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Tumors formed by a highly metastatic human lung cancer cell line are characterized by activated signaling via vascular endothelial growth factor (VEGF)-C through its receptor (VEGFR-3) and aggressive lymph node metastasis. In this study, we examined how these highly metastatic cancers acquired aggressive lymph node metastasis. Compared with their lower metastatic counterparts, the highly metastatic tumors formed by this cell line expressed higher amounts of interleukin (IL)-1α, with similarly augmented expression of IL-1α and IL-1β by tumor stromal cells and of VEGF-A and VEGF-C by tumor-associated macrophages. These tumor-associated macrophages were mainly of the M2 type. Administration of a macrophage-targeting drug suppressed the production of these potent angiogenic and lymphangiogenic factors, resulting in decreased tumor growth, angiogenesis, lymphangiogenesis, and lymph node metastasis. In Matrigel plug assays, the highly metastatic cells formed tumors that were extensively infiltrated by M2-type macrophages and exhibited enhanced angiogenesis and lymphangiogenesis. All of these responses were suppressed by the IL-1 receptor (IL-1R) antagonist anakinra. Thus, the IL-1α-driven inflammatory activation of angiogenesis and lymphangiogenesis seems to provide a highly metastatic tumor microenvironment favorable for lymph node metastasis through cross-talk with macrophages. Accordingly, the IL-1R/M2-type macrophage axis may be a good therapeutic target for patients with this form of lung cancer.

V-ATPases are proton-translocating enzymes, which are found not only in numerous intracellular organelles but also in the plasma membranes of many eukaryotic cells. Using differential display, we have identified one of the proton pump subunit genes, ATP6C, as a cisplatin-inducible gene. Northern blot analysis demonstrated that expression of other members of the subunit is inducible by cisplatin treatment. Proton pump gene expression is also upregulated in 3 independent cisplatin-resistant cell lines but not in vincristine- or etoposide-resistant cell lines. Cellular pH was significantly higher in cisplatin-resistant cells than in sensitive parental cells. In vitro DNA-binding activity of cisplatin was markedly increased in acidic conditions, suggesting that the cytotoxicity of cisplatin is modulated by cellular pH. Furthermore, the proton pump inhibitor bafilomycin can synergistically potentiate the cytotoxicity of cisplatin but not of etoposide or camptothecin. These results indicate that cellular pH is one of the critical parameters for effective cancer chemotherapy with cisplatin.

A nonhistone chromosomal protein, high mobility group (HMG) 1, is ubiquitous in higher eukaryotic cells and binds preferentially to cisplatin-modified DNA. HMG1 also functions as a coactivator of p53, a tumor suppressor protein. We investigated physical interactions between HMG1 and p53 and the influence of p53 on the ability of HMG1 to recognize damaged DNA. Using immunochemical coprecipitation, we observed binding of HMG1 and p53. Interaction between HMG1 and p53 required the HMG A box of HMG1 and amino acids 363-376 of p53. Cisplatin-modified DNA binding by HMG1 was significantly enhanced by p53. An HMG1-specific antibody that recognized the A box of this protein also stimulated cisplatin-modified DNA binding. These data suggest that an interaction with either p53 or antibody may induce conformational change in the HMG1 A box that optimizes DNA binding by HMG1. Interaction of p53 with HMG1 after DNA damage may promote activation of specific HMG1 binding to damaged DNA in vivo and provide a molecular link between DNA damage and p53-mediated DNA repair.

STAT3 is involved in the signal transduction activated by various cytokines and growth factors. We found that the STAT3 gene is overexpressed in cisplatin-resistant cells. We isolated a genomic fragment containing the 5'-portion of the human STAT3 gene using a bubble PCR method. Using the bubble PCR product as a probe, one genomic clone was isolated. The nucleotide sequence of the first exon and the 1800 base pairs (bps) preceding it was determined. The promoter region of the human STAT3 gene is highly homologous to the corresponding region of the mouse STAT3 gene; several potential factor binding sites such as CRE/ATF, SBE, and GC boxes are also well conserved between human and mouse. A transient expression assay using the luciferase reporter gene showed that the sequence from -403 to +102 possesses maximal promoter activity, and transcription of the STAT3 gene was significantly higher in cisplatin-resistant cells than in parental cisplatin-sensitive cells. Deletion of the region between -261 and -167 resulted in significant loss of promoter activity in both parental and cisplatin-resistant cells. In vivo footprint analysis revealed several protein bindings; however, no significant differences were observed between drug-sensitive and drug-resistant cells. MNase digestion revealed that several open or active nucleosomes were only detected in cisplatin-resistant cells. These results suggest that STAT3 promoter function in a highly structured chromatin environment requires a complex interaction of several transcriptional factors.

