Biomedical subjects

Akihiro Murakami

Publications and source records attributed to Akihiro Murakami.

9 recordsLinked to original sources

PubMed2006

Different expression patterns of intact forms of squamous cell carcinoma antigens between normal and malignant cervical squamous epithelial tissues: nondenaturing polyacrylamide gel electrophoretic analysis.

Squamous cell carcinoma antigen (SCCA), a 45-kDa tumor-associated serpin, mainly consists of two highly homologous molecules, SCCA1 and SCCA2, which possess unique proteinase inhibitory properties. Importantly, our previous study demonstrated that an intact structure of SCCAs, and not a cleaved form yielded by interacting with target proteinase, is essential for their function as a serpin. The aim of this study is therefore, to develop a simple method of analyzing expression patterns of intact forms of SCCAs (functional SCCAs) in cervical squamous epithelial tissues and to investigate whether there are any differences in the expression of intact forms of SCCAs between normal and malignant cervical squamous epithelial tissues. We used nondenaturing polyacrylamide gel electrophoresis (PAGE) with immunoblotting. The newly generated antibody, Pab Y2, recognizes only intact form of SCCAs, while the conventional antibody, Mab 27, reacts with the cleaved form of SCCA1 as well as intact forms of SCCAs. Nondenaturing PAGE using Pab Y2 showed that an intact form of SCCAs in the heat-treated tissue extract at 60 degrees C for 2 h was separated into at least five bands, termed as bands A-E from cathode to anode. By comparison with two-dimensional electrophoresis patterns of SCCAs, it was found that the first three bands, i.e. bands A-C, are derived from the intact form of SCCA1, while the other two bands, i.e. band D and E are from the intact form of SCCA2. Specifically, band E, but not band D, of SCCA2 is apparently increased in squamous cell carcinomas compared with normal squamous epithelium. In conclusion, this novel analytical approach will be useful for investigating the different expression patterns of functional SCCAs between normal and malignant cervical squamous epithelial tissues.

Antigens, Neoplasm↗

PubMed2006

Suppression of SCC antigen promotes cancer cell invasion and migration through the decrease in E-cadherin expression.

Squamous cell carcinoma antigen (SCCA) is a useful tumor marker for diagnosis and management of squamous cell carcinoma. Recent studies have shown that SCCA can influence the behavior of cancer cells. It is well known that cell-cell adhesion is an important factor for the progression of cancer. The present study, therefore, was undertaken to investigate the effect of SCCA2 on the cell adhesion related molecule, E-cadherin, and cancer cell behavior. For this purpose, antisense SCCA2 cDNA was transfected into human uterine cancer cell lines, SKG IIIa and SiHa, which express SCCA2. Suppression of SCCA2 expression by antisense SCCA2 cDNA transfection decreased E-cadherin expression and promoted cell migration and invasion as well as the blockage of E-cadherin function by anti-E-cadherin antibody administration. In conclusion, SCCA2 regulates cell migration and invasion via E-cadherin expression, suggesting that SCCA2 may be involved in cancer behavior such as invasion or metastasis.

Antigens, Neoplasm↗

PubMed2005

Promoter analyses of SCC antigen genes.

SCC antigen (SCCA) has been used as a tumor marker for squamous cell carcinoma. Analyses of the SCCA1 and SCCA2 genes, which are almost identical, and their promoters have been reported. Recently it was found that both SCCAs were stimulated by interleukin (IL)-4 and IL-13. Here we analyzed the promoter activity of both SCCAs in the 5'-flanking region, exon 1, and intron 1 to evaluate a putative STAT6 binding site. The addition of intron 1 to the luciferase assay constructs including the 5'-flanking region significantly augmented the promoter activity of both SCCA1 and SCCA2. Furthermore, deletion analyses of intron 1 revealed that a 50-bp fragment of intron 1 that includes putative STAT6 binding site was responsible for the increased promoter activity. Although the sequences of SCCA1 and SCCA2 are very similar in the 5'-flanking region, the analysis of the -337 single nucleotide polymorphism of SCCA2 indicated that this polymorphism may underlie the difference in promoter activity between SCCA1 and SCCA2.

Amino Acid Sequence↗

PubMed2005

Tumor-associated serpin, squamous cell carcinoma antigen stimulates matrix metalloproteinase-9 production in cervical squamous cell carcinoma cell lines.

Squamous cell carcinoma antigen (SCCA) is clinically used as a tumor marker for patients with squamous cell carcinoma of various organs. SCCA1 and its highly homologous molecule, SCCA2, belong to the serine proteinase inhibitors (serpins) family, suggesting that these proteins may be involved in the malignant behavior of squamous cell carcinoma cells. The aim of this study is to functionally characterize these tumor-associated serpins regarding the potential to influence the production of matrix metalloproteinases (MMPs), which play a key role in tumor cell invasion and metastasis. Cervical squamous cell carcinoma cell lines, CaSki cells and SKG-IIIa cells were incubated with SCCA1 or SCCA2 and MMP production was analyzed by gelatin zymography. Both SCCA1 and SCCA2 significantly increased production of proMMP-9, but not proMMP-2. These stimulatory effects were still observed when cells were treated with SCCA mutants lacking the proteinase inhibitory activity of serpins. Furthermore, treatments with various forms of SCCAs, which are generated by interacting with their target proteinases, diminished the stimulatory effect of SCCAs, implying the importance of the conformational structure of SCCAs in the stimulatory effects of SCCAs on proMMP-9 production. In addition, in vitro invasion assay showed that SCCA1 and SCCA2 significantly promoted the activity of cell invasion. It is concluded that SCCAs can alter the invasive phenotype of cervical squamous cell carcinoma cells, probably by stimulating proMMP-9 production, and that intact conformational structure of SCCAs, but not proteinase inhibitory activity of serpins, is required for its stimulatory activity on proMMP-9 production.

