Short-term in vivo testing to discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens using next-generation RNA sequencing, DNA microarray, and qPCR.

Next-generation RNA sequencing (RNA-Seq) has identified more differentially expressed protein-coding genes (DEGs) and provided a wider quantitative range of expression level changes than conventional DNA microarrays. JEMS·MMS·Toxicogenomics group studied DEGs with targeted RNA-Seq on freshly frozen rat liver tissues and on formalin-fixed paraffin-embedded (FFPE) rat liver tissues after 28 days of treatment with chemicals and quantitative real-time PCR (qPCR) on rat and mouse liver tissues after 4 to 48 h treatment with chemicals and analyzed by principal component analysis (PCA) as statics. Analysis of rat public DNA microarray data (Open TG-GATEs) was also performed. In total, 35 chemicals were analyzed [15 genotoxic hepatocarcinogens (GTHCs), 9 non-genotoxic hepatocarcinogens (NGTHCs), and 11 non-genotoxic non-hepatocarcinogens (NGTNHCs)]. As a result, 12 marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) were proposed to discriminate GTHCs from NGTHCs and NGTNHCs. U.S. Environmental Protection Agency studied DEGs induced by 4 known GTHCs in rat liver using DNA microarray and proposed 7 biomarker genes, Bax, Bcmp1, Btg2, Ccng1, Cdkn1a, Cgr19, and Mgmt for GTHCs. Studies involving the use of whole-transcriptome RNA-Seq upon exposure to chemical carcinogens in vivo have also been performed in rodent liver, kidney, lung, colon, and other organs, although discrimination of GTHCs from NGTHCs was not examined. Candidate genes published using RNA-Seq, qPCR, and DNA microarray will be useful for the future development of short-term in vivo studies of environmental carcinogens using RNA-Seq.

Human gastric cancer risk screening: From rat pepsinogen studies to the ABC method.

We examined the development of gastric cancer risk screening, from rat pepsinogen studies in an experimental rat gastric carcinogenesis model induced with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and human pepsinogen studies in the 1970s and 1980s to the recent "ABC method" for human gastric cancer risk screening. First, decreased expression or absence of a major pepsinogen isozyme, PG1, was observed in the rat gastric mucosa from the early stages of gastric carcinogenesis to adenocarcinomas following treatment with MNNG. In the 1980s, decreases in PGI in the human gastric mucosa and serum were identified as markers of atrophic gastritis. In the 1990s, other researchers revealed that chronic infection with Helicobacter pylori (Hp) causes atrophic gastritis and later gastric cancer. In the 2000s, a gastric cancer risk screening method combining assays to detect serum anti-Hp IgG antibody and serum PGI and PGII levels, the "ABC method", was established. Eradication of Hp and endoscopic follow-up examination after the ABC method are recommended to prevent gastric cancer.

Detection of genome-wide low-frequency mutations with Paired-End and Complementary Consensus Sequencing (PECC-Seq) revealed end-repair-derived artifacts as residual errors.

To improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strand-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 7 bp of the library fragments in the 5'-NpCpA-3' and 5'-NpCpT-3' trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 7 bp, PECC-Seq could reduce the sequencing error frequency to mid-10-7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10-8. In mutagen-treated (6 µg/mL methyl methanesulfonate or 12 µg/mL N-nitroso-N-ethylurea) TK6, increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights into further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.

Using FFPE RNA-Seq with 12 marker genes to evaluate genotoxic and non-genotoxic rat hepatocarcinogens.

