Biomedical subjects

Y Otsuki

Publications and source records attributed to Y Otsuki.

At least 19 recordsLinked to original sources

PubMed2006

Electrogene therapy using endostatin, with or without suicide gene therapy, suppresses murine mammary tumor growth and metastasis.

Syngeneic inoculated metastatic mammary cancers received direct intratumoral injection of a plasmid vector containing either endostatin (pEndo) with or without a suicide gene (pHSVtk), pHSVtk alone or control vector once a week for 8 weeks. We applied electrogene transfer to the tumors after each injection and administered ganciclovir (GCV) to pHSVtk-transfected mice using an osmotic minipump. Anticancer efficacy was monitored using a variety of parameters, namely tumor volume, intratumoral microvessel density and DNA synthesis, number of mice with metastasis, and number of sites of metastasis per mouse. Tumor volume was significantly lower in all therapeutic groups, with the most effective growth suppression in the pEndo+pHSVtk/GCV group. Lymph node metastasis was significantly less frequent in all therapeutic groups, whereas the multiplicity of lung metastases was significantly lower only in the pEndo and pEndo+pHSVtk/GCV groups. All therapeutic groups showed significantly lower intratumor microvessel density and DNA synthesis. The pEndo and pEndo+pHSVtk/GCV groups also showed a significant reduction in the numbers of dilated lymphatic vessels containing intralumenal tumor cells. Our data suggest that endostatin electrogene therapy alone or in combination with pHSVtk/GCV suicide gene therapy is more beneficial than suicide gene therapy alone. The observed antimetastatic activity of endostatin may be of high clinical significance in the treatment of metastatic breast cancer.

Adenoviridae↗

PubMed2005

A novel STK11 germline mutation in two siblings with Peutz-Jeghers syndrome complicated by primary gastric cancer.

Patients with Peutz-Jeghers syndrome (PJS) are known to be at risk of gastric cancer (GC), and the STK11 gene is a susceptibility gene for PJS. However, as no cases of PJS with GC in which a STK11 germline mutation has been identified have ever been reported and other susceptibility genes have also been suggested to be involved in PJS, the relation between STK11 germline mutations and GC in PJS is still unknown. In this study, we used sequencing analysis to investigate the STK11, CDH1, and TP53 loci for a germline mutation in two siblings with PJS with primary GC. A novel type of the STK11 germline mutation, c.890delG, encoding a truncated protein (p.Arg297fsX38) was identified, but no germline mutations of the CDH1 and TP53 genes were detected. No inactivation of the wild-type allele by somatic mutation or chromosomal deletion or hypermethylation at the 5'-CpG site of STK11 was detected in the GC. This is the first report of a STK11 germline mutation in a PJS patient with GC and should contribute to establishing correlations between the STK11 germline mutations and GC in PJS patients.

AMP-Activated Protein Kinase Kinases↗

PubMed2003

Identification of c-Jun as bcl-2 transcription factor in human uterine endometrium.

We describe the application of the biomolecular interaction (BIA) technique to detection of the interaction between protein (e.g., c-Jun) and DNA (e.g., two AP-1 motifs from bcl-2 promoter), compared with immunohistochemistry (IHC) of c-Jun. The specific binding assay for the interaction of c-Jun and activating protein-1 (AP-1) motifs was performed using a Biacore 2000 system. Intense immunoreactivity of c-Jun in glandular cells of the human uterine endometrium was observed in the proliferative phase, while c-Jun in stromal cells was expressed throughout the menstrual cycle. In contrast to the IHC of c-Jun, the specific binding of c-Jun to two separate AP-1 motifs in the bcl-2 promoter region was detected only in nuclear extracts of glandular cells, but not in stromal cells, during the proliferative phase. These results indicate that, while transmitting various signals, c-Jun enhances the transcription level of bcl-2, which in turn keeps glandular cells alive and proliferating in normal human endometrium during the proliferative phase. Moreover, the method involving real-time biomolecular interactions such as DNA-protein binding is novel for the study of transcription factors when combined with IHC.

Adult↗

PubMed2002

Cell death and proliferation in Nd-YAG laser, electrocautery, and scalpel wounds on mice skin.

