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Biomedical subjects

H Saya

Publications and source records attributed to H Saya.

At least 55 records · Page 3Linked to original sources
PubMed1998

Highly specific and sensitive detection of malignancy in urine samples from patients with urothelial cancer by CD44v8-10/CD44v10 competitive RT-PCR.

CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism. Overexpression of specific CD44 splice variant, i.e., CD44v8-10, has been found in several human malignancies and is considered to be associated with tumor progression and metastasis. We have demonstrated a novel molecular approach, CD44v8-10/CD44v10 competitive reverse transcription-polymerase chain reaction (CC-RT-PCR) for the detection of cancer cells overexpressing CD44v8-10 by the measurement of the transcriptional level of CD44v8-10 relative to that of CD44v10 (v8-10/v10 ratio). In this study, we initially examined the expression of CD44 splice variants in human urothelial cancers and their adjacent normal urinary tissues by RT-PCR. Any CD44 variant isoforms were barely detectable in normal urinary tissues, whereas CD44v8-10 was predominantly expressed in 23 of the 30 (77%) urothelial cancer specimens. We then applied CC-RT-PCR to spontaneously voided urine samples from patients with 80 urothelial cancer and 50 various benign urologic diseases. The CC-RT-PCR analysis revealed that all of the samples associated with benign diseases presented a predominant expression of the CD44v10 transcript (the v8-10/v10 ratios = 0.00-0.87), whereas 62 of the 80 samples associated with urothelial cancers mostly expressed the CD44v8-10 transcript (the v8-10/v10 ratios > 1.00). In addition, the positivity rate obtained by the CC-RT-PCR analysis was high regardless of the pathological grade of the urothelial cancers, although the sensitivity of the cytological examination declined with decreasing tumor grade. Our findings suggest strongly that CC-RT-PCR is a non-invasive useful tool for the diagnosis of urine samples from patients with urothelial cancer.

Adolescent↗
PubMed1998

Roles of Rho-associated kinase in cytokinesis; mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.

Amino Acid Sequence↗
PubMed1998

Impairment of cell adhesion by expression of the mutant neurofibromatosis type 2 (NF2) genes which lack exons in the ERM-homology domain.

Neurofibromatosis 2 (NF2) is an inherited disorder characterized by a predisposition to multiple intracranial tumors. The protein encoded by the NF2 gene has striking similarities to ezrin, radixin and moesin (ERM) proteins which link membrane proteins to the cytoskeleton. Therefore, it can be speculated that the disruption of cytoskeletal organization by alterations in the NF2 gene is involved in the development of tumors. It has been reported that the majority of NF2 mutations were nonsense or frameshift mutations that result in premature termination of translation. To facilitate the detection of these mutations, we performed protein truncation test and found that 11 of 14 NF2 patients had truncational mutations (79%). Seven of the 11 patients (64%) had a splicing abnormality which lead to absence of exons in the ERM homology domain. To examine the biological significance of the exon-missing mutations in the ERM homology domain, we expressed the wild-type (wt-NF2) and the various mutant NF2s (mu-NF2s) in a fibroblast cell line by using both liposome-mediated transfection and nuclear microinjection of the expression plasmids. The wt-NF2 showed intense punctate staining in the perinuclear cytoplasm in addition to overall staining of the submembranous area, whereas the mu-NF2s lacking exons in the ERM homology domain showed granular staining at the perinuclear region without any accumulation at the submembrane region. Microinjection of wt-NF2 cDNA into the nucleus of VA13 cells revealed that wt-NF2 protein induced a progressive elongation of cell processes. Furthermore, cells that expressed mu-NF2 had decreased adhesion, which resulted in detachment from the substratum. These findings suggested that the exon-missing mutations in the ERM-homology domain may affect cell membrane-cytoskeleton signaling and consequently disrupt cell-to-cell or cell-to-matrix interaction.

Adolescent↗
PubMed1998

Identification of a novel human homolog of the Drosophila dlg, P-dlg, specifically expressed in the gland tissues and interacting with p55.

We have identified a novel human homolog of the Drosophila dlg tumor suppressor gene, termed P-dlg, which has been mapped at chromosome 10q23. Unlike other human dlg homologs, P-dlg is expressed in placenta and various gland tissues but not in brain. The P-dlg protein is localized at the plasma membrane and cytoplasm, and it is expressed in the gland epithelial cells in normal prostate tissue but not in prostate cancer cell lines. Furthermore, we identified interaction between P-dlg and p55 palmitoylated membrane protein by yeast two-hybrid screening. These findings suggest that P-dlg forms a complex with p55 at the plasma membrane and plays roles in maintaining the structure of epithelial cells and transmitting extracellular signals to the membrane and cytoskeleton, which may negatively regulate cell proliferation.

