Masaru Sekijima

Acute oral toxicity of microcystin-LR, a cyanobacterial hepatotoxin, in mice

Microcystin-LR (MCLR) is a hepatotoxic peptide produced by Microcystis aeruginosa, an alga found worldwide in reservoirs for drinking supply; however, acute oral toxicity of purified MCLR remains unknown. Therefore, a single dose of MCLR (more than 95% purity) ranging from 8.0 to 20.0 mg/kg body weight was orally given to female 6-week old BALB/c mice, and lethality and pathological changes were observed. Median lethal dose (LD50) of the orally given MCLR estimated by the up and down method was 10.9 mg/kg, which was 167 times higher than the i.p. LD50 value (65.4 microgram/kg by moving average method). Orally administrated toxin caused primarily hepatocellular injuries with characteristics of hemorrhage and necrosis. In situ end-labeling as well as electron microscopic observation revealed an induction of apoptotic cell death to hepatocytes. These results indicate the lethality of MCLR was much lower in oral dosage than by i.p. administration, but toxic effects are similar. In addition, apoptosis is considered one of major components in MCLR-induced hepatotoxicity.

Identification of expressed genes characterizing long-term survival in malignant glioma patients

Better understanding of the underlying biology of malignant gliomas is critical for the development of early detection strategies and new therapeutics. This study aimed to define genes associated with survival. We investigated whether genes coupled with a class prediction model could be used to define subgroups of high-grade gliomas in a more objective manner than standard pathology. RNAs from 29 malignant gliomas were analysed using Agilent microarrays. We identified 21 genes whose expression was most strongly and consistently related to patient survival based on univariate proportional hazards models. In six out of 10 genes, changes in gene expression were validated by quantitative real-time PCR. After adjusting for clinical covariates based on a multivariate analysis, we finally obtained a statistical significance level for DDR1 (discoidin domain receptor family, member 1), DYRK3 (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 3) and KSP37 (Ksp37 protein). In independent samples, it was confirmed that DDR1 protein expression was also correlated to the prognosis of glioma patients detected by immunohistochemical staining. Furthermore, we analysed the efficacy of the short interfering RNA (siRNA)-mediated inhibition of DDR1 mRNA synthesis in glioma cell lines. Cell proliferation and invasion were significantly suppressed by siRNA against DDR1. Thus, DDR1 can be a novel molecular target of therapy as well as an important predictive marker for survival in patients with glioma. Our method was effective at classifying high-grade gliomas objectively, and provided a more accurate predictor of prognosis than histological grading.

Dimerization and the signal transduction pathway of a small in-frame deletion in the epidermal growth factor receptor

A short, in‐frame deletional mutant (E746‐A750del) is one of the major mutant forms of epidermal growth factor receptor (EGFR) and has been reported to be a determinant of response to EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. However, the biological and pharmacological functions of mutational EGFR remain unclear. To clarify these biological functions of deletional EGFR, we examined the cellular response to EGF ligand stimulation. Dimerization and phosphorylation of EGFR were observed without any ligand stimulation in the 293(D) cells transfected with deletional EGFR as compared with those transfected with wild‐type EGFR (293(W) cells). When the 293(D) cells were exposed to gefitinib, an immunoblotting analysis revealed remarkable inhibition of AKT phosphorylation but not phospho‐p44/42 MAPK. To examine the cellular response in a lung cancer cell line intrinsically expressing deletional EGFR, phospho‐EGFR, and downstream reactions were monitored under EGF stimulation with a beads‐based mulitiplex assay. EGFR and its downstream proteins were constitutively phosphorylated in the PC‐9 cells without any ligand stimulation as compared with A549 lung cancer cells expressing wild‐type EGFR. In conclusion, deletional EGFR is constitutively active and phosphorylates p44/42 MAPK and AKT in the cells, although the fact that the EGFR phosphorylation in the PC‐9 cells is still modulated by EGF stimulation cannot be ignored. Gefitinib‐inhibited phospho‐AKT predominantly in deletional EGFR expressing cells.