The purpose of this study is to compare cell death and proliferation in laser, electrocautery and scalpel wounds on the mice epidermis. Wounds were examined by transmission electron microscopy, the detection of free 3'-OH DNA ends and immunohistochemistry of proliferating cell nuclear antigen (PCNA), inducible nitric oxide synthase (iNOS), keratinocyte growth factor (KGF) and keratinocyte growth factor receptor (KGFR). Reepithelization was first observed 5 days after scalpel and laser incisions and 7 days after electrocautery incision. Ultrastructurally, keratinocytes in both electrocautery and laser wounds showed similar post-apoptotic necrotic changes. Interestingly, dividing cells were often observed 3 days after laser incision. Apoptotic index in electrocautery wounds was higher than in laser wounds, although there was no significant difference in the PCNA expression level between them. The expression of iNOS, KGF and KGFR in laser wounds was more intense than in electrocautery wounds. In scalpel wounds, keratinocytes did not show significant changes in morphology or of markers of cell death and proliferation during the observation period. Therefore, the increase in the number of dividing cells and in the expression level of iNOS, KGF and KGFR may induce earlier and thicker reepithelization in laser wounds than in electrocautery and scalpel wounds.

Animals↗

PubMed2002

Apoptosis and cell proliferation of small intestinal villi in mitomycin C-treated rats.

Mitomycin C (MMC) therapy often causes toxicity affecting the small intestine. We investigated the relationship between pathological manifestations and cell death, or the proliferation of small intestinal villi in rats treated with MMC. The length of the villi, apoptosis, and cell proliferation were evaluated in the small intestine at 3, 7, and 11 days after MMC treatment by the TUNEL method, BrdU-immunohistochemistry, and transmission electron microscopy. In MMC-treated rats, the body weight decreased until day 7 and recovered from day 8, while most rats had watery stools from days 4 to 7. The villi were the shortest on day 7 and were still shorter on day 11 than in the control group. The highest incidence of TUNEL-positive cells in the small intestinal crypts was observed on day 3, and the number decreased thereafter to reach the control level on day 11. The percentage of BrdU-labeled cells was the highest on day 3 and the lowest on day 7, but recovered to the control level on day 11. The clinical symptoms caused by MMC treatment are consistent with the changes of villous length that reflect the viability of stem cells in the small intestinal crypts about 4 days earlier.

Animals↗

PubMed2001

Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1.

The putative tumor metastasis suppressor nm23H1 was originally identified in murine melanomas by subtraction cloning. It displays nucleoside diphosphate kinase activity and regulates cellular events, including growth and development. Recently nm23H1 has been reported to also act as a GTPase-activating protein of the Ras-related GTPase Rad. We attempted to determine whether nm23H1 also regulates Rho-family GTPases. Although we were unable to detect a direct association between nm23H1 and Rho-family GTPases, nm23H1 was shown to be associated with a Rac1-specific nucleotide exchange factor, Tiam1, by interaction with its amino-terminal region in extracts from the cells expressing exogenous Tiam1 and from native tissue. Overexpression of nm23H1 inhibited the Tiam1-induced production of GTP-bound Rac1 and activation of c-Jun kinase. On the other hand, forced overexpression of the wild type, but not the kinase-inactivated mutant of nm23H1, converted the GDP-bound forms of Rac1, Cdc42, and RhoA to their GTP-bound forms in vitro by its nucleoside diphosphate kinase activity, but nm23H1 alone apparently did not produce the GTP-bound form of these GTPases in vivo. These results suggest that nm23H1 negatively regulates Tiam1 and inhibits Rac1 activation in vivo. Moreover, adhesion-stimulated membrane ruffles of Rat1 fibroblasts were reduced by overexpression of nm23H1. Based on these observations, we concluded that we had identified a function of nm23H1 as a regulator of Rac1 and that it may be related to the effect of nm23H1 as a tumor metastasis suppressor.

Cell Line↗

PubMed2001

Apoptosis in human endometrium: apoptotic detection methods and signaling.

It is generally accepted that apoptosis occurs in human endometrium through the late secretory to menstruating phases. We found that Bcl-2 expression showed a cyclic pattern, peaking during the late proliferative phase. The decreased Bcl-2 in human endometrial glandular cells during the secretory phase was consistent with the appearance of apoptotic cells during the same phase. The expression patterns of both Sp-3 and c-Jun in glandular cells were similar to that observed in Bcl-2. Therefore, Sp-3 and c-Jun could be candidates for bcl-2 transcriptional factors in the human endometrium. In contrast to Bcl-2, both Fas and Fas ligand in glandular cells were coexpressed throughout the menstrual cycle. In particular, glandular cells showed the most intense expression of Fas ligand from the secretory to menstruating phases. The activities of caspase-3, -8 and -9 were higher from the secretory to menstruating phases than during the proliferating phase. We therefore conclude that bcl-2 transcription in glandular cells may be promoted by the binding of several transcriptional molecules to bcl-2 promoter, and the translated Bcl-2 blocks the release of cytochrome c from mitochondria to the cytosol during the proliferative phase. During the secretory phase, glandular cells may undergo apoptotsis via both death-receptor and mitochondrial pathways.