Amino Acid Sequence↗
PubMed1998

Association of the myosin-binding subunit of myosin phosphatase and moesin: dual regulation of moesin phosphorylation by Rho-associated kinase and myosin phosphatase.

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. We found that in MDCK epithelial cells, MBS accumulated at the tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling area, where moesin, a member of the ERM (ezrin, radixin, and moesin) family, was localized. Neither membrane ruffling nor an accumulation of moesin and MBS at the free-end plasma membrane was induced when MDCK cells were stimulated with TPA after the microinjection of C3, which ADP-ribosylates and inactivates Rho. MBS was colocalized with moesin at the cell-cell contact sites in MDCK cells. We also found that moesin was coimmunoprecipitated with MBS from MDCK cells. Recombinant MBS interacted with the amino-terminal domains of moesin and ezrin. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward moesin, which was phosphorylated by Rho-kinase. The phosphatase activity was inhibited when MBS was phosphorylated by Rho-kinase. These results suggest that MBS is recruited with moesin to the plasma membrane and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of moesin downstream of Rho.

ADP Ribose Transferases↗
PubMed1998

Rapid identification of Leishmania species from formalin-fixed biopsy samples by polymorphism-specific polymerase chain reaction.

The precise identification and classification of Leishmania species is important for public health surveillance since different species cause different clinical features of the disease. A highly specific polymerase chain reaction (PCR) panel was developed to enable the identification of the five major Leishmania species that cause New World cutaneous leishmaniases. The primers used for this panel were designed to distinguish the polymorphism in sequences of commonly amplified DNA bands of the parasites produced by arbitrarily primed PCR. These polymorphism-specific PCR diagnoses were performed with formalin-fixed biopsy specimens of the leishmanial lesions from four patients in Ecuador and one hamster skin lesion, and these lesions were determined to be caused by Leishmania (Viannia) panamensis, L. (Leishmania) mexicana, and L. (L.) amazonensis. The PCR panel may offer an important and practical approach to the standardized identification of Leishmania species in field examinations.

Animals↗
PubMed1998

Identification of a human homolog of the Drosophila neuralized gene within the 10q25.1 malignant astrocytoma deletion region.

The loss of chromosome 10 is the most frequent genetic alteration found in malignant astrocytomas. In particular, the long arm of chromosome 10 was previously reported to have two or more common deletion regions where tumor suppressor genes may be located. In this study, we performed deletion mapping of 44 malignant astrocytomas using 12 microsatellite markers on chromosome 10q and demonstrated that the minimal common region of loss of heterozygosity (LOH) was present between D10S192 and D10S566 localized at 10q25.1. Subsequently, we have identified a novel gene, termed h-neu, within the region frequently deleted and found that h-neu encodes a protein with strong homology to the Drosophila neuralized (D-neu) protein. Northern blot and RT-PCR analyses revealed that h-neu mRNA was expressed at very low levels in human malignant astrocytoma tissues and the majority of glioma cell lines examined, while normal brains expressed h-neu transcript. Furthermore, DNA sequencing analysis of the h-neu transcript revealed one of the glioma cell lines, U251MG, had a single nucleotide substitution which resulted in an amino acid change from glycine (GGC) to serine (AGC) at codon 253. The D-neu gene is known to serve a critical function in neurogenesis in Drosophila, and loss-of-function mutations produce hyperplasia of primitive neuronal cells. These observations led us to hypothesize that h-neu gene plays a role in determination of cell fate in the human central nervous system and may act as a tumor suppressor whose inactivation could be associated with malignant progression of astrocytic tumors.

Amino Acid Sequence↗
PubMed1998

Overexpression of Bcl-2 in bladder cancer cells inhibits apoptosis induced by cisplatin and adenoviral-mediated p53 gene transfer.

