miR-9-3p plays a tumour-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells.

BACKGROUND: The inactivation of the Hippo pathway lead to TAZ (PDZ-binding motif)/YAP (yes-associated protein) overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). Although there are several reports of microRNA (miR) targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC. METHODS: MicroRNA expression was analysed using the Human miFinder 384HC miScript miR PCR array, and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumour-suppressor miR targeting TAZ. We examined the functional role of miR-9-3p using miR-9-3p mimic and inhibitor in HCC cell lines). RESULTS: In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions. TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. Treatment of miR-9-3p mimic inhibited cell proliferative ability with downregulated phosphorylations of Erk1/2, AKT, and ß-catenin in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared with control in HuH1. CONCLUSIONS: MicroRNA-9-3p was identified as the tumour-suppressor miR targetting TAZ expression in HCC cells.

Interleukin-6 released by colon cancer-associated fibroblasts is critical for tumour angiogenesis: anti-interleukin-6 receptor antibody suppressed angiogenesis and inhibited tumour-stroma interaction.

BACKGROUND: Interleukin-6 (IL-6) has an important role in cancer progression, and high levels of plasma IL-6 are correlated with a poor prognosis in a variety of cancers. It has also been reported that tumour stromal fibroblasts are necessary for steps in cancer progression, such as angiogenesis. There have been few reports of a correlation between fibroblast actions and IL-6 levels. In this study, we examined the correlation between cancer stromal fibroblasts and IL-6 and the utility of IL-6 as a therapeutic target in human colon cancer. METHODS: The expression levels of IL-6 and VEGF of fibroblasts and cancer cell lines were evaluated using real-time PCR and ELISA. The anti-angiogenic effect of inhibiting IL-6 signalling was measured in an angiogenesis model and animal experiment. RESULTS: We demonstrate that stromal fibroblasts isolated from colon cancer produced significant amounts of IL-6 and that colon cancer cells enhanced IL-6 production by stromal fibroblasts. Moreover, IL-6 enhanced VEGF production by fibroblasts, thereby inducing angiogenesis. In vivo, anti-IL6 receptor antibody targeting stromal tissue showed greater anti-tumour activity than did anti-IL6 receptor antibody targeting xenografted cancer cells. CONCLUSION: Cancer stromal fibroblasts were an important source of IL-6 in colon cancer. IL-6 produced by activated fibroblasts induced tumour angiogenesis by stimulating adjacent stromal fibroblasts. The relationship between IL-6 and stromal fibroblasts offers new approaches to cancer therapy.

NOTCH1 expression predicts patient prognosis in esophageal squamous cell cancer.

PURPOSE: The prognosis of patients with esophageal cancer remains poor, and the classification of tumor node metastasis has proven insufficient to predict patient prognosis. Therefore, novel predictive markers of esophageal cancer prognosis are needed. Notch receptors and their ligands have been reported to be upregulated in cervical, lung, colon, renal, and pancreatic cancers, but NOTCH1 expression has not been studied in esophageal cancer. METHODS: Expression of NOTCH1 was quantified by real-time reverse transcription-polymerase chain reaction in 55 primary esophageal squamous cell carcinomas (ESCCs) and their paired normal esophageal mucosa. We then examined the correlations between NOTCH1 expression, clinicopathological factors, and prognosis in patients with ESCC. RESULTS: The probability of overall survival was significantly lower for patients with high NOTCH1 expression (p = 0.0028; log-rank test). Overexpression of NOTCH1 was identified as a significant and independent prognostic factor (p = 0.0061) in patients who had undergone surgical treatment for ESCCs. The hazard ratio for predicting early death was 4.298 (95% confidence interval 1.515-12.195) for high versus low NOTCH1 expression. CONCLUSIONS: Our data indicate that NOTCH1 may be a candidate molecular prognostic marker and a molecular target for the development of an effective therapeutic intervention for patients with ESCC.

Correlation between variable-number tandem-repeat-based genotypes and drug susceptibility in Mycobacterium avium isolates.