Animals↗

PubMed2001

Apoptosis in the normal human amnion at term, independent of Bcl-2 regulation and onset of labour.

This study was designed to detect apoptosis in the human amnion and to elucidate the signalling pathway involved in its regulation. Samples of human amnion were obtained from 34 women (weeks 11-42 of gestation) and studied using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) method with light microscopy (LM) and transmission electron microscopy (TEM). Apoptotic regulators in the samples were studied by immunohistochemistry and caspase activity assay. The TUNEL method with LM demonstrated that the percentage of TUNEL-positive cells in the amniotic epithelium was the highest in weeks 40-41 of gestation (P < 0.05) independent of the onset of labour, and the cells were often detached from the epithelium into the amniotic cavity at term. The TUNEL method with TEM clearly showed the characteristic features of apoptosis such as the nuclear condensed chromatin with abundant free 3'-OH DNA ends, cell shrinkage and a decrease in the number of desmosomes, except for the presence of apoptotic bodies. Fas and Fas ligand (FasL) were constantly expressed on apical membranes of amniotic epithelial cells from weeks 16-27 through to 40-41 of gestation, while no Bcl-2 expression was observed throughout the gestational periods. Activities of caspase-3 and caspase-8, but not of caspase-9, were higher in weeks 40-41 than those from weeks 16-27 of gestation (P < 0.01). We conclude that apoptosis in term amniotic epithelium is independent of Bcl-2 regulation and onset of labour, and may play an important role in the fragility and rupture of human fetal membranes at term.

Amnion↗

PubMed2001

Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene.

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH(2)-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (alpha PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of alpha PIX (DeltaCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated alphaPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted alphaPIX (Delta SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of alphaPIX (Delta CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas alphaPIX (Delta SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the alphaPIX (Delta CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that alphaPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.

Apoptosis↗

PubMed2001

[A case of papillary adenocarcinoma of the ovary that responded favorably to irinotecan hydrochloride and cisplatin after the administration of paclitaxel and carboplatin].

Recently, the standard treatment for advanced ovarian cancer has changed from CP therapy (cyclophosphamide, cisplatin (CDDP)) to TJ therapy (paclitaxel (TXL), carboplatin (CBDCA)). Irinotecan (CPT-11) is one of the derivatives of camptotecin and has been reported to have a high efficacy for ovarian cancer. In one case of ovarian cancer, chemotherapy was applied with CBDCA and TXL. However, after 2 months of six courses of the chemotherapy, CA-125 was elevated. The elevation of tumor marker levels in serum without the recurrent focus forced us to treat the patient with CPT-11 and CDDP for the second line chemotherapy. Tumor marker levels improved at the beginning of the therapy. In conclusion, CPT-11 and CDDP was effective against the recurrence of ovarian cancer treated with TJ therapy.

Adenocarcinoma, Papillary↗

PubMed2000

Xylem-specific expression of wound-inducible rice peroxidase genes in transgenic plants.

A peroxidase gene, poxA, was isolated from a rice (Oryza sativa L.) genomic library. The gene consists of four exons whose combined sequences were identical to that of the prxRPA mRNA whose levels were dramatically stimulated by wounding as well as by treatment of rice shoots with ethephon or UV irradiation [H. Ito, F. Kimizuka, A. Ohbayashi, H. Matsui, M. Honma, A. Shinmyo, Y. Ohashi, A.B. Caplan, R.L. Rodriguez, Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots, Plant Cell Rep. 13 (1994) 361-366]. The temporal and spatial expression properties of the poxA gene promoter as well as that from a second related peroxidase gene, poxN, were analyzed in transgenic tobacco and rice plants using the uidA gene as a reporter. In transgenic tobacco, UV- and wound-responsive cis-elements were located within 144 bp from the translational start codon of the poxA gene. The poxN promoter, however, was inactive in the heterologous host as no significant GUS activity was evident. On the other hand, chimeric uidA genes containing 2.2 kb of the poxA promoter or 1.4 kb of poxN promoter were active in transgenic rice plants. Both peroxidase promoters directed GUS activities in a spatial and tissue specific manner coincident with the expression patterns exhibited by their mRNAs. Histochemical analysis of transgenic rice plants showed that both peroxidase genes are expressed in the vascular bundles of the shoot apex and lamina joint, and in xylem-parenchyma cells of the leaf blade and sheath.