To investigate the effects of the expression of Bcl-2 protein in bladder cancer on the apoptosis induced by cisplatin or adenoviral-mediated p53 gene (Ad5CMV-p53) transfer, we transfected the bcl-2 gene into KoTCC-1, a human bladder cancer cell line that does not express the Bcl-2 protein. The Bcl-2-transfected KoTCC-1 (KoTCC-1/B) exhibited significantly higher resistance to both cisplatin and Ad5CMV-p53 transfer than did either the parental KoTCC-1 (KoTCC-1/P) or the vector-only transfected cell line (KoTCC-1/C). The flow cytometric analysis of the propidium iodide-stained nuclei and DNA fragmentation analysis after cisplatin or Ad5CMV-p53 treatment revealed DNA degradation in both KoTCC-1/P and KoTCC-1/C, whereas KoTCC1/B showed a marked inhibition of DNA degradation. Following the treatment with cisplatin or Ad5CMV-p53, the accumulation of p53 protein was highly detectable for a long period in KoTCC-1/B compared to that in KoTTC-1/P and KoTCC-1/C. Furthermore, the cisplatin and Ad5CMV-p53 treatments each reduced the volume of the subcutaneous tumors established in nude mice formed by KoTCC-1/P or KoTCC-1/C; in contrast, their reductive effects on the tumors formed by KoTCC-1/B were significantly suppressed. The intraperitoneal tumor cell implantation model revealed that the prognoses of mice injected with KoTCC-1/B were significantly inferior to those of the mice injected with either KoTCC-1/P or KoTCC-1/C after treatment with cisplatin or Ad5CMV-p53. These findings suggest that the expression of Bcl-2 in bladder cancer cells interferes with the therapeutic effects of cisplatin and Ad5CMV-p53 through the inhibition of the apoptotic pathway.

Adenoviruses, Human↗
PubMed1998

Molecular detection of cancer cells by competitive reverse transcription-polymerase chain reaction analysis of specific CD44 variant RNAs.

BACKGROUND: CD44 is a cell surface glycoprotein implicated in such diverse biologic processes as lymphocyte activation and homing, extracellular matrix adhesion, and cellular migration. Primary transcripts of the CD44 gene can be alternatively spliced to produce a variety of messenger RNA (mRNA) species. The standard form of CD44 mRNA contains sequences from at least 20 genomic exons; variant mRNAs contain sequences from one or more additional exons (v1-10). Predominant expression of a specific CD44 variant, i.e., CD44v8-10, in several human carcinomas has been described previously. In this study, we developed a novel molecular approach for detecting cancer cells that overexpress CD44v8-10 mRNA. METHODS: After finding that CD44v8-10 was predominantly expressed in non-small-cell lung and bladder carcinomas and that CD44v10 was predominantly expressed in leukocytes, we developed a competitive reverse transcription-polymerase chain reaction assay (CC-RT-PCR) that allows quantification of the relative expression of these two mRNA species in clinical specimens (i.e., determination of a v8-10/v10 ratio). CC-RT-PCR analysis was applied to pleural effusion specimens from patients with benign or malignant lung diseases as well as to spontaneously voided urine samples from patients with benign or malignant urologic diseases. RESULTS: Fifty two of 54 samples from patients with benign diseases expressed CD44v10 predominantly (v8-10/v10 ratio < or = 0.65), whereas 46 of 61 samples from patients with malignant diseases expressed CD44v8-10 predominantly (v8-10/v10 ratio > 1.00) (two-sided P < .001). CC-RT-PCR detected predominant expression of CD44v8-10 in cytologically negative samples from 11 patients who were later diagnosed with malignant disease. CONCLUSIONS: CC-RT-PCR analysis of CD44v8-10 expression could be an important adjunct to cytologic examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusions and urine.

Breast Neoplasms↗
PubMed1998

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms.

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.

Adult↗
PubMed1998

The involvement of calpain-dependent proteolysis of the tumor suppressor NF2 (merlin) in schwannomas and meningiomas.

Neurofibromatosis type 2 (NF2) protein, also known as merlin or schwannomin, is a tumor suppressor, and NF2 is mutated in most schwannomas and meningiomas. Although these tumors are dependent on NF2, some lack detectable NF2 mutations, which indicates that alternative mechanisms exist for inactivating merlin. Here, we demonstrate cleavage of merlin by the ubiquitous protease calpain and considerable activation of the calpain system resulting in the loss of merlin expression in these tumors. Increased proteolysis of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.

Base Sequence↗
PubMed1998

Monitoring adenoviral p53 transduction efficiency by yeast functional assay.

Monitoring the transduction efficiency is of paramount importance in gene therapy. To monitor adenovirus-mediated wild-type p53 gene transfer, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast. Selective amplification of cellular and viral p53 transcripts followed by quantitative assessment of mutant p53 content with the assay permits measurement of the wild-type p53 transduction efficiency into SF-188, U251MG and HUG31 glioblastoma cells. One reverse transcription primer tracks the wild-type/mutant ratio of endogenous p53 mRNA (P2), and the other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1). Following infection of cell lines homozygous for mutant p53, the apparent transduction efficiency calculated (tau 0 = [P1-P2]/[1 + P2]) correlated with the level of p21 expression. Transduction efficiency in heterozygous wild-type/mutant HUG31 cells increased linearly with multiplicity of infection (MOI) for tau 0 values between 0.5 and 5.9, and admixture of normal cell-derived RNA produced only a modest reduction in tau 0 value, in keeping with theoretical predictions. These results suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy.

