Yoshiaki Yura

PPARγ ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor, whose activation has been linked to several physiologic pathways including those related to the regulation of insulin sensitivity. Here, we investigate effects of PPARgamma specific ligands, rosiglitazone and pioglitazone, on formation of nitrotyrosine and increased expression of inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and intercellular adhesion molecule-1 (ICAM-1) in adjuvant-induced murine arthritis. Administration of rosiglitazone or pioglitazone (30 mg/kg, p.o.) significantly inhibited the adjuvant-induced increase in formation of nitrotyrosine and expression of iNOS on both ankle and temporomandibular joints. Rosiglitazone also inhibited the adjuvant-induced expression of M30 positive cells, as a marker of apoptosis, in the joint tissues. In addition, treatment with rosiglitazone or pioglitazone (30 microM) inhibited lipopolysaccharide plus tumor necrosis factor (TNF)-alpha-induced protein expression of iNOS, cyclooxygenase-2, ICAM-1 and nitrotyrosine formation in RAW 264 cells, a murine macrophage-like cell line. Rosiglitazone or pioglitazone inhibited increase in phosphorylated I-kappaB (pI-kappaB) expression, as an index of activation of nuclear factor (NF)-kappaB, in both joint tissues and RAW264 cells. Furthermore, in PPARgamma-transfected HEK293 cells, rosiglitazone inhibited the TNF-alpha-stimulated response using NF-kappaB-mediated transcription reporter assay. These results indicate that PPARgamma ligands may possess anti-inflammatory activity against adjuvant-induced arthritis via the inhibition of NF-kappaB pathway.

Search for specific markers of neoplastic epithelial duct and myoepithelial cell lines established from human salivary gland and characterization of their growth in vitro

The neoplastic epithelial duct cells human salivary gland (HSG) and myoepithelial cells human pleomorphic adenoma (HPA) established from human salivary gland were examined by the immunoperoxidase method for the presence of specific antigens such as carcioembryonic antigen (CEA), S‐100 protein, secretory component (SC), lactoferrin (LF), and myosin. Isolation of the cells and their morphologic features were reported previously. Consequently, the presence of CEA, SC, and LF in the HSG cells was demonstrated. The HPA cells were identified to express the specific antigens reactive to anti‐S‐100 protein, anti‐myosin and anti‐CEA sera in addition to the presence of oxytocin receptor. When the two cell lines were co‐cultured in monolayer culture or within the sponge matrix, a large number of ductlike or tubular structures were formed in an optimal ratio of 1:2 in HSG and HPA cells, whereas the cultures of HSG cells only grew with occasional formation of ductlike structure. In addition, in HSG and HPA cells in an area with their contact in the mixed cultures, CEA staining was intensified as compared with the culture of HSG or HPA cells only and further S‐100 protein was detected in HSG cells, whereas S‐100 protein was not detected in the culture of HSG cells only. These findings strongly suggest that the intercalated duct and myoepithelial cells from human salivary gland propagate with their interaction together in the expression of specific antigens such as CEA and S‐100 protein or in the morphogenesis of salivary gland epithelial cells.

Generation of cells with phenotypes of both intercalated duct-type and myoepithelial cells in human parotid gland adenocarcinoma clonal cells grown in athymic nude mice

SummaryA neoplastic epithelial cell line was established from nude mice tumors grown after transplantation of surgical specimens from a human parotid gland adenocarcinoma. This cell line, which had ultrastructure similarities to salivary intercalated duct cells, was found by immunohistochemical techniques to contain amylase, but myosin was not detected. Ultrastructurally, cells of an intermediate type between intercalated ductal and myoepithelial cells were found in the transplanted tumors. Moreover, the expression of myosin in addition to the presence of amylase was detected in the tumors. These findings indicate that some transplanted tumor cells appear to be differentiating towards myoepithelial cells.