Journal Article↗

PubMed2000

The synergistic effects of hyperthermia and anticancer drugs on induction of apoptosis.

We studied the synergistic effects of hyperthermia and anticancer drugs on induction of apoptosis in lung cancer cells (LK-2 and LU-65A) using in situ end-labeling of DNA, the DNA fragmentation assay, and transmission electron microscopy. A few apoptotic cells were detected only when both cell lines were heated at relatively high temperature (44 degrees C). Moderate numbers of apoptotic cells were observed when both cell lines were incubated with high concentrations (30 or 40 microM) of anticancer drug. Compared with hyperthermia or anticancer drug alone, the combined treatment induced many apoptotic cells in both cell lines, even in the cells treated with lower concentrations (6 or 8 microM) of anticancer drugs following mild hyperthermia (43 degrees C). In regard to kinetics of apoptotic cells induced by treatment, the maximum induction of apoptosis by the combined treatment was higher than that of hyperthermia or anticancer drug alone in both cell lines, although the time of the peak of apoptotic index differed among the three treatments. Therefore, "hyperthermo-chemotherapy" may reduce the required dosage of anticancer drug and decrease the temperature of hyperthermia on induction of apoptosis.

Antineoplastic Agents↗

PubMed2000

The relationship between apoptosis and splenocyte depletion in rats following ethanol treatment.

Splenocyte depletion observed in chronic ethanol-treated rats (ETRs) was studied in relation to apoptosis. The rats were fed with ethanol in a Liber-DeCarli liquid diet (36% of total calories as ethanol) for 7 weeks. Spleens of ETRs and control rats were examined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, immunohistochemistry using anti-rat p53 and RM4 (specific for macrophages) monoclonal antibodies, and transmission electron microscopy (TEM). The splenic white pulp in ETRs decreased in size and showed a moth-eaten appearance because of the severe depletion of splenocytes. Most TUNEL-positive cells aggregated into clusters or nests and were not isolated in the white pulp of ETRs. The site of RM4 immunoreactivity was consistent with that of clusters of TUNEL-positive cells. The p53 immunoreactivity was observed in apoptotic splenocytes that were isolated or phagocytosed by macrophages. TEM study revealed the increase in tingible body macrophages phagocytosing apoptotic splenocytes in their cytoplasm in ETRs. Chronic ethanol intake certainly induces apoptosis in splenic white pulps, and tingible body macrophages act as both sentinels and scavengers of apoptotic splenocytes expressing p53.

Animals↗

PubMed2000

Expressions of Fas ligand and other apoptosis-related genes and their prognostic significance in epithelial ovarian neoplasms.

Expression of apoptosis-related proteins, bcl-2, Bax, Fas and Fas ligand (L), in ovarian epithelial neoplasms together with its clinical relevance was examined by immunohistochemistry. They included 36 cases with adenoma, 33 with low potential malignancy (LPM) and 63 with carcinomas. bcl-2 expression was observed in 14 of 36 cases (39%) with adenoma, five of 33 (15%) with LPM (P< 0.05) and 12 of 63 (19%) with carcinoma (P < 0.05). Cases with bcl-2 expression showed more favourable prognosis than those without, but the difference was not statistically significant. There was no difference in frequency of Bax and Fas expression between each histologic category. Fas L expression was observed in one of 36 cases (3%) with adenoma, but in 12 of 33 (36%) with LPM (P < 0.001) and 42 of 63 (67%) with carcinoma (P < 0.0001). In carcinomas, cases expressing Fas L showed a less favourable prognosis than those without (P = 0.02). Density of CD8+ lymphocytes, possibly cytotoxic T-cells, was higher in serous carcinoma with negative Fas L expression than those with positive Fas L expression. These findings suggest that Fas L expressing carcinomas induce apoptosis in infiltrating CTL with Fas expression, and escape from immune surveillance.

Adenoma↗

PubMed2000

[Preoperative bronchial arterial infusion of docetaxel for squamous cell carcinoma of the lung--clinical evaluation and correlation between anti-tumor effect and apoptosis].

