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.

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.

2-GHz band CW and W-CDMA modulated radiofrequency fields have no significant effect on cell proliferation and gene expression profile in human cells.

We investigated the mechanisms by which radiofrequency (RF) fields exert their activity, and the changes in both cell proliferation and the gene expression profile in the human cell lines, A172 (glioblastoma), H4 (neuroglioma), and IMR-90 (fibroblasts from normal fetal lung) following exposure to 2.1425 GHz continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) RF fields at three field levels. During the incubation phase, cells were exposed at the specific absorption rates (SARs) of 80, 250, or 800 mW/kg with both CW and W-CDMA RF fields for up to 96 h. Heat shock treatment was used as the positive control. No significant differences in cell growth or viability were observed between any test group exposed to W-CDMA or CW radiation and the sham-exposed negative controls. Using the Affymetrix Human Genome Array, only a very small (< 1%) number of available genes (ca. 16,000 to 19,000) exhibited altered expression in each experiment. The results confirm that low-level exposure to 2.1425 GHz CW and W-CDMA RF fields for up to 96 h did not act as an acute cytotoxicant in either cell proliferation or the gene expression profile. These results suggest that RF exposure up to the limit of whole-body average SAR levels as specified in the ICNIRP guidelines is unlikely to elicit a general stress response in the tested cell lines under these conditions.

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.

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.

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.

High-accuracy prediction of carcinogenicity by global quantitative analysis of post-translational modifications in a 28-day in vivo rat study.

A global quantitative analysis of post-translational modifications (PTMs) of distinct proteins was executed at the proteomic level using two-dimensional fluorescence differential gel electrophoresis. We evaluated the effects of 66 chemical compounds, including 15 genotoxic carcinogens, 28 non-genotoxic carcinogens, and 23 non-carcinogens, in the male F344 rat liver in a 28-day repeated dose study. In the master gel of rat liver protein, we identified 728 spots by hybrid quadrupole time-of-flight mass spectrometry. They collapsed into 356 distinct proteins. Of these, 126 were represented by two or more spots in the 2-D gel. We calculated the logarithmic ratio of volume changes of all 1028 combinations generated from 126 proteins and investigated the relevance to carcinogenicity. This quantitative proteomic study revealed the existence of several PTMs characteristic of carcinogens that may play an important role in early stage of carcinogenicity. Prediction of carcinogenicity from PTM data gave a higher concordance (92.4%) than prediction from protein expression data (74.2%). This novel approach holds great promise as a way of revealing the roles of charge modifications and molecular weight variations of proteins in biological processes.

A comparative study of gene expression profiles in rat liver after administration of alpha-hexachlorocyclohexane and lindane.

Alpha-hexachlorocyclohexane (alpha-HCH) is a stereoisomer of gamma-HCH, the active ingredient of lindane (> 99% gamma-HCH). In the present study, cDNA microarray technology was employed to identify changes in gene expression associated with toxicity in livers of male Fischer 344 rats after treatment with alpha-HCH (2, 20 mg/kg/day) and lindane (1, 10 mg/kg/day) by daily oral gavage for up to 28 days. Liver samples were obtained after 1, 3, 7, 14 and 28 days and compared for gene expression profiles. The dose of the alpha-HCH was higher than that of lindane and toxicity was greater, but the numbers of probe sets with differences in expression were fewer for the alpha-HCH-treated group except on Day 3. Only very few probe sets with differences in expression overlapped between alpha-HCH and lindane at each time point and the gene expression profiles were very dissimilar. Important liver-based differences in expression between alpha-HCH and lindane might possibly account for hepato-carcinogenicity of alpha-HCH.

Sequential occurrence of non-small cell and small cell lung cancer with the same EGFR mutation.

We report a case of small cell lung cancer (SCLC) developing after prolonged treatment (more than 2 years) for primary adenocarcinoma of the lung, and we show that both the SCLC and non-small cell lung cancer (NSCLC) tissues obtained from the same site share the same deletion in exon 19 of EGFR. This case suggests that the activating EGFR mutations may confer the pathogenesis of a subset of SCLC.

Optimization of an animal test protocol for toxicogenomics studies (I); requirement study of a protocol.

Toxicogenomics is a promising new tool for prediction of chemical toxicities including carcinogenicity in a relatively short period. However, it is important to develop a reliable animal test protocol for toxicogenomics studies. The preparation of RNA and tissues is also crucial, since it greatly influences outcomes of gene expression analysis. In the present study, we examined an animal test protocol by comparing gene expression data from different conditions and proposed a reliable animal test protocol for toxicogenomic studies. With regard to the preparation of tissues and RNA, here we present evidence that quality of RNA and tissues is well-preserved even after freezer storage for up to 2.5 years. Gene expression levels were compared using a GeneChip System (RGU34A, Affymetrix, Santa Clara, CA, USA) between RNA samples that were freshly prepared, stored at -80 degrees C or re-prepared from tissue kept at -20 degrees C. None showed degradation and no significant differences in expression were evident among the three sets of samples. The data demonstrate that gene expression analysis by DNA microarray is suitable for RNA or tissues that have been stored at an appropriate temperature.