INTRODUCTION: Various challenges have been overcome with regard to applying 'omics technologies for chemical risk assessments. Previously we published results detailing targeted mRNA sequencing (RNA-Seq) on a next generation sequencer using intact RNA derived from freshly frozen rat liver tissues. We successfully discriminated genotoxic hepatocarcinogens (GTHCs) from non-genotoxic hepatocarcinogens (NGTHCs) using 11 selected marker genes. Based on this, we next attempted to use formalin-fixed paraffin-embedded (FFPE) pathology specimens for RNA-Seq analyses. FINDINGS: In this study we performed FFPE RNA-Seq to compare a typical GTHC, 2-acetylaminofluorene (AAF) to genotoxicity equivocal p-cresidine (CRE). CRE is used as a synthetic chemical intermediate, and this compound is classified as an IARC 2B carcinogen and is mutagenic in S. typhimurium, which is non-genotoxic to rat livers as assessed by single strand DNA damage analysis. RNA-Seq was used to examine liver FFPE samples obtained from groups of five 10-week-old male F344 rats that were fed with chemicals (AAF: 0.025% and CRE: 1% in food) for 4 weeks or from controls that were fed a basal diet. We extracted RNAs from FFPE samples and RNA-Seq was performed on a MiniSeq (Illumina) using the TruSeq custom RNA panel. AAF induced remarkable differences in the expression of eight genes (Aen, Bax, Btg2, Ccng1, Gdf15, Mbd1, Phlda3 and Tubb4b) from that in the control group, while CRE only induced expression changes in Gdf15, as shown using Tukey's test. Gene expression profiles for nine genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Mbd1, Phlda3, and Plk2) differed.between samples treated with AAF and CRE. Finally, principal component analysis (PCA) of 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) using our previous Open TG-GATE data plus FFPE-AAF and FFPE-CRE successfully differentiated FFPE-AAF, as GTHC, from FFPE-CRE, as NGHTC. CONCLUSION: Our results suggest that FFPE RNA-Seq and PCA are useful for evaluating typical rat GTHCs and NGTHCs.

Preparation of the standard cell lines for reference mutations in cancer gene-panels by genome editing in HEK 293 T/17 cells.

BACKGROUND: Next Generation Sequencer (NGS) is a powerful tool for a high-throughput sequencing of human genome. It is important to ensure reliability and sensitivity of the sequence data for a clinical use of the NGS. Various cancer-related gene panels such as Oncomine™ or NCC OncoPanel have been developed and used for clinical studies. Because these panels contain multiple genes, it is difficult to ensure the performance of mutation detection for every gene. In addition, various platforms of NGS are developed and their cross-platform validation has become necessity. In order to create mutant standards in a defined background, we have used CRISPR/Cas9 genome-editing system in HEK 293 T/17 cells. RESULTS: Cancer-related genes that are frequently used in NGS-based cancer panels were selected as the target genes. Target mutations were selected based on their frequency reported in database, and clinical significance and on the applicability of CRISPR/Cas9 by considering distance from PAM site, and off-targets. We have successfully generated 88 hetero- and homozygous mutant cell lines at the targeted sites of 36 genes representing a total of 125 mutations. CONCLUSIONS: These knock-in HEK293T/17 cells can be used as the reference mutant standards with a steady and continuous supply for NGS-based cancer panel tests from the JCRB cell bank. In addition, these cell lines can provide a tool for the functional analysis of targeted mutations in cancer-related genes in the isogenic background.

Evaluation of 12 mouse marker genes in rat toxicogenomics public data, Open TG-GATEs: Discrimination of genotoxic from non-genotoxic hepatocarcinogens.

Previously, we proposed 12 marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb4b) to discriminate mouse genotoxic hepatocarcinogens (GTHC) from non-genotoxic hepatocarcinogens (NGTHC). This was determined by qPCR and principal component analysis (PCA), as the aim of an in vivo short-term screening for genotoxic hepatocarcinogens. For this paper, we conducted an application study of the 12 mouse marker genes to rat data, Open TG-GATEs (public data). We analyzed five typical rat GTHC (2-acetamodofluorene, aflatoxin B1, 2-nitrofluorene, N-nitrosodiethylamine and N-nitrosomorpholine), and not only seven typical rat NGTHC (clofibrate, ethanol, fenofibrate, gemfibrozil, hexachlorobenzene, phenobarbital and WY-14643) but also 11 non-genotoxic non-hepatocarcinogens (NGTNHC; allyl alcohol, aspirin, caffeine, chlorpheniramine, chlorpropamide, dexamethasone, diazepam, indomethacin, phenylbutazone, theophylline and tolbutamide) from Open TG-GATEs. The analysis was performed at 3, 6, 9 and 24 h after a single administration and 4, 8, 15 and 29 days after repeated administrations. We transferred Open TG-GATEs DNA microarray data into log2 data using the "R Project for Statistical Computing". GTHC-specific dose-dependent gene expression changes were observed and significance assessed with the Williams test. Similar significant changes were observed during 3-24 h and 4-29 days, assessed with Welch's t-test, except not for NGTHC or NGTNHC. Significant differential changes in gene expression were observed between GTHC and NGTHC in 11 genes (except not Tubb4b) and between GTHC and NGTNHC in all 12 genes at 24 h and 10 genes (except Ccnf and Mbd1) at 29 days, per Tukey's test. PCA successfully discriminated GTHC from NGTHC and NGTNHC at 24 h and 29 days. The results demonstrate that 12 previously proposed mouse marker genes are useful for discriminating rat GTHC from NGTHC and NGTNHC from Open TG-GATEs.