Little is known about the correlation between genotype and drug susceptibility in Mycobacterium avium (Mav) strains isolated from patients with Mav infections. To examine whether drug susceptibility profile of Mav is associated with genotype, we carried out variable-number tandem-repeat (VNTR) typing and drug susceptibility testing for Mav isolates from Japanese with nodular-bronchiectasis (NB)-type and cavitary disease (CA)-type diseases. We performed M. avium tandem repeat (MATR)-VNTR typing and drug susceptibility testing by the broth dilution method, using macrolides, rifamycins, ethambutol, isoniazid, aminoglycosides, and quinolones, for Mav isolates from patients with NB and CA-type diseases (NB-Mav and CA-Mav). Based on the VNTR genotyping, the Mav strains were grouped into three clusters. There was no difference with respect to the distribution of NB-Mav and CA-Mav among the clusters. We observed a strong association between VNTR genotype and susceptibility to quinolones (levofloxacin, moxifloxacin, gatifloxacin, sitafloxacin, and garenoxacin) and ethambutol. There was essentially no significant difference in drug susceptibility between NB- and CA-Mav strains, although NB-Mav was somewhat more resistant to fluoroquinolones, especially gatifloxacin, than CA-Mav. There was a significant association between VNTR genotype and susceptibility to quinolones and ethambutol in Mav isolates from Japanese patients.

Human melanoma antigen AH is an autoantigenic ganglioside related to GD2.

AH antigen, initially defined by an antibody present in a melanoma patient, is a cell surface antigen found on approximately 65% of melanoma cell lines. Absorption analysis indicates that AH is a differentiation antigen marking normal and malignant cells of neuroectodermal origin. The AH determinant has been found to be related to GD2 ganglioside.

Comparative study for the virulence of Mycobacterium avium isolates from patients with nodular-bronchiectasis- and cavitary-type diseases.

Mycobacterium avium (Mav) lung infections, called nodular-bronchiectasis (NB)-type M. avium complex (MAC) disease, are globally increasing. To elucidate whether there are unusual populations of Mav, causing NB-type disease rather than cavitary (CA)-type disease, we compared the virulence of Mav isolates from patients with NB-type (NB-Mav) and those from CA-type (CA-Mav) diseases, based on intracellular growth in various types of human cells. Five strains each of NB-Mav and CA-Mav were compared with each other for their invasiveness and ability to intracellularly replicate in various types of cultured cells of human origin. The two types of Mav isolates showed a similar ability, on average, to replicate in macrophages and lung epithelial cells. Moreover, they showed a similar ability to induce the production of reactive nitrogen intermediates and reactive oxygen intermediates by macrophages and susceptibility to antimicrobial molecules. Therefore, it appears that there is no essential difference in virulence in terms of infectivity to human macrophages and lung cells between Mav strains isolated from NB-MAC disease and those from CA-MAC disease. These findings indicate the importance of further studies to elucidate the mechanism for the establishment of NB-type MAC diseases based on host immunological conditions rather than the pathogenic nature of MAC organisms themselves.

Sentinel node biopsy for follicular tumours of the thyroid gland.

BACKGROUND: It is difficult to differentiate histologically between benign and malignant follicular-type tumours of the thyroid gland. The present study evaluated whether sentinel lymph node (SLN) biopsy was helpful in obtaining the correct diagnosis of malignant follicular-type tumours, as metastasis to the lymph nodes is sufficient evidence for a diagnosis of cancer. METHODS: SLN biopsy was performed for 37 follicular-type tumours suspected to be malignant on the basis of preoperative examinations, but for which the diagnosis had not been confirmed. RESULTS: SNs were identified in 32 of 37 procedures. SLN metastases were found in four of 12 patients with a malignant tumour identified by intraoperative frozen-section analysis. There was one false-negative but no false-positive diagnoses. Frozen-section analysis of the tumour itself identified only one follicular cancer. The accuracies of preoperative imaging methods were lower than that of SLN detection. CONCLUSION: Detection of SLN metastasis was helpful in diagnosing follicular thyroid cancer and thus enabling one-stage surgery.

Integrin-linked kinase activity is associated with interleukin-1 alpha-induced progressive behavior of pancreatic cancer and poor patient survival.

Cancer cell adhesion and invasion into extracellular matrix are regulated by integrin-linked kinase (ILK) activity in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. In this study, we demonstrated that ILK and beta(1)-integrin play important roles in interleukin (IL)-1alpha-induced enhancement of adhesion and invasion of pancreatic cancer cells through p38 mitogen-activated protein kinase (MAPK) signaling pathway and activator protein-1 (AP-1) activation. Alteration of ILK kinase activity controlled IL-1alpha-induced p38 MAPK phosphorylation and its downstream AP-1 activation with subsequent regulation of pancreatic cancer cell adhesion and invasion. Overexpressed ILK enhances the IL-1alpha-induced p38 MAPK phosphorylation more strongly through glycogen synthase kinase 3 (GSK-3) activation, and subsequently induces AP-1 activation, which promotes aggressive capabilities of pancreatic cancer cells. In contrast, knockdown of ILK kinase activity inhibits the IL-1alpha-induced activation of MAPK/AP-1 pathway via inhibition of GSK-3 phosphorylation. In immunohistochemical analysis, statistically significant association between strong expression of ILK and poor prognosis of pancreatic cancer patients were observed, and strong expression of ILK in cancerous tissues can be a significant prognostic indicator of pancreatic cancer patients. Our results suggest that ILK is involved with aggressive capability in pancreatic cancer and that these regulations can be helpful to understand biological processes for a better translational treatment for pancreatic cancer patients.