In the past, as a posterior decompression surgery for OPLL, Iaminectomy was perfomed. Howerver, postoperative follow-up was made difficult by such negative factors as instability of the operated part, abnormal alignment, scar formation, etc. Therefore, to counteract these negative results, a variety of Iaminoplasty are being tried in recent years. We have performed Iaminoplasty both by the Kirita and Hirabayashi methods at our hospital since 1983, and we hereby report our evaluation of the operative results and the postoperative complications.With the Hirabayashi method, we observed the limitation of the range of movement in the posterior flexion. We could observe no difference in the improvement rate of the clinical condition when using different operative techniques. Postoperatively, we had no case where OPLL's growth could be clearly observed.In case of the unilateral existence of ossifications with a high rate of stenosis, we thought it was necessary to practice sufficient caution at the time of operation in order to prevent complications.

Dilute hydrogen fluoride (DHF) is the final clean widely used after the re­moval of particles, metals, and organic contaminants from the Si surface for oxidation film removal conducted in atmospheric pressure at room tempera­ture. However, although there has been concern about readhesion of particles and metals because of the exposure of the Si surface, with the trend towards high purity hydrogen fluoride and hydrogen fluoride—ammonium fluoride wa­ter solution mixtures (sometimes called buffered hydrogen fluoride), contam­ination from chemical solutions has been controlled to the utmost. Also, it has been reported that by controlling the particle and Si zeta potentials by the addition of surfactants to buffered hydrogen fluoride and other solutions, particle adhesion to the Si surface can be prevented [1, 2], and, furthermore, the Si surface roughness can be controlled [3].

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.

Neovascularization is often required for rapid growth of solid tumors and also limits vascular metastasis of tumor cells. Neovascularization-targeting agents are a recent innovation that may be a novel means of anticancer therapy. These antiangiogenic drugs have been developed by targeting cell proliferation of vascular endothelial cells, basement-membrane-degrading enzymes, angiogenic factors/receptors, extracellular matrix, angiogenesis signaling, and cell-cell/cell-matrix interactions. In this report, we describe how tumor angiogenesis occurs and how antiangiogenic agents are developed.

Our previous studies imply that tumor necrosis factor-alpha (TNF-alpha) and epidermal growth factor (EGF) might share a common signal transduction pathway in human omental microvascular endothelial (HOME) cells. Exposure of cultured HOME cells to TNF-alpha for 10 min enhanced EGF receptor phosphorylation at a rate comparable to EGF. Apparent phosphorylation of tyrosine residues was observed in addition to serine/threonine of the EGF receptor by EGF, but only a slightly if any tyrosine phosphorylation by TNF-alpha. In vitro kinase activity of EGF receptor was also enhanced by TNF-alpha as well as by EGF. Furthermore, expression of the c-fos gene was enhanced in response to either EGF or TNF-alpha. Pretreatment of HOME cells with EGF for 12 h almost completely blocked the induction of the c-fos gene by EGF and partially blocked the c-fos induction by TNF-alpha. TNF-alpha-induced c-fos gene expression appeared to be partly due to its transactivation of EGF receptor. EGF and TNF-alpha could enhance c-fos gene expression when protein kinase C was down-regulated by phorbol ester myristate (PMA). Gel retardation assay with the NF-kappa B consensus sequence showed that NF-kappa B binding activity was dramatically activated by TNF-alpha, but not by EGF or PMA. The binding of another transcription factor, AP-1 (Jun/Fos), was enhanced by EGF, TNF-alpha, and PMA, whereas TNF-alpha could still activate AP-1 after longer exposure to EGF. TNF-alpha-induced activation of c-fos gene appears to be mediated through pleiotropic mechanisms and partly through a common signal with EGF, possibly through EGF receptor in microvascular endothelial cells.