Using RNA-Seq with 11 marker genes to evaluate 1,4-dioxane compared with typical genotoxic and non-genotoxic rat hepatocarcinogens.

It has long been unclear whether 1,4-dioxane (DO) is a genotoxic hepatocarcinogen (GTHC). Therefore, the present study aimed to evaluate rat GTHCs and non-genotoxic hepatocarcinogens (NGTHCs) via selected gene expression patterns in the liver, as determined by next generation sequencing-targeted mRNA sequencing (RNA-Seq) and principal component analysis (PCA). Previously, we selected 11 marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) to discriminate GTHCs and NGTHCs. In the present study, we quantified changes in the expression of these genes following DO treatment, and compared them with treatment with two typical rat GTHCs, N-nitrosodiethylamine (DEN) and 3,3'-dimethylbenzidine·2HCl (DMB), and a typical rat NGTHC, di(2-ethylhexyl)phthalate (DEHP). RNA-Seq was conducted on liver samples from groups of five male, 10-week-old F344 rats after 4 weeks' feeding of chemicals in the water or the food. Rats in the control group were given water and a basal diet. Significant changes in gene expression in experimental groups compared with the control group were observed in eight genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Phlda3 and Plk2), as shown by Tukey's test. Gene expression profiles of the 11 genes under DO treatment differed significantly from those with DEN and DMB, as well as DEHP. Gene expression profiles with DO treatment differed partially from those with typical GTHCs for five genes (Bax, Btg2, Cdkn1a, Lrp1 and Plk2) and were substantially different from treatment with a typical NGTHC (DEHP) for nine genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Mbd1, Phlda3 and Tubb4b) as determined by Tukey's test. Finally, PCA successfully differentiated GTHCs from DEHP and DO with the 11 genes. The present results suggest that RNA-Seq and PCA are useful to evaluate rat typical GTHCs and typical NGTHCs. DO was suggested to result in a different intermediate gene expression profile from typical GTHCs and NGTHC.

Collaborative studies in toxicogenomics in rodent liver in JEMS·MMS; a useful application of principal component analysis on toxicogenomics.

Toxicogenomics is a rapidly developing discipline focused on the elucidation of the molecular and cellular effects of chemicals on biological systems. As a collaborative study group of Toxicogenomics/JEMS·MMS, we conducted studies on hepatocarcinogens in rodent liver in which 100 candidate marker genes were selected to discriminate genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens. Differential gene expression induced by 13 chemicals were examined using DNA microarray and quantitative real-time PCR (qPCR), including eight genotoxic hepatocarcinogens [o-aminoazotoluene, chrysene, dibenzo[a,l]pyrene, diethylnitrosamine (DEN), 7,12-dimethylbenz[a]anthracene, dimethylnitrosamine, dipropylnitrosamine and ethylnitrosourea (ENU)], four non-genotoxic hepatocarcinogens [carbon tetrachloride, di(2-ethylhexyl)phthalate (DEHP), phenobarbital and trichloroethylene] and a non-genotoxic non-hepatocarcinogen [ethanol]. Using qPCR, 30 key genes were extracted from mouse livers at 4 h and 28 days following dose-dependent gene expression alteration induced by DEN and ENU: the most significant changes in gene expression were observed at 4 h. Next, we selected key point times at 4 and 48 h from changes in time-dependent gene expression during the acute phase following administration of chrysene by qPCR. We successfully showed discrimination of eight genotoxic hepatocarcinogens [2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitsosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane] from four non-genotoxic hepatocarcinogens [1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, DEHP and furan] using qPCR and principal component analysis. Additionally, we successfully identified two rat genotoxic hepatocarcinogens [DEN and 2,6-dinitrotoluene] from a nongenotoxic-hepatocarcinogen [DEHP] and a non-genotoxic non-hepatocarcinogen [phenacetin] at 4 and 48 h. The subsequent gene pathway analysis by Ingenuity Pathway Analysis extracted the DNA damage response, resulting from the signal transduction of a p53-class mediator leading to the induction of apoptosis. The present review of these studies suggests that application of principal component analysis on the gene expression profile in rodent liver during the acute phase is useful to predict genotoxic hepatocarcinogens in comparison to non-genotoxic hepatocarcinogens and/or non-carcinogenic hepatotoxins.