Significance of additional high-dose cytarabine in combination with cyclophosphamide plus total body irradiation regimen for allogeneic stem cell transplantation.

The combination of cyclophosphamide (CY) and total body irradiation (TBI) has been used as a standard conditioning regimen for allogeneic transplantation. Several studies showed an advantage of adding high-dose cytarabine (HDCA) to this regimen. To clarify the significance of additional HDCA, we conducted a retrospective multicenter study and compared the clinical results of these two regimens. From June 1985 to March 2003, 219 patients with hematological malignancies underwent allogeneic transplantation after conditioning with CY+TBI 12Gy (n=73) or CA+CY+TBI 12Gy (n=146). Engraftment, overall survival, transplant-related mortality (TRM), relapse rate and incidence of graft-versus-host disease (GVHD) were compared according to risks and donors. Addition of HDCA had no impact on the relapse rate in all subgroups, and it was associated with lower TRM among standard-risk patients after related transplantation, and with higher TRM and worse survival among standard-risk patients after unrelated transplantation. The incidence of acute GVHD was not significantly different between the two regimens, and HDCA resulted in a higher incidence of chronic GVHD among standard-risk patients after related transplantation. In summary, addition of HDCA is not beneficial for high-risk patients, and is not recommended for standard-risk patients receiving unrelated transplantation.

A new technique for intestinal isoperistaltic anastomosis utilizing a linear stapler for enlargement after anastomosis performed with a circular stapler.

BACKGROUND: The high incidence of anastomotic stenosis after gastrointestinal surgery using circular staplers is a major problem. In response, we have developed a new technique that uses a linear stapler to enlarge an anastomotic opening made using a circular stapler. METHODS: Anastomoses were created by the new technique or by the conventional approach using a circular stapler in pig small intestine. The method was also applied in treatment of a colon cancer patient. RESULTS: The area of the anastomotic opening obtained with the new technique was more than 3 times that in the control (p < 0.001), with no significant difference between the methods in a leak test. Follow-up of the patient undergoing surgery with this approach revealed an uneventful course with a widely patent anastomosis confirmed after 3 months. CONCLUSIONS: This procedure provides a larger anastomotic opening than conventional anastomosis with circular staplers, without impairing the integrity of the anastomosis.

Impairment of DYRK2 augments stem-like traits by promoting KLF4 expression in breast cancer.

Whereas accumulating studies have supported the cancer stem cell theory, a specific therapy targeting a cancer stem cell subpopulation has not been established. Here, we show that dual-specificity tyrosine phosphorylation-kinase 2 (DYRK2) is a novel negative regulator for formation of breast cancer stem cells. Downregulation of DYRK2 promotes cancer stem-like traits in vitro, tumourigenesis in vivo and the proportion of the cancer stem cell population in human breast cancer tissues. We found that Krupple-like factor 4 (KLF4) serves as a key mediator of DYRK2's control over the cancer stem phenotype. Reduced DYRK2 expression increases KLF4 expression, which induces cancer stem-like properties. We identified androgen receptor (AR) as a transcription factor binding to the KLF4 promoter region; this process is dependent on DYRK2 kinase activity. Our findings delineate a mechanism of cancer stem cell regulation by the DYRK2-AR-KLF4 axis in breast cancer. Targeting of this pathway may be a promising strategy against breast cancer stem cells.

Production of a novel monoclonal antibody, JT-95, which can detect antigen of thyroid carcinoma.