Optimization of an animal test protocol for toxicogenomics studies (ii); a cross-laboratory gene expression analysis.

Toxicogenomics is a promising new tool for prediction of chemical toxicities including carcinogenicity in a relatively short period. However, it is important to develop a reliable animal test protocol for toxicogenomics studies. The preparation of RNA and tissues is also crucial, since it greatly influences outcomes of gene expression analysis. We proposed an animal test protocol for toxicogenomics studies. In the present study, we examined an animal test protocol by comparing biological and gene expression data from different laboratories running identical in vivo studies on the same microarray platform. The results gave good correspondence in all three laboratories at the level of biological responses and gene expression, especially for genes whose expression changes were quite large. As the fold change or the signal values become smaller, however, discrepancies occur in gene expression data. For example, one laboratory shows an opposite directional change to the other two or no change. The results of hierarchical clustering and principal component analysis (PCA) demonstrated all samples from the three laboratories being clearly divided between control and treatment. Examination of the reproducibility of gene expression data across laboratories using the proposed animal test protocol thus confirmed only minor differences, which was expected to present no problems for gene expression analysis.

Quantitative proteomic analysis of rat liver for carcinogenicity prediction in a 28-day repeated dose study.

The potential of quantitative proteomic analysis to predict carcinogenicity of chemical compounds was investigated. Using 2D-DIGE, we analyzed the effects of 63 chemical compounds on protein expression in the rat liver after 28 daily doses. Types of carcinogens were categorized depending on the species and organ specificity. The carcinogen characteristic proteins for each classification were identified by Welch's t value. For evaluation of the predictive concordance we used support vector machines. The rat hepatic carcinogen-specific classification gave higher concordance than the other classification. The generalization performance was measured by leave-one-out cross-validation. For genotoxic and non-genotoxic compounds, a concordance of 79.3 and 76.5%, respectively, was obtained by the top 30 ranked proteins with Welch's t value. Furthermore, we found that the increase of the expression level of the stress response proteins as the common feature of poorly predicted chemical compounds in the leave-20%-out cross-validation. Quantitative proteomics could be promising technique for developing biomarker panels that can be used for carcinogenicity prediction. The list of proteins identified in this study and the zoomed gel images of the top ranked proteins in statistic analysis are provided in Supplementary Data.

Differences in gene expression profiles in the liver between carcinogenic and non-carcinogenic isomers of compounds given to rats in a 28-day repeat-dose toxicity study.

Some compounds have structural isomers of which one is apparently carcinogenic, and the other not. Because of the similarity of their chemical structures, comparisons of their effects can allow gene expression elicited in response to the basic skeletons of the isomers to be disregarded. We compared the gene expression profiles of male Fischer 344 rats administered by daily oral gavage up to 28 days using an in-house oligo microarray. 2-Acetylaminofluorene (2-AAF), 2,4-diaminotoluene (2,4-DAT), 2-nitropropane (2-NP), and 2-nitro-p-phenylenediamine (2-NpP) are hepatocarcinogenic. However, their isomers, 4-acetylaminofluorene (4-AAF), 2,6-diaminotoluene (2,6-DAT), 1-nitropropane (1-NP), and 4-nitro-o-phenylenediamine (4-NoP), are non-hepatocarcinogenic. Because of the limited carcinogenicity of 2-NpP, we attempted to perform two-parametric comparison analyses with (1) a set of 4 isomers: 2-AAF, 2,4-DAT, 2-NP, and 2-NpP as "carcinogenic", and 4-AAF, 2,6-DAT, 1-NP, and 4-NoP as "non-carcinogenic"; and (2) a set of 3 isomers: 2-AAF, 2,4-DAT, and 2-NP, as "carcinogenic", and 4-AAF, 2,6-DAT, and 1-NP as "non-carcinogenic". After ratio filtering and Welch's approximate t-test analysis, 54 and 28 genes were selected from comparisons between the sets of 3 and 4 isomers, respectively, for day 28 data. Using hierarchical clustering analysis with the 54 or 28 genes, 2-AAF, 2,4-DAT, and 2-NP clustered into a "carcinogenic" branch. 2-NpP was in the same cluster as 4-NoP and 4-AAF. This clustering corresponded to the previous finding that 2-NpP is not carcinogenic in male Fischer 344 rats, which indicates that comparing the differences in gene expression elicited by different isomers is an effective method of developing a prediction system for carcinogenicity.

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.

Plasma MIP-1beta levels and skin toxicity in Japanese non-small cell lung cancer patients treated with the EGFR-targeted tyrosine kinase inhibitor, gefitinib.