Y-box binding protein-1 (YB-1) plays an important role in tumor progression and drug resistance. This study examined whether YB-1 is involved in the alteration of response to endocrine therapy in ER-positive breast cancer cells. MCF7 cells that stably expressed YB-1 (MCF7-YB-1) and vector control cells (MCF7-vector) were established. These cells were used to analyze the expression of the factors related to the ER and growth factor receptor signaling pathways, response to the antiestrogens (tamoxifen and fulvestrant), and estrogen responsive element (ERE) activity. The effect of knocking down endogenous YB-1 expression was tested in wild-type MCF7 cells. In addition, the expression of the YB-1 and the factors related to the ER and growth factor receptor signaling pathways were evaluated in the clinical breast cancers treated with preoperative chemotherapy. The expression of HER2, AIB1, p-Erk and c-Myc were increased in the MCF7-YB-1 cells. In contrast, knocking down of YB-1 decreased the expression of these factors, but increased the expression of ERα in the wild-type MCF7 cells. Furthermore, sensitivity to antiestrogens was decreased in the MCF7-YB-1 in comparison to those in the MCF7-vector cells. In the MCF7-YB-1 cells, the expression levels of p-Erk and c-Myc were continuously upregulated when the cells were treated either with tamoxifen or fulvestrant. The ERE activity was decreased in the MCF7-YB-1 cells in comparison to the MCF7-vector cells, and the ERE activity of the MCF7-YB-1 cells was inhibited by fulvestrant at a lower concentration than that which inhibited the ERE activity of the MCF7-vector cells. In the ER-positive clinical breast cancers treated with preoperative chemotherapy, significantly more of the specimens that showed increased or positive nuclear YB-1 expression after the chemotherapy were positive for HER2 expression. These data suggest that alteration of YB-1 may modify the crosstalk between the ER pathway and HER2 pathway in ER-positive breast cancer cells, and consequently may alter the response to endocrine therapy in these cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-15.

Tumor necrosis factor-alpha (TNF-alpha) produced morphological changes from a cobblestone-like shape into a spindle shape in human omental microvascular endothelial (HOME) cells and also a drastic rearrangement of actin filaments. Expression of beta-actin gene was diminished in HOME cells treated with TNF-alpha for 24 h. Northern blot analysis of the beta-actin gene demonstrated that the cellular level of beta-actin mRNA was decreased at 6-12 h after exposure to TNF-alpha. However, there appeared to be no changes in cellular mRNA levels of beta-tubulin, fibronectin, laminin B1, laminin B2, and laminin binding protein genes after treatment with TNF-alpha. Nuclear run-on assays showed increased transcription of the low-density lipoprotein receptor gene, but not of the beta-actin gene. These data suggested that the TNF-alpha-induced inhibition of beta-actin gene expression was not due to altered transcription activity. The degradation rates of beta-actin, plasminogen activator inhibitor-1, and epidermal growth factor receptor mRNAs were examined in the presence of actinomycin D. beta-Actin mRNA was found to be specifically destabilized in TNF-alpha-treated HOME cells, while other mRNA species were not. Coadministration of cycloheximide blocked the TNF-alpha-induced degradation of beta-actin mRNA. The TNF-alpha-induced destabilization of beta-actin mRNA and rearrangement of actin filaments are discussed in relation to the morphological changes in human microvascular endothelial cells.