An active alternative splicing isoform of human mitochondrial 8-oxoguanine DNA glycosylase (OGG1).

Eight alternatively spliced isoforms of human 8-oxoguanine DNA glycosylase (OGG1) (OGG1-1a, -1b, -1c, -2a, -2b, -2c, -2d and -2e) are registered at the National Center for Biotechnology Information (NCBI). OGG1-1a is present in the nucleus, whereas the other seven isoforms are present in the mitochondria. Recombinant OGG1-1a has been purified and enzyme kinetics determined. OGG1(s) in mitochondria have not been fully characterized biochemically until recently. The major mitochondrial OGG1 isoform, OGG1-2a (also named ß-OGG1), has also been expressed and purified; however, its activity is unresolved. Recently, we purified recombinant mitochondrial OGG1-1b and found that it was an active OGG1 enzyme. We reported its enzyme kinetics and compared the results with those of OGG1-1a. The reaction rate constant of OGG1-1b 8-oxoG glycosylase activity (k g) was 8-oxoG:C > > 8-oxoG:T > > 8-oxoG:G > 8-oxoG:A and was similar to that of OGG1-1a under single-turnover conditions ([E] > [S]). Both OGG1-1b and OGG1-1a showed high specificity towards 8-oxoG:C. The reaction rate constant of OGG1-1b N-glycosylase/DNA lyase activity (k gl) was 8-oxoG:C > 8-oxoG:T ≃ 8-oxoG:G > > 8-oxoG:A and that of OGG1-1a was 8-oxoG:C > 8-oxoG:T, 8-oxoG:G and 8-oxoG:A. The k gl of OGG1-1b and OGG1-1a is one order of magnitude lower than the corresponding k g value. OGG1-1b showed an especially low k gl towards 8-oxoG:A. Comparable expression of OGG1-1a and OGG1-1b was detected by RT-PCR in normal human lung tissue and lung cell lines. These results suggest that OGG1-1b is associated with 8-oxoG cleavage in human lung mitochondria and that the mechanism of this repair is similar to that of nuclear OGG1-1a. Currently, the other five mitochondrial OGG1 isoforms have not been isolated. I summarize information on OGG1 isoform mRNAs, coding DNA sequences and amino acid sequences that are archived by the National Center for Biotechnology Information.

Enzyme kinetics of an alternative splicing isoform of mitochondrial 8-oxoguanine DNA glycosylase, ogg1-1b, and compared with the nuclear ogg1-1a.

Eight alternatively spliced isoforms of human 8-oxoguanine DNA glycosylase (OGG1) (OGG1-1a to -1c and -2a to -2e) are registered in the National Center for Biotechnology Information. OGG1(s) in mitochondria have not yet been fully characterized biochemically. In this study, we purified mitochondrial recombinant OGG1-1b protein and compared its activity with nuclear OGG1-1a protein. The reaction rate constant (kg ) of the 7,8-dihydro-8-oxoguanine (8-oxoG) glycosylase activity of OGG1-1b was 8-oxoG:C >> 8-oxoG:T >> 8-oxoG:G > 8-oxoG:A (7.96, 0.805, 0.070, and 0.015 min(-1) , respectively) and that of the N-glycosylase/DNA lyase activity (kgl ) of OGG1-1b was 8-oxoG:C > 8-oxoG:T ≃8-oxoG:G >> 8-oxoG:A (0.286, 0.079, 0.040, and negligible min(-1) , respectively). These reaction rate constants were similar to those of OGG1-1a except for kgl against 8-oxoG:A. APEX nuclease 1 was required to promote DNA strand breakage by OGG1-1b. These results suggest that OGG1-1b is associated with 8-oxoG cleavage in human mitochondria and that the mechanism of this repair is similar to that of nuclear OGG1-1a.