Antigens, Neoplasm↗

PubMed2004

Severe calcification of mucocutaneous and gastrointestinal tissues induced by high dose administration of vitamin D in a puppy.

A three-month-old male Bull Terrier was referred to the Animal Medical Centre, Nihon University with chief complaints of subacute emesis and lethargy. Severe leukocytosis, high CRP, hypercalcemia and hypochloremia were detected. Moreover, severe calcification of gingival mucosa and abdominal skin, and abnormalities of the skeletal system were discerned. Abdominal X-ray and endoscopic examination revealed ulcer and hemorrhage on the mucosal membrane of the stomach. This might have been due to injections of high dose vitamin D at 3 and 2 weeks ago by another practioner, according to the detailed history of medication. After two months, a gastrointestinal and skin disorder disappeared, although calcification of the stomach membranes remained and abnormality of the skeletal system had worsened. Therefore, vitamin D should be carefully administrated to a puppy.

Animals↗

PubMed2004

Regulation of squamous cell carcinoma antigen production by E-cadherin mediated cell-cell adhesion in squamous cell carcinoma cell line.

Squamous cell carcinoma antigen (SCCA) is a useful tumor marker for diagnosis and management of squamous cell carcinoma. It is well known that cell-cell adhesion is important for progression of cancer. However, it is not clarified whether cell-cell adhesion affects SCCA production in squamous cell carcinoma. The present study was, therefore, undertaken to investigate whether E-cadherin-mediated cell-cell adhesion affects SCCA production in squamous cell carcinoma of the uterine cervix. SKG-IIIa cells or CaSki cells, cervical squamous cell carcinoma cell lines, were treated with anti-E-cadherin antibodies (1 microg/ml) up to 72 h. The cells were dissociated, and SCCA content in the cytosol and SCCA mRNA levels were significantly decreased compared to the control group treated with mouse IgG. Secondly, the signaling pathway for SCCA production mediated by E-cadherin was examined. Phosphatidylinositol (PI) 3-kinase is a well-known mediator of E-cadherin-mediated biological events. The treatment with a PI 3-kinase inhibitor suppressed SCCA production in SKG-IIIa cells. It is concluded that E-cadherin mediated cell-cell adhesion maintains SCCA production through PI 3-kinase in squamous cell carcinoma.

Antibodies, Neoplasm↗

PubMed2003

Electrophoretic analysis of the cleaved form of serpin, squamous cell carcinoma antigen-1 in normal and malignant squamous epithelial tissues.

The aim of this study was to detect the cleaved form of serine proteinase inhibitor (serpin), squamous cell carcinoma (SCC) antigen-1 in normal and malignant squamous epithelial tissues, which implies the presence of its target proteinase. The cleaved SCC antigen-1 in normal squamous epithelium was identified as a single spot with pI 6.35 and M(r) 40,000 by two-dimensional electrophoresis (2-DE) combined with immunoblotting. Interestingly, the cleaved form showed different biochemical properties in heat stability or immunoreactivity with a monoclonal antibody for SCC antigen (Mab 426) compared to intact SCC antigen-1. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of tissue extracts showed an abundant 40 kDa band of cleaved SCC antigen-1 in tumor tissue compared to normal tissue. Among the potential target proteinase of SCC antigen-1, immunoblotting analyses revealed that cathepsin L2 was remarkably overexpressed in tumor tissue, while cathepsin L was expressed in both normal and tumor tissues. These findings indicate that SCC antigen-1 interacts with specific endogenous proteinases such as cathepsins L and L2 in physiological and pathological states of squamous epithelium.

Antigens, Neoplasm↗

PubMed2002

A new conformational epitope generated by the binding of recombinant 70-kd protein and U1 RNA to anti-U1 RNP autoantibodies in sera from patients with mixed connective tissue disease.

OBJECTIVE: To establish an enzyme-linked immunosorbent assay (ELISA) using a complex of in vitro-transcribed U1 RNA and recombinant 70-kd, A, and C proteins (C-ELISA) to detect anti-U1 RNP antibodies reactive in double immunodiffusion (DID), but not in ELISA using the proteins alone (P-ELISA). METHODS: Sera from 196 patients with mixed connective tissue disease were used to test reactivity in P- and C-ELISAs, and the specificity of the sera was also tested by DID and immunoprecipitation (IP). RESULTS: In P-ELISA, 15 of 196 sera positive for anti-U1 RNP in DID did not react, while all sera reacted in C-ELISA. The reactivity of 15 sera to the U1 RNA was tested by IP and ELISA, and only 3 sera reacted with the U1 RNA. These results indicated that the increased reactivity in C-ELISA was not due to the U1 RNA itself. We confirmed that the 70-kd and A proteins were bound directly to the U1 RNA by IP using antibodies to His-tag, and we tested the reactivity of the sera to the U1 RNA-70-kd protein complex and the U1 RNA-A protein complex by IP. All sera reacted with the U1 RNA-70-kd protein complex, and 1 sample reacted with the U1 RNA-A protein complex. CONCLUSION: These results suggest that some anti-U1 RNP-positive sera specifically recognize the conformational structure altered by the binding of U1 RNA to the proteins, and the ELISA using U1 RNA and recombinant proteins is as useful as the DID method for detecting anti-U1 RNP antibodies.

Autoantibodies↗

PubMed1997

DNA bending of pSC101 ori induced by Rep, a replication initiator protein and IHF.

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees ). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (Rep(IHF)) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep(IHF) monomer for iterons was similar to that of wild-type Rep and bent DNA only 14 degrees more strongly than did the wild-type Rep. Rep(IHF)-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is discussed.

Journal Article↗