Kazuo Honda

Abnormal expression of BRCA1 and BRCA1‐interactive DNA‐repair proteins in breast carcinomas

Breast cancer is one of the most common malignancies among women. The molecular mechanisms involved in breast carcinogenesis, however, remain to be elucidated. Although somatic mutation of BRCA1 is rare, BRCA1 protein expression is reduced in about 30% of sporadic breast carcinomas (Yoshikawa et al., Clin. Cancer Res., 5:1249–1261, 1999), indicating its possible involvement even in sporadic breast carcinogenesis. Among the BRCA1‐interactive proteins are hRAD51 (a human homologue of Escherichia coli rec A protein), BARD1 (BRCA1‐associated RING domain 1) and p53, all of which are involved in DNA repair. We have analyzed the expression patterns of the hRAD51, BARD1 and p53 proteins in five breast cancer cell lines, including a BRCA1‐deficient cell line, and in 179 breast cancer tissue samples from Japanese women, including 113 sporadic, 47 hereditary (i.e., BRCA1 status unknown), and 19 BRCA1‐associated cases. Of the 179 breast carcinomas, fifty‐four (30%) exhibited reduced hRAD51 expression, and sixty‐two (35%) exhibited p53 overexpression. On the other hand, reduced expression level of BARD1, and of hMSH2 and hMLH1, which are components of DNA mismatch‐repair pathway and are involved in colorectal carcinogenesis, was observed respectively in only 10 (6%), 8 (5%) and 3 (2%) cases. The overall frequency of sporadic breast carcinomas with abnormal expression of either BRCA1 or the BRCA1‐interactive proteins was 67% (76/113). These results indicate that there may be an important role for the BRCA1‐associated DNA‐repair pathway, not only in BRCA1‐associated breast carcinomas, but also in sporadic breast carcinomas. Int. J. Cancer 88:28–36, 2000.

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze‐drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long‐term storage conditions and analyses of freeze‐dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)‐PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze‐dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT‐PCR analyses, respectively. Overexpression of c‐erbB‐2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze‐dried human breast cancer tissues. Although macromolecules in freeze‐dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

Myoglobin gene expression attenuates hepatic ischemia reperfusion injury

BACKGROUNDnCellular functions are maintained by a continuous supply of ATP, which is supplied efficiently by mitochondrial oxidative phosphorylation. Since myoglobin, found in cardiac myocytes and red skeletal muscle, but not in the liver, facilitates oxygen diffusion under low oxygen conditions and enhances oxidative phosphorylation, this study seeks to enhance hepatic ATP levels and attenuate ischemia-reperfusion injury in rodent livers by adenovirus-mediated myoglobin expression.nnnMATERIAL AND METHODSnAfter infecting Hep3B and rodent livers with adenovirus carrying CMV promoter sequences linked to the human myoglobin gene (AdCMVMyo), reverse transcriptase-PCR and immunodetection for myoglobin, and cellular and hepatic ATP levels were examined. The effect of myoglobin was evaluated in a hepatic ischemia-reperfusion model in the rat.nnnRESULTSnMyoglobin expression was confirmed in Hep3B and rat livers after AdCMVMyo infection. The ATP levels in Hep3B cells and C57BL/6 mice livers 72 h after AdCMVMyo transfection were significantly higher than control levels and those after adenovirus-mediated beta-galactosidase transfection. Finally, expression of myoglobin attenuated ischemia-reperfusion injury in the rat liver.nnnCONCLUSIONnThese results indicate that myoglobin gene transfer to the liver enhanced ATP levels both in vitro and in vivo and might be a novel strategy to reduce ischemia-reperfusion injury.

Gene transfer to the rat biliary tract with the HVJ-cationic liposome method

BACKGROUND/AIMSnThe ability to transfer foreign genes into the biliary tract would be useful for the treatment of biliary tract diseases, including cancer, cystic fibrosis and other genetic diseases. To introduce a foreign gene precisely into the rat biliary epithelial cells, we developed a new technique, inserting a polyethylene catheter into the common bile duct through the papilla of Vater by use of a fusigenic cationic liposome with hemagglutinating virus of Japan (HVJ-cationic liposome).nnnMETHODSnTransfection efficiency was estimated with the use of FITC-oligonucleotides (FITC-ODNs) and cDNA of beta-galactosidase (pCAG-lacZ).nnnRESULTSnFITC-ODNs encapsulated in HVJ-cationic liposome were effectively transfected into cell nuclei of human cholangiocellular carcinoma in vitro after a 30-min incubation as compared with the simple application of naked FITC-ODNs. After in vivo injection of FITC-ODNs using the HVJ-cationic liposome method through the papilla of Vater, fluorescence accumulation was observed only in the epithelial cells of the biliary tract, but not in the parenchymal cells of the liver. Beta-galactosidase expression was observed in the biliary epithelial cells 3 days after the transfection of pCAG-lacZ and was also detected at 14 days, but not at 28 days, without obvious cytotoxicity.nnnCONCLUSIONSnHVJ-cationic liposome-mediated gene transfer to the biliary tract via the papilla of Vater is a minimally-invasive and an effective gene-delivery method for site-specific targeting to the epithelial cells of the biliary tract, which could be applied to the treatment of human biliary tract diseases.