No Chronic oral toxicity of a low dose of microcystin-LR, a cyanobacterial hepatotoxin, in female BALB/c mice

Chronic oral toxicity of a low dose of microcystin‐LR (MCLR) was examined in female BALB/c mice for 18 months. Six‐week‐old female mice received 20 μg/L of the toxin in drinking water, which is about 200‐fold higher than the level in contaminated drinking water. Control mice received water alone. Mortality, clinical signs, body weights, and food and water consumptions were recorded during the study. Examinations on hematology, serum biochemistry, necropsy, organ weights, and histopathology were performed at months, 3, 6, 12, and 18. The immunohistochemical distribution of MCLR was examined in the liver at these time points. Mean cumulative MCLR intake after 18 months was estimated at 35.5 μg per mouse. The present test indicates that administration of a low dose of MCLR in drinking water resulted in neither chronic toxicity nor accumulation of the toxin in the liver. Based on previous epidemiological studies and the present chronic toxicity test, we recommend 0.01 μg/L as an maximum acceptable level for microcystins in drinking water, applying a safety factor of more than 1000. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 45–55, 1999

Association of epidermal growth factor receptor (EGFR) gene mutations with EGFR amplification in advanced non-small cell lung cancer

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the response to EGFR tyrosine kinase inhibitors in patients with non‐small cell lung cancer (NSCLC). Increased EGFR copy number has also been associated with sensitivity to these drugs. However, given that it is often difficult to obtain sufficient amounts of tumor tissue for genetic analysis from patients with advanced NSCLC, the relationship between these two types of EGFR alterations has remained unclear. We have now evaluated EGFR mutation status both by direct sequencing and with a high‐sensitivity assay, the Scorpion‐amplification‐refractory mutation system, and have determined EGFR copy number by fluorescence in situ hybridization (FISH) analysis in paired tumor specimens obtained from 100 consecutive patients with advanced NSCLC treated with chemotherapy. EGFR mutations or FISH positivity (EGFR amplification or high polysomy) were apparent in 18% (18/100) and 32% (32/100) of patients, respectively. The Scorpion‐amplification‐refractory mutation system was more sensitive than direct sequencing for the detection of EGFR mutations. Furthermore, EGFR mutations were associated with EGFR amplification (P = 0.009) but not with FISH positivity (P = 0.266). Our results therefore suggest the existence of a significant association between EGFR mutation and EGFR amplification in patients with advanced NSCLC. (Cancer Sci 2008; 99: 2455–2460)

Discrimination of Carcinogens by Hepatic Transcript Profiling in Rats Following 28-day Administration

This study aimed at discriminating carcinogens on the basis of hepatic transcript profiling in the rats administrated with a variety of carcinogens and non-carcinogens. We conducted 28-day toxicity tests in male F344 rats with 47 carcinogens and 26 non-carcinogens, and then investigated periodically the hepatic gene expression profiles using custom microarrays. By hierarchical cluster analysis based on significantly altered genes, carcinogens were clustered into three major groups (Group 1 to 3). The formation of these groups was not affected by the gene sets used as well as the administration period, indicating that the grouping of carcinogens was universal independent of the conditions of both statistical analysis and toxicity testing. Seventeen carcinogens belonging to Group 1 were composed of mainly rat hepatocarcinogens, most of them being mutagenic ones. Group 2 was formed by three subgroups, which were composed of 23 carcinogens exhibiting distinct properties in terms of genotoxicity and target tissues, namely nonmutagenic hepatocarcinogens, and mutagenic and nonmutagenic carcinogens both of which are targeted to other tissues. Group 3 contained 6 carcinogens including 4 estrogenic substances, implying the group of estrogenic carcinogens. Gene network analyses revealed that the significantly altered genes in Group 1 included Bax, Tnfrsf6, Btg2, Mgmt and Abcb1b, suggesting that p53-mediated signaling pathway involved in early pathologic alterations associated with preceding mutagenic carcinogenesis. Thus, the common transcriptional signatures for each group might reflect the early molecular events of carcinogenesis and hence would enable us to identify the biomarker genes, and then to develop a new assay for carcinogenesis prediction.

1950 MHz IMT-2000 field does not activate microglial cells in vitro

Given the widespread use of the cellular phone today, investigation of potential biological effects of radiofrequency (RF) fields has become increasingly important. In particular, much research has been conducted on RF effects on brain function. To examine any biological effects on the central nervous system (CNS) induced by 1950 MHz modulation signals, which are controlled by the International Mobile Telecommunication-2000 (IMT-2000) cellular system, we investigated the effect of RF fields on microglial cells in the brain. We assessed functional changes in microglial cells by examining changes in immune reaction-related molecule expression and cytokine production after exposure to a 1950 MHz Wideband Code Division Multiple Access (W-CDMA) RF field, at specific absorption rates (SARs) of 0.2, 0.8, and 2.0 W/kg. Primary microglial cell cultures prepared from neonatal rats were subjected to an RF or sham field for 2 h. Assay samples obtained 24 and 72 h after exposure were processed in a blind manner. Results showed that the percentage of cells positive for major histocompatibility complex (MHC) class II, which is the most common marker for activated microglial cells, was similar between cells exposed to W-CDMA radiation and sham-exposed controls. No statistically significant differences were observed between any of the RF field exposure groups and the sham-exposed controls in percentage of MHC class II positive cells. Further, no remarkable differences in the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) were observed between the test groups exposed to W-CDMA signal and the sham-exposed negative controls. These findings suggest that exposure to RF fields up to 2 W/kg does not activate microglial cells in vitro.