Gefitinib (Iressa() is an orally active, selective EGFR tyrosine kinase inhibitor that blocks signal transduction pathways. Skin toxicity has been reported to be the major toxicity observed in patients treated with the EGFR-targeted tyrosine kinase inhibitors, such as gefitinib and erlotinib. Although the mechanisms underlying the development of the skin toxicity remain to be precisely clarified, immunological mechanisms are considered to be involved. We examined the correlations between the plasma levels of several cytokines and the risk of development of adverse events, especially skin toxicity, induced by the administration of gefitinib as first-line monotherapy in non-small cell lung cancer (NSCLC) patients. Paired plasma samples were obtained from a total 28 patients of non-small cell lung cancer; the first before the initiation of gefitinib administration (250 mg/day) (24 patients) and the second 2 or 4 weeks after the initiation of treatment (23 patients). The plasma concentrations of 17 major cytokines were measured using a bead-based multiplex assay. The median concentrations of eight of these cytokines before the start of treatment ranged from 0.06 (IL-5) to 58.26 (MIP-1beta) (microg/ml). The concentrations of the remaining nine cytokines were under the detectable limit (<0.01 microg/ml) in more than 50% of the samples. Comparisons of the levels before and after treatment showed no significant differences for any of the cytokines measured. The MIP-1beta levels were significantly lower in the patients with skin toxicity (16/24) as compared with those in the patients not showing any skin toxicity (59.1+/-10.5 versus 119.0+/-36.8; p=0.042 by the two-sample t-test). The K-Nearest Neighbor Prediction (K=3) showed the classification rate to be 75% for the prediction sets containing MIP-1beta, IL-4 and IL-8. There were no significant associations between the levels of any of the cytokines measured and any other parameters, including the tumor response to the drug. In conclusion, the plasma MIP-1beta level may be a useful predictor of the development of skin toxicity in patients receiving gefitinib treatment.

Large scale in vitro experiment system for 2 GHz exposure.

A beam formed radiofrequency (RF) exposure-incubator employing a horn antenna, a dielectric lens, and a culture case in an anechoic chamber is developed for large scale in vitro studies. The combination of an open type RF exposure source and a culture case through which RF is transmitted realizes a uniform electric field (+/-1.5 dB) in a 300 x 300 mm area that accommodates 49 35 mm diameter culture dishes. This large culture dish area enables simultaneous RF exposure of a large number of cells or various cell lines. The RF exposure source operates at 2142.5 MHz corresponding to the middle frequency of the downlink band of the International Mobile Telecommunication 2000 (IMT-2000) cellular system. The dielectric lens, which has a gain of 7 dB, focuses RF energy in the direction of the culture case and provides a uniform electric field. The culture case is sealed and connected to the main unit for environmental control, located outside the anechoic chamber, via ducts. The temperature at the center of the tray, which contains the culture dishes in the culture room, is maintained at 37.0 +/- 0.2 degrees C by air circulation. In addition, the appropriate CO2 density and humidity supplied to the culture case realizes stable long-term culture conditions. Specific absorption rate (SAR) dosimetry is performed using an electric field measurement technique and the Finite Difference Time Domain (FDTD) calculation method. The results indicate that the mean SAR of the culture fluid at the bottom of the 49 (7 x 7 array) culture dishes used in the in vitro experiments is 0.175 W/kg for an antenna input power of 1 W and the standard deviation of the SAR distribution is 59%. When only 25 culture dishes (5 x 5 array) are evaluated, the mean SAR is 0.139 W/kg for the same antenna input power and the standard deviation of the SAR distribution is 47%. The proliferation of the H4 cell line in 72 h in a pair of RF exposure-incubators reveals that the culture conditions are equivalent to those of a common CO2 incubator.

Elliptically polarized magnetic fields do not alter immediate early response genes expression levels in human glioblastoma cells.

Expression of immediate early response genes such as c-fos, c-jun, and c-myc in response to 1-500 microT resultant (r) 60 Hz elliptically polarized (EP) magnetic fields (MFs), typical of environmental MFs polarization under overhead power lines, was analyzed in both at transcriptional and translational levels using human glioblastoma (T98G) cells. Pseudo synchronized T98G cells at G1 phase were exposed to EP-MFs (1, 20, 100, and 500 microTr) for up to 3 h, but produced no statistical difference (P>0.05) in the levels of expression ratio at both the transcriptional and translational levels at 30 min for c-fos and c-jun and at 180 min for c-myc after serum stimulation. In addition, exposure of T98G cells to linearly (vertical and horizontal) and/or circularly polarized MFs (500 microTr) produced no significant change (P>0.05) in the expression ratio at both transcriptional and post-transcriptional levels. Thus, there was no evidence that linearly or rotating polarized MFs enhanced early response gene expression in these studies. These results suggest that environmental MFs at 1-500 microT flux density are unlikely to induce carcinogenesis through a mechanism involving altered expression of the immediate early response genes.