Oncolytic Gene Therapy Combined with Double Suicide Genes for Human Bile Duct Cancer in Nude Mouse Models

BACKGROUNDnThe prognosis of bile duct cancer is quite poor because of the low resection rate and the tolerance of the cancer to chemotherapy and radiotherapy. We investigated the feasibility of an oncolytic adenovector with two suicide genes for the treatment of bile duct cancer.nnnMATERIALS AND METHODSnWe developed a new conditionally replicating adenovirus (AxE1CAUT) with the uracil phosphoribosyltransferase (UPRT) gene and the herpes simplex virus thymidine kinase (HSV-tk) gene, and compared its antitumor effects with a replication defective adenovector (AxCAUT) that has both the UPRT and HSV-tk genes. We evaluated the effects of these adenoviruses with 5-fluorouracil (5-FU) and/or ganciclovir (GCV) on human cholangiocarcinoma cells (HuCCT1, with mutant p53) in vitro and in vivo.nnnRESULTSnThe drug sensitivity of HuCCT1 cells to 5-FU and/or GCV was increased with an increase in the multiplicity of infection (MOI). The antitumor effect increased when 5-FU and GCV were given at the same time. Subcutaneous tumors of nude mice directly injected with AxCAUT showed a higher response to 5-FU/GCV than 5-FU or GCV alone, but there was no difference between AxCAUT and AxE1CAUT. However, AxE1CAUT with 5-FU/GCV produced a decrease in tumor weight and better survival than AxCAUT in a peritoneal dissemination model infected by intraperitoneal administration of the adenovectors.nnnCONCLUSIONnOncolytic double suicide gene therapy is effective against human cholangiocarcinoma cells in nude mouse models.

Downregulation of Cytokine Release by Heat Preconditioning of Livers Used for Transplantation in Rats

We evaluated the changes in the levels of released cytokines following heat preconditioning of the livers used in rat liver transplantation. The donor rats in the heat preconditioning (HP) group were subjected to heat preconditioning 48 h before graft harvesting. The liver isografts were preserved in Euro-Collins solution for 8 h, and then transplanted orthotopically. The one-week survival rate of the HP group was significantly better than that of the control (C) group. The serum levels of interleukin-6 and interleukin-10 were significantly lower in the HP group than in the C group. Histological staining revealed that the stagnation of red blood cells and infiltration of neutrocytes were reduced in the HP group. The expression of intercellular adhesion molecule-1 was decreased around the central vein in the HP group, as revealed by immunohistochemistry. These results indicate that heat preconditioning downregulates cytokine release and reduces the frequency of microcirculation disorders.

Clinical Features of Early Myocardial Rupture of Acute Myocardial Infarction

We assessed the clinical features of patients with myocardial rupture within 48 to 72 hours, defined as early myocardial rupture, after percutaneous coronary intervention (PCI) for ST-segment elevation acute myocardial infarction (STEMI). Six patients (4 men, 66 ± 13 years) with early myocardial rupture were identified from 1252 consecutive patients undergoing PCI for STEMI. We evaluated the degree of microvascular reperfusion using thrombolysis in myocardial infarction (TIMI) myocardial perfusion (TMP) grade and a resolution of sum of ST-segment elevation in a 12-lead electrocardiogram (ECG). Time from PCI to myocardial rupture was 11 ± 7 hours. All patients showed TMP grade 0 or 1 and an increase in sum of ST-segment elevation after PCI (1.9 ± 0.5 vs 2.5 ± 0.7 mV; P = .032), suggesting severely failed reperfusion at the level of microcirculation as the common feature to develop early myocardial rupture after PCI for STEMI.

Critical role of systemic inflammation in patients with ST-segment elevation myocardial infarction complicated with renal dysfunction

side branch stent for the treatment of bifurcation lesions: a two center registry analysis. Catheter Cardiovasc Interv 2011;77(6):798–806. [7] Grundeken MJ, Asgedom S, Damman P, et al. Six-month and one-year clinical outcomes after placement of a dedicated coronary bifurcation stent: a patient-level pooled analysis of eight registry studies. EuroIntervention 2013;9(2):195–203. [8] Cutlip DE,Windecker S,Mehran R, et al. Clinical end points in coronary stent trials: a case for standardized definitions. Circulation 2007;115(17):2344–51. [9] Editors Of The Heart Group Journals. Statement on matching language to the type of evidence used in describing outcomes data. Int J Cardiol 2013;166(1):1.