Differential gene expression profiling between genotoxic and non-genotoxic hepatocarcinogens in young rat liver determined by quantitative real-time PCR and principal component analysis.

We recently successfully discriminated mouse genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens via selected gene expression profiling in the mouse liver based on quantitative real-time PCR (qPCR) and statistical analysis using principal component analysis (PCA). In the present study, we applied these candidate marker genes to rat hepatocarcinogens in the rat liver. qPCR analysis of 33 genes was conducted on liver samples from groups of 4 male 4-week-old F344 rats at 4 and 48 h after a single oral administration of chemicals [2 genotoxic hepatocarcinogens: diethylnitrosamine and 2,6-dinitrotoluene; a non-genotoxic hepatocarcinogen: di(2-ethylhexyl)phthalate; and a non-genotoxic non-hepatocarcinogen: phenacetin]. Thirty-two genes exhibited significant changes in their gene expression ratios (experimental group/control group) according to statistical analysis using the Williams' test and the Dunnett's test. The changes appeared to be greater at 4h than at 48 h. Finally, statistical analysis via PCA successfully differentiated the genotoxic hepatocarcinogens from the non-genotoxic hepatocarcinogen and the non-genotoxic non-hepatocarcinogen at 4h based on 16 genes (Ccnf, Ccng1, Cyp4a10, Ddit4l, Egfr, Gadd45g, Gdf15, Hspb1, Igfbp1, Jun, Myc, Net1, Phlda3, Pml, Rcan1 and Tubb2c) and at 48 h based on 10 genes (Aen, Ccng1, Cdkn1a, Cyp21a1, Cyp4a10, Gdf15, Igfbp1, Mdm2, Phlda3 and Pmm1). Eight major biological processes were extracted from a gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oxidative stress, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response, which signals through a Tp53-mediated pathway and leads to the induction of apoptosis. Immunohistochemical analyses for the expression of Cdkn1a and Hmox1 proteins and the level of apoptosis measured by the TUNEL assay in the liver confirmed the aforementioned results. The present results showed that mouse candidate marker genes are applicable for differentiating genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens examined in this paper in the rat liver.

Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR.

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.

Identification of BC005512 as a DNA damage responsive murine endogenous retrovirus of GLN family involved in cell growth regulation.

Genotoxicity assessment is of great significance in drug safety evaluation, and microarray is a useful tool widely used to identify genotoxic stress responsive genes. In the present work, by using oligonucleotide microarray in an in vivo model, we identified an unknown gene BC005512 (abbreviated as BC, official full name: cDNA sequence BC005512), whose expression in mouse liver was specifically induced by seven well-known genotoxins (GTXs), but not by non-genotoxins (NGTXs). Bioinformatics revealed that BC was a member of the GLN family of murine endogenous retrovirus (ERV). However, the relationship to genotoxicity and the cellular function of GLN are largely unknown. Using NIH/3T3 cells as an in vitro model system and quantitative real-time PCR, BC expression was specifically induced by another seven GTXs, covering diverse genotoxicity mechanisms. Additionally, dose-response and linear regression analysis showed that expression level of BC in NIH/3T3 cells strongly correlated with DNA damage, measured using the alkaline comet assay,. While in p53 deficient L5178Y cells, GTXs could not induce BC expression. Further functional studies using RNA interference revealed that down-regulation of BC expression induced G1/S phase arrest, inhibited cell proliferation and thus suppressed cell growth in NIH/3T3 cells. Together, our results provide the first evidence that BC005512, a member from GLN family of murine ERV, was responsive to DNA damage and involved in cell growth regulation. These findings could be of great value in genotoxicity predictions and contribute to a deeper understanding of GLN biological functions.

Genome-wide gene expression profile of jejunal pouch mucosa as a gastric substitute.