Purpose: The Y-box binding protein 1 (YB-1) regulates expression of P-glycoprotein (P-gp) encoded by the MDR1 gene. There have been no previous studies regarding the involvement of YB-1 in the development of resistance to paclitaxel. The present study was performed to examine how paclitaxel affects the localization and expression of YB-1 in breast cancer.Experimental Design: We evaluated the expression and localization of YB-1 and P-gp in breast cancer tissues obtained from 27 patients before and after treatment with paclitaxel. The effect of paclitaxel on localization of cellular YB-1 was examined by using GFP-YB-1. Interaction of YB-1 with the Y-box motif of the MDR1 promoters was studied by EMSA. The effects of paclitaxel on MDR1 promoter activity were examined by luciferase assay.Results: Of 27 breast cancer tissues treated with paclitaxel, nine (33%) showed translocation of YB-1 from the cytoplasm to the nucleus together with increased expression of P-gp during the course of treatment. Twelve breast cancer tissues (44%) showed neither translocation of YB-1 nor increased expression of P-gp. Nuclear translocation of YB-1 was correlated significantly with increased expression of P-gp (P=0.0037). Confocal analysis indicated that paclitaxel induced nuclear translocation of green fluorescent fused YB-1 in MCF7 cells. Furthermore, binding of YB-1 to the Y-box of MDR1 promoter was increased in response to treatment with paclitaxel. In addition, MDR1 promoter activity was significantly up-regulated by paclitaxel in MCF7 cells (p< 0.001).Conclusions: The results of the present study suggested that YB-1 may be involved in the development of resistance to paclitaxel in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1141.

High mobility group box chromosomal protein 1 (HMGB1) was recently identified as a late proinflammatory mediator of endotoxin lethality, which amplifies and sustains the inflammatory processes via macrophage/monocyte activation by extracellular release of HMGB1. However, it was originally described as a nonhistone nuclear protein to bind and distort the structure of DNA and to regulate transcription through association with several transcription factors. Here we investigated the effect of intracellular HMGB1 on transcriptional gene regulation of an immediate early proinflammatory cytokine IL-1β in transient transfection studies using HMGB1 expression vector (pcDNA3HMGB1) and murine macrophage cells RAW264.7. When pcDNA3HMGB1 was introduced into RAW264.7 cells with pGL3HT reporter containing the -131 to +12 minimal IL-1β gene promoter, IL-1β promoter activity was significantly induced by expression of HMGB1. The gene promoter possesses two important transcription factor binding motifs, one for PU.1, a myeloid and B cell-specific transcription factor that belongs to the ETS family, and the other a binding site for NF-IL6. To verify the functional role of intracellular HMGB1 in transactivation of the IL-1β promoter, a PU.1 expression vector and/or pcDNA3HMGB1 were cotransfected into PU.1-deficient murine thymocytes EL4 cells along with pGL3HT reporter. As a result, HMGB1 synergized with PU.1 to transactivate the IL-1β promoter, but not HMGB1 alone. This argument was supported by our GST-pulldown data, which demonstrated direct physical interaction of HMGB1 with PU.1. In addition, deletion of the PU.1 winged helix-turn-helix DNA-binding domain significantly inhibited the association of PU.1 with HMGB1. To determine whether HMGB1 could affect PU.1 DNA-binding affinity, we performed electrophoretic mobility shift assay using a radiolabeled IL-1β -59 to +12 promoter element (DT), recombinant PU.1 (rPU.1) and GST-HMGB1. Two complexes with slower and faster mobilities were generated by the addition of HMGB1 to a mixture of rPU.1 and DT probe. The two complexes were abrogated by preincubation with anti-PU.1 Ab, while anti-HMGB1 Ab reacted only with the complex with a slower mobility, indicating that the complex with a slower mobility formed by addition of HMGB1 contained both HMGB1 and PU.1, while the band with a faster mobility contained only PU.1. From the present study, we propose that intracellular HMGB1 might function as a coactivator in PU.1-mediated transcriptional activation, which facilitate access of PU.1 to specific DNA targets. The fact that PU.1 is a transcription factor essential to macrophages/monocyte-specific proinflammatory cytokine genes further raise the possibility that interaction of PU.1 with HMGB1 may play an important role in the inflammation cascade in macrophages/monocytes.