Therapy for oral squamous cell carcinoma by tegafur and streptococcal agent OK-432 in combination with radiotherapy: Association of the therapeutic effect with differentiation and apoptosis in the cancer cells

Twenty patients with oral squamous cell carcinoma having mainly stage II or III lesions without distant metastasis, were treated with tegafur and streptococcal agent, OK-432, in combination with radiotherapy. As a consequence, 16 cases among the treated 20 cases showed complete remission by this therapy alone. Especially, we have found that the squamous cell carcinoma arising in non-keratinizing oral epithelium rather than in keratinizing oral epithelium has better response to this therapy. Among the 16 cases with complete remission (CR) by the current therapy, 10 cases were histopathologically diagnosed as well-differentiated squamous cell carcinoma and six cases as moderately differentiated squamous cell carcinoma. When we examined immunohistochemically the expres-sion of various antigens such as proliferating cell nuclear antigen (PCNA), p53 and LeY or the presence of DNA fragmentation by nick-end labelling in the biopsy materials taken at the first visit to our clinic from 20 patients treated with the current therapy, the CR group showed a significantly increased LeY expres-sion level ( p< 0.05) and DNA fragmentation rate ( p< 0.05) as compared with the partial response (PR, n= 3) + no change (NC, n= 1) group. On the other hand, the CR group with respect to PCNA expression level was significantly decreased as compared with the PR + NC group ( p< 0.05). From these findings, it can be considered that the therapy for oral squamous cell carcinoma by UFT and OK-432 in combination with radiotherapy is very effective, which may be associated with differentiation or apoptosis in oral squamous carcinoma cells. In addition, we present the clinical findings and results of immunohistochemical staining for the biopsy materials obtained from four CR cases treated with the current therapeutic method.

Inhibition of herpes simplex virus replication by genistein, an inhibitor of protein-tyrosine kinase

SummaryGenistein, an inhibitor of protein-tyrosine kinase, inhibited the replication of herpes simplex virus type 1 (HSV-1) at genistein concentrations of more than 25 µM, whereas the related compounds, which do not inhibit protein-tyrosine kinases, did not affect the replication of HSV-1. In the presence of genistein, the phosphorylation of tyrosine residues in specific viral polypeptides was markedly reduced. These results indicate that the phosphorylation of tyrosine residues in viral polypeptides may be essential for the replication of HSV-1.

Effect of dibutyryl cyclic AMP on morphologic features and biologic markers of a human salivary gland adenocarcinoma cell line in culture

Dibutyryl cyclic AMP (dB‐cAMP) has marked effects on the growth, morphologic features, and biologic markers of a human salivary gland adenocarcinoma cell line in culture. A cell line with ultrastructure and biologic markers specific to the intercalated duct cells of human salivary glands was cultivated in the presence of dB‐cAMP. Major alterations, such as process formation and expression of myofilaments and oxytocin receptor in addition to myosin and S‐100 protein, were observed in those cells with a phenotype similar to myoepithelial cells. Both the anchorage‐independent and anchorage‐dependent growths were markedly suppressed in the presence of dB‐cAMP. After the removal of dB‐cAMP from the culture, the treated cells returned rapidly to the phenotype of the untreated cells. These findings indicate that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line occurs in growth medium containing dB‐cAMP.

Effects of 5-fluorouracil and the combination of 5-fluorouracil and human leukocyte interferon on human salivary gland adenocarcinoma cell line in culture

The growth inhibitory effects of 5-fluorouracil (5-Fu) and the combination of 5-Fu and human leukocyte interferon (HuIFN-alpha) on the human salivary gland adenocarcinoma cell line HSG in culture were examined by measuring colony formation in the semi-solid agar medium and cell proliferation in the monolayer culture. As a consequence, the colony-forming ability of HSG cells in the agar medium containing 5-Fu was found to be markedly inhibited as compared with the untreated control. The concentration of 5-Fu yielding 50% inhibition of colony-forming ability of HSG cells under the presence of 5-Fu was 0.08 micrograms/ml. When the growth inhibitory effects of the combination of 5-Fu and HuIFN-alpha on HSG cells were examined, the colony forming ability of HSG cells was synergistically inhibited, whereas effects of the combined treatment on HSG cells in the monolayer culture were less sensitive than those on the colony formation. These findings strongly suggest that the combination of 5-Fu and HuIFN-alpha or 5-Fu alone is selectively effective on the neoplastic cells of HSG cell population but not on the non-neoplastic cells.