As an induction therapy for advanced lung cancer, a patient received bronchial arterial infusion of 30 mg/m2 of docetaxel (TXT). Two weeks after infusion, although the tumor size was not reduced, the central necrotic area was significantly increased. There were no adverse effects. Nineteen days after infusion, we performed a right lower lobectomy and lymph nodal dissection. The resected specimen was examined by immunohistochemistry and biochemistry. The anti-tumor effects were evaluated clinically and the correlation between arterial TXT infusion therapy and apoptosis was studied. Hematoxylin and eosin staining demonstrated moderately differentiated squamous cell carcinoma associated with central necrosis, and cells with chromatin condensation scattered in both the necrotic areas and the margin of this area. It is thought that these findings were the result of this therapy, rather than just the self-necrosis seen in an untreated group.

Aged↗

PubMed1999

alphaPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase.

p21-activated kinase (PAK) is a common effector protein of the small GTPases Cdc42 and Rac, leading to the activation of downstream mitogen activated protein kinases. PAK also mediates polarized cytoskeletal changes induced by these GTPases. The recently identified PAK-interacting exchange factor (PIX) acts as a guanine nucleotide exchange factor on Rac, and colocalizes with PAK in a focal complex, but little is known about the associated signaling cascades, including upstream activators of PIX. In this study, we show that one of the isoforms of PIX, alphaPIX, is activated by signaling cascades from the platelet-derived growth factor (PDGF) receptor and EphB2 receptor, and from integrin-induced signaling through phosphatidylinositol 3-kinase (PI3-kinase). alphaPIX is activated by forming a complex with these receptors either via association with PAK and Nck, or direct association with the p85 regulatory subunit of PI3-kinase. Synthetic phosphoinositide and membrane targeted PI3-kinase augmented the alphaPIX activity in vivo. In Xenopus, aggregates of mesodermal cells derived from embryos microinjected with alphaPIX significantly increased the peripheral spreading on fibronectin substrate in response to PDGF through PI3-kinase. These results indicate that alphaPIX is activated by PI3-kinase, and is involved in the receptor mediated signaling leading to the activation of the kinase activity of PAK, and the migration of mesodermal cells on extracellular matrix.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Fas ligand, Fas antigen and Bcl-2 expression in human endometrium during the menstrual cycle.

In this study, we investigate Fas ligand expression in the human endometrium during the menstrual cycle in relation to Fas antigen and Bcl-2 expression, using immunoelectron microscopy and Western blotting. Endometrial samples were obtained from 54 pre-menopausal non-pregnant women who underwent laparotomies for benign diseases. The Fas ligand, as well as the Fas antigen, were expressed on the surface of endometrial glandular cells throughout the menstrual cycle, whereas Bcl-2 showed a cyclic expression pattern, peaking during the late proliferative phase. A noteworthy finding was that both the Fas ligand and the Fas antigen were localized on Golgi apparatuses and vesicles, in addition to the cell membranes, during the late proliferative phase. These results indicate that the Fas ligand and Fas antigen which are localized on Golgi apparatus and vesicles during the late proliferative phase are incorporated into the cell membranes during the secretory phase, and are co-expressed on the cell membranes of endometrial glands throughout the menstrual cycle. The factors regulating Fas-mediated apoptosis in the human endometrium, including the level of expression of the Fas ligand and Bcl-2 are discussed.

Adult↗

PubMed1998

Apoptosis and tumor angiogenesis in cervical cancer after preoperative chemotherapy.

The correlation between apoptosis and tumor angiogenesis in uterine cervical cancer treated by preoperative intraarterial infusion chemotherapy (IAC) was investigated. Cervical cancer samples surgically obtained from 12 patients (stages Ib-IIIb) receiving IAC and from 10 patients (stages Ib-IIb) receiving no chemotherapy and biopsy specimens from the 12 patients before IAC were examined. The apoptotic index (AI) was determined with an in situ end-labeling assay. Intratumoral microvessel density (IMVD) and thymidine phosphorylase (dThdPase) expression were evaluated immunohistochemically using anti-CD34 and anti-dThdPase antibodies. AIs were higher in the 8 patients with complete or partial response to IAC than they were in the 4 nonchemoresponsive patients and in the 10 patients who received no chemotherapy (P < 0.01) and were inversely related to IMVDs (r = 0.724; P < 0.01). AIs and IMVDs after IAC were higher and lower than those before IAC (P < 0.01), respectively. The expression of dThdPase, which has angiogenic activity, was markedly decreased after IAC. These results suggest that the antitumor effects of IAC are closely associated with apoptotic cell death, which may be influenced in part by the extent of tumor angiogenesis inhibition.

Adult↗