Adenoviridae↗
PubMed1998

Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes.

BACKGROUND: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. RESULTS: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. CONCLUSION: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Cell Differentiation↗
PubMed1998

Significant correlation of nitric oxide synthase activity and p53 gene mutation in stage I lung adenocarcinoma.

Nitric oxide (NO) and its derivatives can directly cause DNA damage and mutation in vitro and may play a role in the multistage carcinogenic process. It has been reported that NO induces mutation in the p53 tumor suppressor gene; we therefore analyzed the relationship between NO synthase (NOS) activity and p53 gene status in early-stage lung adenocarcinoma. Surgical samples were classified into two categories: 14 lung adenocarcinomas with high NOS activity (>25 pmol/min/g tissue, category A), and 16 with low NOS activity (<25 pmol/min/g tissue, category B). A yeast functional assay for p53 mutations disclosed a red colony that corresponded to a mutation in the p53 gene in 8 cases (57.1%) in category A and 3 cases (18.8%) in category B, the frequency being significantly higher in the former (P<0.05). A p53 DNA sequence analysis revealed that 5 of the 8 p53 mutation-positive samples in category A had a G:C-to-T:A transversion, which is reported to be a major target of NO. The mechanism of carcinogenesis of adenocarcinoma is not fully understood, but these results suggest that an excess of endogenously formed NO may induce a p53 gene mutation containing mainly G:C-to-T:A transversion in the early stage of lung adenocarcinoma. Our results suggest that NO has potential mutagenic and carcinogenic activity, and may play important roles in human lung adenocarcinoma.

Adenocarcinoma↗
PubMed1998

Expression of CD44 splicing isoforms in lung cancers: dominant expression of CD44v8-10 in non-small cell lung carcinomas.

We examined CD44 isoform expression in 138 frozen tissue samples, which included primary lung carcinomas, adjacent non-tumorous lung tissues and benign lung diseases, by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical analyses. CD44v8-10 mRNA and protein were dominantly expressed in non-small cell lung carcinomas (NSCLC), while non-tumorous tissues principally expressed CD44s and small cell lung carcinomas (SCLC) expressed either CD44s or no detectable CD44. These results indicate that CD44v8-10 is the dominant splicing isoform in NSCLC and can be practically utilized as a diagnostic marker and therapeutical target in NSCLC.

Aged↗
PubMed1998

Loss of merlin-p85 protein complex in NF2-related tumors.

The NF2 tumor suppressor gene product, designated merlin, belongs to the family of molecules that links membranous protein with the cytoskeleton. We have previously shown that merlin was co-immunoprecipitated with a cellular protein, p85, in cultured cell. To analyze the alteration of merlin and associated proteins in surgical specimens, we developed a new method for biotin-labeling of whole cellular proteins. Screening of tumor tissues using our method showed that none of malignant gliomas and half of the NF2-related tumors had altered p85 and merlin. Our detection method seems useful for the screening of merlin alterations in NF2-related tumors.

Avian Proteins↗
PubMed1998

Interaction between CD44 and hyaluronic acid regulates human prostate cancer development.

PURPOSE: This study was designed to investigate the significance of the interaction between CD44 and hyaluronic acid in the development of human prostate cancer. MATERIALS AND METHODS: We transfected the standard CD44 isoform (CD44s) cDNA into PC3, a human prostate cancer cell line that barely expresses CD44s protein. The effects of the reintroduction of CD44s into PC3 cells on the ability to bind hyaluronic acid (HA) were analyzed by the cell adhesion assay and by the cell migration assay. The in vitro growth rate of CD44s transfected PC3 was measured by using the MTT assay. We then evaluated the in vivo tumor development of CD44s transfected PC3 cells by subcutaneous, intravenous, and intraperitoneal injection models in athymic nude mice. RESULTS: The introduction of CD44s in PC3 cells markedly enhanced the binding and migration of these cells to HA, but not to other extracellular matrix molecules. In vitro growth of CD44s-transfected PC3 was found to be significantly decreased. In addition, the CD44s-transfected PC3 cells also demonstrated reduced tumorigenicity and metastatic potential in in vivo experimental models. CONCLUSIONS: These findings suggest that the CD44s downregulation plays an important role in the development of human prostate cancer, in part through reduction of the ability to bind HA.

Down-Regulation↗