Effects of Protein Tyrosine Kinase Inhibitors on the Replication of Herpes Simplex Virus and the Phosphorylation of Viral Proteins

The effect of protein tyrosine kinase (PTK) inhibitors on the replication of herpes simplex virus (HSV) was examined. Tyrphostins AG17, AG213, AG490, and AG555, and herbimycin A all inhibited the plaque formation of HSV type 1 (HSV-1) in Vero cells, but AG17, AG490, and AG555 exhibited a more selective antiviral effect. In the presence of 0.4 microM AG17, the virus production 24 h after infection was decreased to 7.7% of the untreated control level. Even if the treatment was started 12 h after the initiation of infection, the viral titer was reduced by 82.4%, compared with the untreated control level. In HSV-1-infected cells ICPs 6, 17/18, 19/20, and 25 were tyrosine-phosphorylated proteins. The synthesis and phosphorylation of these proteins were inhibited by AG17, and suppression of ICP 19/20, which were identified as the UL47 gene products, was the greatest. In contrast, the in vitro autophosphorylation of viral proteins was not affected by this PTK inhibitor. These results indicate that tyrphostin may represent a novel class of inhibitors of HSV-1, and that the viral proteins which have phosphorylated tyrosine residues and are suppressed by AG17 most significantly are the products of the UL47 gene, the tegument proteins VP13/14.

Induction of cells with a myoepithelial cell phenotype by treatment with dibutyryl cyclic AMP in human salivary adenocarcinoma cells grown in athymic nude mice

SummaryThe adenocarcinoma produced by transplantation into nude mice of a neoplastic human salivary intercalated duct cell line was treated with 0.1 ml of minimal essential medium (MEM) containing dibutyryl cyclic AMP (dB-cAMP) at a final concentration of 1 mM daily for 28 days and examined morphologically and immunohistochemically. The dB-cAMP treatment resulted in a marked suppression of tumor growth. In addition, tumor nests with a myoepithelial cell phenotype characterized by the presence of microfilament systems reactive with antimyosin and anti-S-100 protein sera were often observed in the treated tumors, but not in untreated controls. These findings lead us to suggest that neoplastic intercalated duct cells can be induced to differentiate into myoepithelial cells and that levels of cAMP within the cells may regulate this cytodifferentiation.

Therapeutic effect of human fibroblast interferon on premalignant lesions arising in oral mucosa. A pilot study.

Human fibroblast interferon (HuIFN-beta) was topically administered to 20 premalignant lesions histopathologically showing epithelial dysplasia such as leukoplakia and lichen planus which arose in the oral mucosa. HuIFN-beta was prepared in the water-soluble gel form containing 2% carboxymethylcellulose, 45% glycelin, 0.1 M citrate buffer (pH 4.5) and 0.2% SDS as stabilizing agents. This preparation was found to be effective for herpetic gingivostomatitis and zostal lesions arising along the intercostal nerve. Thus, the HuIFN-beta preparation (10(4) to 5 X 10(3) IU) was applied to the oral mucosal lesion for 1 h twice a week. The lesion with topical administration of HuIFN-beta was covered tightly with the mucosal bandage which was coated with carboxymethylcellulose, glycelin and CaCl2 on vinyl acetate matrix. The 14 oral lesions with erosion or ulcer formation accompanied by severe pain by touch, had complete remission after approximately 10 successive applications of this preparation. Although subjective symptoms such as irritation pain in the other 6 patients with severe hyperkeratotic lesion subsided, white coatings and streaks could not be completely removed by this therapy. No other side-effects excluding slight pain and reddish swelling which occurred intermittently during HuIFN-beta administration were observed.