BACKGROUND/AIMS: Gastric cancer is the most common cancer in Japan. Genome-wide gene expression in the jejunal pouch mucosa was examined using a DNA microarray and quantitative real-time PCR (qPCR) to evaluate the safety, especially with regard to carcinogenic changes, of the jejunal pouch in patients who showed long-term survival. METHODOLOGY: Biopsy samples of jejunal pouch and jejunal conduit were collected from four patients who had undergone gastrectomy 9 to 13 years previously. Total RNA was extracted, amplified to give complementary RNA, labeled with Cyanine 3-CTP and hybridized with a whole human genome oligo microarray (44k). Gene expression was confirmed partly by qPCR. RESULTS: Although some changes in the expression of 417 reported cancer genes were observed with the DNA microarray, crucial changes related to small intestinal adenocarcinoma were not observed. Changes in the expression of eight genes related to small intestinal adenocarcinoma were also not detected by qPCR. CONCLUSIONS: Crucial changes in the expression of genes related to small intestinal adenocarcinoma were not observed in the jejunal pouch of these four patients with gastric cancer by either DNA microarray or qPCR. The present results support the safety of the use of a jejunal pouch with a food pooling function in patients who show long-term survival after gastrectomy.

Discrimination analysis of human lung cancer cells associated with histological type and malignancy using Raman spectroscopy.

The Raman spectroscopic technique enables the observation of intracellular molecules without fixation or labeling procedures in situ. Raman spectroscopy is a promising technology for diagnosing cancers-especially lung cancer, one of the most common cancers in humans-and other diseases. The purpose of this study was to find an effective marker for the identification of cancer cells and their malignancy using Raman spectroscopy. We demonstrate a classification of cultured human lung cancer cells using Raman spectroscopy, principal component analysis (PCA), and linear discrimination analysis (LDA). Raman spectra of single, normal lung cells, along with four cancer cells with different pathological types, were successfully obtained with an excitation laser at 532 nm. The strong appearance of bands due to cytochrome c (cyt-c) indicates that spectra are resonant and enhanced via the Q-band near 550 nm with excitation light. The PCA loading plot suggests a large contribution of cyt-c in discriminating normal cells from cancer cells. The PCA results reflect the nature of the original cancer, such as its histological type and malignancy. The five cells were successfully discriminated by the LDA.

Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR.

BACKGROUND: Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD), squamous cell carcinoma (SQ), large cell carcinoma (LC), and small cell carcinoma (SC). Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR). RESULTS: We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA) and a normal control lung cell line (MRC-9). From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L). Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA) of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2). The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression. CONCLUSIONS: These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results confirm that qPCR and PCA analysis provide a useful tool for characterizing cancer cell subtypes, and we discuss the possible clinical applications of this approach.

Light sheet direct Raman imaging technique for observation of mixing of solvents.

The light sheet direct Raman (LSDR) imaging technique is used to obtain wide scope, simultaneous images of samples emitting Raman scattered light, without mapping their point-to-point Raman scattering intensities. A prototype system consisting of a background-free electronically tuned Ti:sapphire laser (BF-ETL), band-pass (BP) filters, and a charge-coupled device (CCD) detector is developed in the present study. The LS excitation method enables us to obtain a wide field of Raman view. The BF-ETL allows us to obtain direct Raman images with multiple Raman bands without the need for rearranging the optical settings. The system is used to observe the mixing of pure solvents: carbon tetrachloride (CCl(4)) and chloroform (CHCl(3)), and ethylene glycol (EG) and polyethylene glycol (PEG). LSDR images are successfully obtained within an exposure time of 0.5 s. EG and PEG, whose Raman spectra appear similar, can be distinguished clearly in the images, suggesting that the system has high spectral resolution.

Gastric carcinogenesis by N-Methyl-N-nitrosourea is enhanced in db/db diabetic mice.