Allograft was used on a patient who was diagnosed as rheumatoid arthritis with severe acetabular protrusion. The acetabular deficiency was reconstructed with the allograft in bipolar hip arthroplasty. We employed a freezed femoral head as the allograft.Roentgenographically, bone trabeculae were passing the graft-acetabular junction in medio-inferior region.Bone scans showed increased activity in the allograft near the host-donor junction at an early stage and maintain high activity up to date.Partial incorporation and high radioactivity suggest that this allograft will be incorporated completely at the early date.

Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase (MAP3K), is ubiquitously expressed and situated in an important upstream position of many signal transduction pathways. ASK1 plays a pivotal role in stressor-induced cell survival and inflammatory reactions. To ascertain the regulatory functions of ASK1 in bile duct ligation (BDL)–induced liver injury, we examined the net effects of ASK1 depletion on hepatic necroinflammation and/or fibrosis. We subjected C57BL/6 wild-type (WT) or ASK1 -deficient ( ASK1 −/− ) mice to sham or BDL surgery for 14 days. In day 3 BDL animals, ASK1 −/− mice had significantly fewer bile infarcts along with more reduced interlobular or portal inflammatory infiltrate of various immune cells, including neutrophils, compared with WT mice in which ASK1 expression was markedly activated. Morphologically apoptotic hepatocytes or cholangiocytes were negligible in both the sham and BDL animals. In contrast, ASK1 −/− mice had significantly less proliferating activity of not only hepatocytes but also large cholangiocytes than WT mice. Day 14 BDL ASK1 −/− mice manifested potential antifibrogenic aspects of ASK1 deficiency, characterized by significantly fewer activated peribiliary fibrogenic cells and peribiliary fibrosis. These observations indicate that ASK1-mediated hepatic necroinflammation and proliferation, but not apoptosis, are closely linked to liver fibrosis and fibrogenesis. A specific ASK1 pathway blocker or inhibitor might offer a therapeutic strategy against human cholestatic diseases.

Background WNT/β-catenin signaling plays an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF); however, the role of WNT10A via transforming growth factor (TGF)-β signaling remains unclear. Methods We evaluated the expression of WNT10A and TGF-β in bleomycin (BLM)-treated mice and the interactions between TGF-β or BLM and WNT10A in vitro. Additionally, we investigated IPF patients who underwent video-assisted thoracoscopic surgery to determine whether the WNT10A expression is related to the survival. Results Increased WNT10A and TGF-β expressions were noted in the BLM-treated mice. Real-time PCR and luciferase reporter assays demonstrated the levels of WNT10A and collagen in the fibroblasts cells to increase after TGF-β administration. Conversely, WNT10A siRNA treatment inhibited the synthesis of collagen in the transfected fibroblasts cells. A Kaplan-Meier survival analysis demonstrated a tendency toward a poor survival among the IPF patients with a WNT10A-positive expression compared to those with a negative expression (Hazard ratio 5.351, 95 % CI 1.703-16.82; p = 0.0041). An overexpression of WNT10A was found to be significantly predictive of an acute exacerbation of IPF (AE-IPF) (Odds ratio 13.69, 95 % CI 1.728-108.5; p = 0.013). Conclusions WNT10A plays an important role in the pathogenesis of IPF via TGF-β activation and it may also be a sensitive predictor for the onset of an AE-IPF.

Several p73 variants have been reported with different carboxy-terminal structures and transcriptional activities. We showed that p73gamma had stronger transactivation activity than the other splicing variants such as alpha, beta and delta by analysing p21 promoter activity in human prostate cancer PC3 cells. The transactivation activity of p73gamma was similar to that of p53 and was enhanced by co-transfection with p300/CBP-associated factor (PCAF). In vitro pull-down assay, p73 variants were able to bind to PCAF with a similar extent. However, in vivo co-immunoprecipitation assays showed that p73gamma interacted preferentially with PCAF. Neither in vitro-translated nor in vivo-immunoprecipitated p73gamma were able to bind to oligonucleotides containing the p53 consensus binding site. However, p73gamma acetylated by PCAF restored DNA binding activity. Differential functions of p73 variants are supposed to be regulated by the structural differences of carboxy-terminal region. Our results revealed that p21 promoter activity was affected by differential interactions of p73 variants with PCAF and its acetylation.