tropane alkaloids from the root to the aerial parts.