POLQ Overexpression Is Associated with an Increased Somatic Mutation Load and PLK4 Overexpression in Lung Adenocarcinoma.

DNA Polymerase Theta (POLQ) is a DNA polymerase involved in error-prone translesion DNA synthesis (TLS) and error-prone repair of DNA double-strand breaks (DSBs). In the present study, we examined whether abnormal POLQ expression may be involved in the pathogenesis of lung adenocarcinoma (LAC). First, we found overexpression of POLQ at both the mRNA and protein levels in LAC, using data from the Cancer Genome Atlas (TCGA) database and by immunohistochemical analysis of our LAC series. POLQ overexpression was associated with an advanced pathologic stage and an increased total number of somatic mutations in LAC. When H1299 human lung cancer cell clones overexpressing POLQ were established and examined, the clones showed resistance to a DSB-inducing chemical in the clonogenic assay and an increased frequency of mutations in the supF forward mutation assay. Further analysis revealed that POLQ overexpression was also positively correlated with Polo Like Kinase 4 (PLK4) overexpression in LAC, and that PLK4 overexpression in the POLQ-overexpressing H1299 cells induced centrosome amplification. Finally, analysis of the TCGA data revealed that POLQ overexpression was associated with an increased somatic mutation load and PLK4 overexpression in diverse human cancers; on the other hand, overexpressions of nine TLS polymerases other than POLQ were associated with an increased somatic mutation load at a much lower frequency. Thus, POLQ overexpression is associated with advanced pathologic stage, increased somatic mutation load, and PLK4 overexpression, the last inducing centrosome amplification, in LAC, suggesting that POLQ overexpression is involved in the pathogenesis of LAC.

The hypocholesterolemic effects of Cistanche tubulosa extract, a Chinese traditional crude medicine, in mice.

The roots of Cistanche (C.) tubulosa (Orobanchaceae), a parasitic plant that grows in the Taklamakan desert, are traditionally used as medicines and foods in China. We prepared aqueous ethanol extract (CTE) from the roots of C. tubulosa and its hypocholesterolemic effect was evaluated. Using gene chip and RT-PCR analysis of the livers of mice given CTE (400 mg/kg) for 14 days, we found mRNA expression of molecules related to cholesterol transport [apolipoprotein B and very low density lipoprotein (VLDL) receptor] and metabolism [cytochrome P450 side chain cleave (SCC) and steroid 5alpha-reductase 2] were up-regulated. The administration of CTE (400 mg/kg) for 14 days significantly suppressed serum cholesterol elevation in high cholesterol diet-fed mice. The mRNA expressions of VLDL receptor and cytochrome P450 SCC were significantly enhanced. In addition, acteoside, a major constituent of CTE, was found to enhance the mRNA expressions of apolipoprotein B, VLDL receptor, and cytochrome P450 SCC in HepG2 hepatocytes. These results suggest that CTE affects the mRNA expressions of molecules related to cholesterol transport and metabolism and exhibits hypocholesterolemic activity in diet-induced hypercholesterolemia mice. