Biomedical subjects

Hajime Shimizu

Publications and source records attributed to Hajime Shimizu.

12 recordsLinked to original sources

PubMed2006

Functional implications of the IL-6 signaling pathway in keloid pathogenesis.

The molecular mechanism(s) behind keloid pathogenesis remains unclear. Previously by global gene expression analysis of keloid fibroblasts (KFs), we implicated the IL-6 signaling pathway in keloid pathogenesis. Here, we determine a functional role of IL-6 signaling in keloid scars. Primary cultures of KFs and surrounding nonlesional fibroblasts (NFs) were subjected to induction or inhibition of IL-6 or its specific receptor IL-6 receptor alpha (IL-6R alpha) and detection of their effects on extracellular matrix gene expression. The levels of gp130 and several downstream targets in IL-6 signaling were also examined. IL-6 secretion was significantly higher in KFs than NFs. Addition of IL-6 peptide to NFs culture or inhibition of IL-6 or its receptor IL-6R alpha by their corresponding antibodies in KFs culture revealed a dose-dependent increase or decrease in collagen type I alpha 2 and fibronectin 1 mRNAs, respectively. Induction of IL-6 by IL-1beta peptide and stimulation by IL-6 peptide in NFs, or inhibition of IL-6 or IL-6R alpha in KFs cultures demonstrated a dose-dependent increase or decrease in procollagen I synthesis, respectively. The mRNA and protein expressions of gp130 and several downstream targets in IL-6 signaling (JAK1, STAT3, RAF1, and ELK1) were upregulated in KFs versus NFs. Our results indicate that IL-6 signaling may play an integral role in keloid pathogenesis and provide clues for development of IL-6 receptor blocking strategies for therapy or prophylaxis of keloid scars.

Adult↗

PubMed2005

Global gene expression analysis of keloid fibroblasts in response to electron beam irradiation reveals the involvement of interleukin-6 pathway.

Keloid is a dermal fibroproliferative lesion of unknown etiology that commonly recurs after surgical excision. Post-operative adjuvant electron beam (EB) irradiation has been successfully used to reduce keloid recurrences. To provide new insights into the molecular mechanism behind the effect of EB irradiation, we used a cDNA microarray screening of more than 5000 genes to assess early changes in gene expression between EB-irradiated and non-irradiated keloid and non-lesional dermal fibroblasts. Primary fibroblast cultures from keloid and associated non-lesional dermis obtained from five patients were exposed to 15 Gy EB irradiation and analyzed after 15 min incubation. Early response to EB irradiation showed that 96 (1.8%) genes were modulated 2-fold or more in keloid fibroblasts. Upregulated genes accounted for 29.2% (28 genes), whereas downregulated genes comprised 70.8% (68 genes), indicating a silencing of many genes in keloid fibroblasts after EB irradiation. Many of the downregulated genes play roles in the enhancement of cell proliferation and extracellular matrix production, whereas several of the upregulated genes involves in the promotion of apoptosis and extracellular matrix (ECM) degradation. Using emerging bioinformatic tools and further corroboration, the interleukin 6 (IL-6) signaling pathway was found to be mainly involved in EB irradiation response. We also showed co-expression of IL-6 and its specific receptor (IL-6Ralpha) in keloid fibroblasts that points to the existence of an IL-6 autocrine loop in these cells. These results suggested that at the molecular level, EB irradiation might hinder keloid formation by regularizing disturbances in the homeostatic equilibrium between inducer and inhibitor activities in the matrix system most likely through the IL-6 pathway. Our study provides clues for the molecular mechanism(s) behind the beneficial effect of EB irradiation in reducing keloid recurrences and may help develop alternative strategies for the therapy and prophylaxis of this lesion.

Adult↗

PubMed2004

Comparative study between DNA copy number aberrations determined by quantitative microsatellite analysis and clinical outcome in patients with stomach cancer.

PURPOSE: We detected the relative DNA copy numbers (RCNs) at target loci in patients with stomach cancer with quantitative microsatellite analysis. We additionally clarified the relationship between DNA copy number aberrations and the clinical outcome of the patients. EXPERIMENTAL DESIGN: Fresh frozen samples were obtained from 30 patients who had undergone surgery for stomach cancer. Seven microsatellite loci in chromosomes 8q, 16q, and 20q and one gene-specific locus (ZNF217) were selected as the target loci. The DNA copy number was obtained relatively to a pooled reference consisting of six microsatellite primer sets selected from the regions where few aberrations have been reported in comparative genomic hybridization analysis. On the basis of the TaqMan PCR system, the internal probes used were carrying donor (6-carboxyfluorescein) and acceptor (6-carboxytetramethylrhodamine) fluorescent molecules complementary to CA repeats in the microsatellite markers and to one gene-specific oligomer in the gene-specific marker. RESULTS: Chromosome 8q gain, 20q gain, and 16q loss were detected in 18 (60.0%), 8 (26.7%), and 13 (43.3%) cases, respectively. Gains in the RCNs of D8S1801 and D8S1724 were most frequently found (36.7%). There was a significant correlation between the loss of D16S3026 and reduced survival duration (P = 0.0158), and the simultaneous aberrations of D8S1801 gain and D16S3026 loss (double marker positive) was significantly associated with reduced survival duration (P = 0.0008). According to Cox proportional hazards model, the double marker positive was a significant and independent factor indicating an unfavorable prognostic factor (relative risk, 17.176; 95% confidence interval, 2.782-106.026; P = 0.0022). CONCLUSION: RCN aberrations in tumor tissues determined by quantitative microsatellite analysis enable identification of the prognostic factors that correlate with clinical outcome of the patients with stomach cancer.

Aged↗

PubMed2004

Effect of ionizing irradiation on human esophageal cancer cell lines by cDNA microarray gene expression analysis.

To provide new insights into the molecular mechanisms underlying the effect of irradiation on esophageal squamous cell carcinomas (ESCCs), we used a cDNA microarray screening of more than 4,000 genes with known functions to identify genes involved in the early response to ionizing irradiation. Two human ESCC cell lines, one each of well (TE-1) and poorly (TE-2) differentiated phenotypes were screened. Subconfluent cells of each phenotype were treated with single doses of 2.0 Gy or 8.0 Gy irradiations. After a 15 min incubation time-point, the cells were collected and analyzed. Compared with non-irradiated cells, many genes revealed at least 2-fold upregulation or downregulation at both doses in well or poorly differentiated ESCC cells. The common upregulated genes in well and poorly differentiated cell types at both irradiation doses included SCYA5, CYP51, SMARCD2, COX6C, MAPK8, FOS, UBE2M, RPL6, PDGFRL, TRAF2, TNFAIP6, ITGB4, GSTM3, and SP3 and common downregulated genes involved NFIL3, SMARCA2, CAPZA1, MetAP2, CITED2, DAP3, MGAT2, ATRX, CIAO1, and STAT6. Several of these genes were novel and not previously known to be associated with irradiation. Functional annotations of the modulated genes suggested that at the molecular level, irradiation appears to induce a regularizing balance in ESCC cell function. The genes modulated in the early response to irradiation may be useful in our understanding of the molecular basis of radiotherapy and in developing strategies to augment its effect or establish novel less hazardous alternative adjuvant therapies.

Carcinoma, Squamous Cell↗

PubMed2004

Pladienolides, new substances from culture of Streptomyces platensis Mer-11107. III. In vitro and in vivo antitumor activities.

We have discovered seven novel 12-membered macrolides, pladienolides A to G, from Streptomyces platensis Mer-11107, with pladienolide B the most potently inhibiting hypoxia induced-VEGF expression and proliferation of the U251 cancer cell line. A growth inhibitory study using a 39-cell line drug-screening panel demonstrated that pladienolide B has strong antitumor activities in vitro. A COMPARE analysis reveals that it has a unique antitumor spectrum that sets it apart from anticancer drugs currently in clinical use. This result suggests that pladienolide B has a novel mechanism of action. A series of xenograft studies were conducted to evaluate the in vivo potency of pladienolides. Pladienolide B extensively inhibited tumor growth in xenograft models. In the most sensitive model, using BSY-1 xenografts, tumors were completely regressed by administration of pladienolide B. For the reason of their novel mechanism of action and excellent in vivo efficacy, pladienolides appear to have major potential for use in cancer treatment.

Animals↗

PubMed2003

Alpha6beta1 integrin induces proteasome-mediated cleavage of erbB2 in breast cancer cells.

ErbB2 and alpha6 integrin have been implicated in malignancy of breast cancer cells. Here we have determined the influence of alpha6beta1 integrin on erbB2 signaling in anchorage-independent growth, using MDA-MB435 breast cancer cells. Firstly, we transfected the cells with erbB2 cDNA, and isolated cells with high or low levels of alpha6beta1 integrin by cell sorting (alpha6H-ErbB and alpha6L-ErbB). We found that an erbB ligand, heregulin beta1, enhanced growth activity of alpha6L-ErbB cells, but not alpha6H-ErbB cells. Secondly, we established cells expressing a beta4 integrin deletion mutant (beta4-deltacyt), which selectively inhibited alpha6beta1 integrin expression and adhesion to laminin-1. Again, heregulin beta1 enhanced the growth of erbB2 cDNA-transfected beta4-deltacyt cells, but not mock cells. Western blot analysis revealed that heregulin beta1 stimulated phosphorylation of Akt and its downstream molecules, GSK3beta and p70S6kinase, and that the extent of phosphorylation was greater in ErbB2/beta4-deltacyt cells than ErbB2/mock cells. Furthermore, we found that the erbB2 cytoplasmic domain was truncated in ErbB2/mock cells, which was independent of ligand stimulation and adhesion, and was suppressed by proteasome inhibitors. These results suggest that alpha6beta1 integrin inhibits erbB2 signals by inducing proteasome-dependent proteolytic cleavage of the erbB2 cytoplasmic domain, and may thereby contribute to the regulation of tumor growth.

Breast Neoplasms↗

PubMed2003

Construction of a gamete-enriched gene pool and RNAi-mediated functional analysis in Dictyostelium discoideum.

Macrocysts in Dictyostelium discoideum possess prototypic features of sexual reproduction and are useful for understanding the basic mechanisms of the reproductive process. Here, we randomly analyzed 1,071 gamete cDNAs, and then constructed a gamete-specific subtraction library, FC-IC. Nucleotide sequences of all 903 FC-IC clones were determined and clustered into 272 independent genes. Expression analysis based on real-time RT-PCR revealed 67 gamete-enriched genes, among which those involved in 'signal transduction' and 'multicellular organization' are prevalent. One of them, FC-IC0003, appeared also to be mating-type specific, and was named gmsA. RNAi-mediated silencing as well as disruption of gmsA reduced the cellular competency for sexual cell fusion, indicating the involvement of this gene in the sexual development of D. discoideum.

Amino Acid Sequence↗

PubMed2002

Molecular cytogenetic characterization of drug-resistant leukemia cell lines by comparative genomic hybridization and fluorescence in situ hybridization.

Resistance to chemotherapeutic drugs is one of the major difficulties encountered during cancer chemotherapy. To detect genomic aberrations underlying the acquired drug resistance, we examined three cultured human myelomonocytic leukemia cell sublines each resistant to adriamycin (ADR), 1-beta-1-D-arabinofuranosylcytosine (ara-C), or vincristine (VCR), using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), RT-PCR, and western blot techniques. Chromosomes 7, 10 and 16 most conspicuously showed frequent aberrations among the resistant sublines as compared to the parental KY-821 cell line. In ADR-resistant cells, gains at 7q21, 16p12, 16p13.1-13.3, 16q11.1-q12.1, and losses at 7p22-pter, 7q36-qter, 10p12, 10p11.2-pter, 10q21-q25, 10q26-qter were notable. In ara-C-resistant cells, no remarkable gain or loss on chromosome 7, but losses at 10p14-pter, 10q26-qter and 16p11.2-p11.3 were observed. In VCR-resistant cells, gain at 7q21 and losses at 10p11-p13, 10p15 and 16p11.2-p13.3 were found. FISH identified amplified signals for the MDR-1 gene located at 7q21.1 in ADR- and VCR- but not ara-C-resistant cells, and for the MRP-1 gene located at 16p13.1 in ADR-resistant cells. These findings were validated at the mRNA and protein levels. Overlapping of the amplified MRP-1 gene with MDR-1 gene may play a critical part in the acquisition of resistance to ADR. Resistance to ara-C excluded MDR-1 gene involvement and highlighted other key genes such as MXR gene. Several other genes putatively involved in the development of drug resistance might lie in other aberrated chromosomal regions.

Antimetabolites, Antineoplastic↗

PubMed2002

Genome-wide screening of laser capture microdissected gastric signet-ring cell carcinomas.

Gastric signet-ring cell carcinoma comprises a distinct category of gastric cancers and has been reported to have poor prognosis. In an attempt to define genetic changes involved in the pathogenesis of this lesion in an in vivo state, we isolated signet-ring cell carcinoma cells from freshly fixed smears of tumor tissues of 7 primary gastric signet-ring cell carcinomas by laser capture microdissection and applied comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes. Frequent chromosomal gains were detected on 2q, 5p, 7q, 14q and 20q, each in 6/7 cases, on 9q, 12q, 17q, and 19q, each in 5/7 cases, and on 18p in 4/7 cases. Frequent losses were observed on 6p and 17p, each in 5/7 cases, on 6q, and 21p, each in 4/7 cases, and on 3p, 8p and 8q, each in 3/7 cases. Losses on 6p have rarely been observed in conventional types of gastric carcinomas reported in the literature. These data provide the first evidence for the occurrence of specific genomic aberrations in gastric signet-ring cell carcinomas. Our observation of frequent losses on 6p chromosomal arm may provide novel abnormalities of potential significance in gastric signet-ring cell carcinomas, suggesting the involvement of genes residing in this region in the genesis of the disease.

Adenocarcinoma↗

PubMed2002

Comparative genomic hybridization study of genetic changes associated with vindesine resistance in esophageal carcinoma.

The acquisition of drug-resistance is a major problem for cancer patients undergoing chemotherapy. To clarify genetic alterations in cancer cells that develop drug-resistance, comparative genomic hybridization (CGH) was applied to esophageal squamous cell carcinoma cell lines (SH-1V1, SH-1V2, SH-1V4 and SH-1V8) and chemoresistance-related genes in altered chromosomal regions were evaluated. These cell lines were derived from the parental SH-1 cell line, after multiple steps of selection by an increasing exposure to vindesine. SH-1V8 cells were strongly resistant to vindesine. DNA copy number at 16p which includes the MRP (multidrug resistance related protein) gene was markedly increased in all cell lines examined. Increased DNA copy numbers were found at the regions of 5q31-32, 10q11.1-23, and 14q32-qter in SH-1V8 cells that acquired resistance to other drugs as well. Both SH-1V4 and SH-1V8 showed increased DNA copy numbers at 7q11.1-22, 16q12.1-qter, 19p13.2-13.3, 19q11-13.2 and 20q13.1-qter. The chromosomal region of 7q11.1-22 including MDR-1 (multidrug resistance-1) gene was highly amplified in SH-1V4 and SH-1V8. Amplification of the MRP region suggests the prerequisite of developing resistance to vindesine, and further amplification of MDR-1 may play a critical role in acquiring drug-resistance. Several unknown genes related to the induction of chemoresistance might be concealed in other altered chromosomal regions.

Chromosome Aberrations↗

PubMed2002

Aberrant expression of integrin and erbB subunits in breast cancer cell lines.

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.

Breast Neoplasms↗

PubMedDate not supplied

Role of cdk4, p16INK4, and Rb expression in the prognosis of bronchioloalveolar carcinomas.

BACKGROUND: The p16(INK4) protein has been identified as a potent inhibitor of cyclin-dependent kinase (cdk)4 by blocking cdk4-mediated phosphorylation of the tumor suppressor retinoblastoma (Rb) protein, thus allowing Rb-mediated growth suppression. OBJECTIVES: Loss of p16(INK4) has been associated with a poor cancer prognosis, but its potential significance in bronchioloalveolar carcinomas (BACs) has not been explored. METHODS: We examined immunohistochemical expression of p16(INK4), cdk4, and Rb proteins in 38 BACs and correlated their expression levels with known clinicopathological features of the disease. RESULTS: All BACs expressed cdk4, while 89 and 82% expressed p16(INK4) and Rb proteins, respectively. None of the clinicopathological factors correlated with p16(INK4), cdk4, or Rb expression separately. A low p16(INK4)/cdk4 ratio was significantly associated with a high disease stage (p = 0.04), and the ratio tended to be lower in mucinous than nonmucinous tumors. BACs with a low p16(INK4)/cdk4 ratio showed significantly higher Rb expression levels (p = 0.02). Univariable survival analyses showed a significantly lower 5-year survival probability in patients with a high stage (p = 0.002) or low p16(INK4)/cdk4 ratio (p = 0.01). CONCLUSIONS: The results suggest a role of the cdk4/p16(INK4) pathway in the prognosis of BACs. Further studies are warranted to clarify whether a low p16(INK4)/cdk4 ratio may identify tumors that are destined to behave unfavorably.

Adenocarcinoma, Bronchiolo-Alveolar↗