In 2005, a Japanese epidemiological study showed that increase in plasma glucose levels is a risk factor for gastric cancer. However, no animal model has hitherto shown any association between diabetes mellitus and neoplasia in the stomach. Diabetic (db/db) mice have obese and diabetic phenotypes, including hyperglycemia, because of disruption of the leptin receptor. In the present study, effects of hyperglycemia and/or hyperinsulinemia on the development of proliferative lesions were therefore examined in db/db mice given N-methyl-N-nitrosourea (MNU). A total of 120 mice were assigned to four groups: Group A, 40 db/db mice with MNU; Group B, 40 + /db mice with MNU; Group C, 30 misty (wild-type) mice with MNU; Group D, 10 db/db mice without MNU. MNU was given at 60 ppm in drinking water for 20 weeks. Subgroups of animals were sacrificed at weeks 21 and 30 and blood samples were collected to measure glucose, insulin, leptin, and adiponectin concentrations. The removed stomachs were fixed in formalin, and embedded in paraffin for histological examination and immunohistochemistry. At week 30 in Groups A, B, C and D, hyperplasia was observed in 100, 79, 57, and 0%, and dysplasia in 91, 43, 71, and 0%, respectively. Adenocarcinomas and pepsinogen-altered pyloric glands (PAPG), putative preneoplastic lesions, were observed only in Group A, at an incidence of 45%. The serum levels of insulin and leptin were also elevated in Group A. Gastric carcinogenesis by MNU was enhanced in db/db mice, possibly in association with hyperinsulinemia and hyperleptinemia.

Dose-dependent alterations in gene expression in mouse liver induced by diethylnitrosamine and ethylnitrosourea and determined by quantitative real-time PCR.

We examined the dose-dependency of gene expression changes for 51 genes in mouse liver treated with two N-nitroso genotoxic hepatocarcinogens, diethylnitrosamine (DEN) and ethylnitrosourea (ENU) by quantitative real-time PCR (qPCR). DEN (3, 9, 27 and 80mg/kg bw) or ENU (6, 17, 50 and 150mg/kg bw) was injected intraperitoneally into groups of five male 9-week-old B6C3F(1) mice and the livers were dissected after 4h and 28 days. Total RNA from pooled livers was reverse-transcribed to cDNA and the amount of each gene was quantified by qPCR. Results were analyzed by hierarchical and k-means clustering and ingenuity pathway analysis (IPA). The most characteristic result was a similar dose-dependency of gene expression changes with DEN and ENU. Twenty-one genes exhibited a distinct dose-dependent increase in expression at 4h for both carcinogens [Bax, Btg2, Ccng1, Cdkn1a, Cyp4a10, Cyp21a1, Fos, Gadd45b, Gdf15, Hmox1, Hspb1, Isg20l1, Jun, Mbd1, Mdm2, Myc, Net1, Plk2, Ppp1r3c, Rcan1 and Tubb2c], although the increase in gene expression due to ENU was generally weaker than that due to DEN. Only Gdf15 showed a dose-dependent increase in expression at 28 days for both carcinogens. The differences between DEN and ENU were in the expression of additional genes (7 for DEN and 8 for ENU). IPA extracted five gene networks: Network-1 included genes related to cancer and cell cycle arrest and associated with Bax, Btg2, Ccng1, Cdkn1a, Gadd45b, Gdf15, Hspb1, Mdm2 and Plk2 and Network-2 was related to DNA replication, recombination, repair and cell death and associated with Cyp21a1, Gdf15, Ppp1r3c, Rcan1 and Tubb2c. The present results show a distinct dose-dependency of gene expression changes induced by DEN and ENU. These changes were associated with cancer, cell cycle arrest, DNA replication, recombination, repair and cell death and were seen not only at 4h but also, for some, at 28 days after administration.

Fluorescence-suppressed Raman technique for quantitative analysis of protein solution using a micro-Raman probe, the shifted excitation method, and partial least squares regression analysis.

A practical Raman analyzing technique with suppression of the strong fluorescent background in order to obtain quantitative information is proposed in the present study. The technique is based on the shifted excitation method and partial least squares regression (PLSR) analysis. The Raman system consists of a single Raman spectrometer, a background-free electrically tunable Ti:Sapphire laser (BF-ETL), and a micro-Raman probe (MRP). The system allows one to obtain reliable shifted excitation Raman spectra with a simple operation. The PLSR analysis successfully provides quantitative information from the obtained spectra with the suppression of random noise including photon shot noise. The present study demonstrates that the technique is effective for extracting quantitative information concealed behind a fluorescent background that is more than 200 times stronger than the Raman signal.