Monoclonal antibody (MAb) JT-95 was produced by immunization of mice with membrane fractions of a human thyroid carcinoma. Immuno-histochemical staining has demonstrated that the antigen recognized by JT-95 is strongly expressed in 95 (95%) of 100 cases of papillary carcinomas and in 3 (75%) of 4 cases of follicular carcinomas. In benign diseases of the thyroid gland, MAb JT-95 reacted with 0 (0%) of 39 adenomas, 1 (4%) of 21 adenomatous goiters, 0 (0%) of 8 hyperthyroidism specimens, and 3 (38%) of 8 chronic thyroiditis specimens. The antigen detected by MAb JT-95 has an apparent Mr 250,000 in thyroid carcinomas. Moreover, circulating antigen in thyroid carcinoma patients was detected by MAb JT-95 in an ELISA and in Western blotting. The circulating antigen has a Mr 105,000. MAb JT-95 conjugated with (131) I was administrated to nude mice bearing a human thyroid carcinoma. JT-95 131I accumulation at the transplanted tumor was visualized by autoradiography with 2.68-14.75-fold higher levels detected at the xenograft compared to that for normal organs. Based on these data, MAb JT-95 may be useful in the diagnosis detection and therapy of thyroid carcinoma.

Tumor necrosis factor expression by human ovarian carcinoma in vivo.

Tumor necrosis factor (TNF) is a cytokine produced by monocytes and other cells with selective cytolytic activity against some but not all tumor cells. Cellular resistance to the cytolytic effects of TNF has been reported to be associated with autocrine production of TNF by the target cells. The purpose of this study was to determine whether or not human tumors produce tumor necrosis factor in vivo. Ovarian carcinoma tissue from 25 patients with ovarian carcinoma was examined for the presence of TNF. Four of 5 ascites fluid specimens and tissue sections of 16 of 20 patients were positive for TNF by immunoperoxidase staining. The source of the immunoreactive protein was further examined by in situ hybridization studies. TNF mRNA was detectable in each of the ascites specimens and 7 of 16 tissue sections that were positive by immunoperoxidase staining. These findings suggest that TNF is produced by some human tumors in vivo and that the association between TNF production and resistance to TNF antitumor action may be clinically relevant.

Human urinary macrophage colony-stimulating factor reduces the incidence and duration of febrile neutropenia and shortens the period required to finish three courses of intensive consolidation therapy in acute myeloid leukemia: a double-blind controlled study.

PURPOSE: To determine whether macrophage colony-stimulating factor (M-CSF) reduces the incidence and duration of febrile neutropenia during three courses of intensive consolidation therapy and whether it shortens time to complete consolidation therapy. PATIENTS AND METHODS: In 198 adult patients with acute myeloid leukemia (AML) in complete remission (CR), M-CSF (8 x 10(6) U/d) or placebo was administered from 1 day after the end of each consolidation chemotherapy for 14 days. RESULTS: The duration and incidence of febrile neutropenia was significantly reduced by 34% (P = .00285) and 17% (P = .02065), respectively, in 88 assessable patients in the M-CSF group compared with those in 94 assessable patients in the placebo group. Patients in the M-CSF group had 565 days and 133 episodes of febrile neutropenia during 7,901 days at risk, while patients in the placebo group had 977 days and 185 episodes during 9,077 days at risk. The median period required to finish the three courses of consolidation therapy was 93 days in the M-CSF group, which was significantly shorter than 110 days in placebo group (P = .0050). In the M-CSF group, the recovery of neutrophils and platelets was significantly faster (P = .0348 and P = 0.0364, respectively), the administration of systemic antimicrobial agents tended to be less (P = .0839), and the frequency of platelet transfusion (P = .0259) and the total volume of transfused platelets (P = .0292) were significantly less. However, there was no significant difference in the disease-free survival. CONCLUSION: M-CSF significantly reduced the incidence and duration of febrile neutropenia during the intensive consolidation therapy, and shortened the time to complete consolidation chemotherapy in AML.

Detection of tumor necrosis factor gene expression at a cellular level in human acute myeloid leukemias.

Tumor necrosis factor (TNF) is a Mr 17,000 cytokine produced by macrophages. We have recently demonstrated that TNF is also produced by transformed human epithelial cells. The present studies have examined TNF expression in human myeloid leukemic cells. We have monitored TNF expression at a cellular level using alkaline phosphatase detection of a biotinylated TNF cDNA probe in situ. Using this approach, TNF transcripts were detectable in HL-60 cells induced along the monocytic lineage by phorbol ester but not in uninduced cells. The specific detection of TNF RNA at a cellular level was supported by the absence of histochemical staining in RNase-treated cells and when using biotinylated pBR322 plasmid without insert. These studies were extended to preparations of purified acute myeloblastic leukemia cells. The results demonstrate that TNF is expressed in myeloblasts in eight of nine patients with AML. In each preparation of myeloblasts with detectable TNF RNA, transcripts were present at 89-98% of the cells. The identification of TNF RNA in situ was also associated with the detection of TNF protein in leukemic blasts by indirect immunofluorescence. Moreover, the detection of TNF protein in these preparations of myeloblasts was confirmed by immunoblotting. However, using this approach to examine AML cells before and after purification indicated that TNF expression is induced as a result of the enrichment procedures. Thus, certain populations of purified myeloid leukemic cells are capable of expressing TNF at both the RNA and protein levels.