Plasma Concentrations of Angiogenesis-related Molecules in Patients with Pancreatic Cancer

BACKGROUND Anti-angiogenic agents are now being clinically evaluated for the treatment of pancreatic cancer and a detailed investigation of the angiogenic profile of pancreatic cancer is needed. The aim of this study was to evaluate the plasma concentrations of angiogenesis-related molecules in patients with pancreatic cancer, compared with those with other diseases. METHODS Plasma samples obtained from 45 patients with pancreatic cancer were analyzed and compared with those from 9 patients with pancreatitis, 16 patients with benign hepatobiliary diseases and 58 patients with colorectal cancers. The plasma levels of angiogenesis-related molecules including angiopoietin-2, follistatin, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin-8, leptin, platelet-derived growth factor beta polypeptide, platelet endothelial cell adhesion molecule-1 and vascular endothelial growth factor were determined using an antibody suspension bead arrays system. RESULTS The plasma levels of all the angiogenesis-related molecules were not increased in patients with pancreatic cancer, compared with those with pancreatitis and benign hepatobiliary diseases, whereas the levels of those with colorectal cancer were markedly increased. The plasma interleukin-8 concentration was significantly elevated in patients with distant metastases and was associated with a poor treatment outcome of chemotherapy in patients with pancreatic cancer. CONCLUSIONS The plasma levels of angiogenesis-related molecules were not elevated in patients with pancreatic cancer, compared with those with benign diseases or colorectal cancer. The plasma interleukin-8 level may be a novel biomarker for the response to chemotherapy in patients with pancreatic cancer and warrants further prospective study.

Integrated analysis of whole genome exon array and array‐comparative genomic hybridization in gastric and colorectal cancer cells

Whole genome‐scale integrated analyses of exon array and array‐comparative genomic hybridization are expected to enable the identification of unknown genetic features of cancer cells. Here, we evaluated this approach in 22 gastric and colorectal cancer cell lines, focusing on protein kinase genes and genes belonging to the cadherin–catenin family. Regarding alternative splicing patterns, several cancer cell lines predominantly expressed isoform 1 of protein kinase A catalytic subunit beta (PRKACB). Paired gastric cancer specimens demonstrated that isoform 1 of PRKACB was a novel cancer‐related variant transcript in gastric cancers. In addition, the exon array analysis clearly identified exon 3 or exon 3–4 skipping in catenin beta 1, a short intron insertion with exon 9 skipping in CDH1, and a deletional transcript of CDH13. These abnormal transcripts were shown to have arisen from small genomic deletions. Meanwhile, an integrated analysis of 11 gastric cancer cell lines revealed that four cell lines amplified fibroblast growth factor receptor 2, with truncated forms observed in two of the cell lines. Gene amplification, and not the truncated form, was found to determine the sensitivity to a fibroblast growth factor receptor inhibitor, indicating that our cell line panel might be useful for cell‐based evaluations of specific inhibitors. Using an integrated analysis, we identified several abnormal transcripts and genomic alterations in gastric and colorectal cancer cells. Our approach might enable genetic changes to be identified more efficiently, and the present results warrant further investigation using clinical samples and integrated analyses. (Cancer Sci 2012; 103: 221–227)

New Short Term Prediction Method for Chemical Carcinogenicity by Hepatic Transcript Profiling Following 28-Day Toxicity Tests in Rats

We have previously shown the hepatic gene expression profiles of carcinogens in 28-day toxicity tests were clustered into three major groups (Group-1 to 3). Here, we developed a new prediction method for Group-1 carcinogens which consist mainly of genotoxic rat hepatocarcinogens. The prediction formula was generated by a support vector machine using 5 selected genes as the predictive genes and predictive score was introduced to judge carcinogenicity. It correctly predicted the carcinogenicity of all 17 Group-1 chemicals and 22 of 24 non-carcinogens regardless of genotoxicity. In the dose-response study, the prediction score was altered from negative to positive as the dose increased, indicating that the characteristic gene expression profile emerged over a range of carcinogen-specific doses. We conclude that the prediction formula can quantitatively predict the carcinogenicity of Group-1 carcinogens. The same method may be applied to other groups of carcinogens to build a total system for prediction of carcinogenicity.