Cloning, sequencing and expressing the carotenoid biosynthesis genes, lycopene cyclase and phytoene desaturase, from the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 in Escherichia coli.

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and beta-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% (Erwinia herbicola), 37.4% (Erwinia uredovora) and 22.9% (Synechococcus sp.), and those between phytoene desaturase genes are 50.3% (E. herbicola), 54.7% (E. uredovora) and 39.6% (Rhodobacter capsulatus).

Phylogenetic analysis of a novel sulfate-reducing magnetic bacterium, RS-1, demonstrates its membership of the delta-Proteobacteria.

Most of the 16S ribosomal RNA gene of a sulfate-reducing magnetic bacterium, RS-1, was sequenced, and phylogenetic analysis was carried out. The results suggest that RS-1 is a member of the delta-Proteobacteria, and it appears to represent a new genus. RS-1 is the first bacterium reported outside the alpha-Proteobacteria that contains magnetite inclusions. RS-1 therefore disrupts the correlation between the alpha-Proteobacteria and possession of magnetite inclusions, and that between the delta-Proteobacteria and possession of greigite inclusions. The existence of RS-1 also suggests that intracellular magnetite biomineralization is of multiple evolutionary origins.

Effect of dietary linoleate/alpha-linolenate balance on experimentally induced gastric injury in rats.

Rats were fed diets containing different ratios of linoleate (18:2 n-6) to alpha-linolenate (18:3 n-3), and the severity of gastric injury induced by ethanol, ischemia/reperfusion and water-immersion stress was compared. On decreasing the 18:2 n-6/18:3 n-3 ratios in the diets, the proportion of arachidonic acid (20:4 n-6) decreased and that of eicosapentaenoic acid (20:5 n-3) increased in the phospholipids of the gastric mucosa, which was associated with decreased mucosal prostaglandin E2 content. Mucosal injury in all the three experimental models was exacerbated significantly in the diet group fed 18:2 n-6/18:3 n-3 ratio of 0.25 (perilla oil) as compared with the groups fed dietary oils with 18:2 n-6/18:3 n-3 ratios of 2.7 (mixture of perilla and safflower oils) and 127 (safflower oil). This adverse effect induced by perilla oil diet was not observed when rats were pretreated with a mild irritant (20% ethanol) prior to challenge with a strong irritant (absolute ethanol). Furthermore, an 18:2 n-6/18:3 n-3 ratio of as low as 1 was found to be in a safe range in the water-immersion stress ulcer model. Thus, oils with very low n-6/n-3 ratios, for example perilla oil, could be used without the risk of the observed adverse effects on experimental gastric injury in people of industrialized countries ingesting foods with n-6/n-3 ratios of above 4. A decrease in the n-6/n-3 ratios to 2 or below is still recommended for the prevention of chronic diseases in the elderly related to atherosclerosis and inflammation.

Posttreatment nadir M-protein level is a stronger discriminator of survival following plateau attainment than is percent reduction in M-protein in patients with IgG myeloma.

We conducted a retrospective study of patients with IgG or IgA myeloma who attained plateau to evaluate the relationships between survival and posttreatment nadir M-protein levels and between survival and the response to treatment evaluated by the percent reduction in M-protein. Of the 127 patients comprising 92 IgG and 35 IgA myeloma patients with disease stages II or III, 51 (40.2%) attained plateau. For IgG myeloma patients who attained plateau, survival time was not affected by the percent reduction in M-protein (median survival, 59.5 months for responding patients versus 54.4 months for nonresponding patients, P = .6910). Posttreatment nadir M-protein level, however, did affect survival time (median survival, 61.2 months for <3000 mg/dL versus 25.7 months for >3000 mg/dL, P = .0439). These findings suggest that the posttreatment nadir M-protein level is a stronger discriminator of survival following plateau attainment than the percent reduction of M-protein in patients with IgG myeloma.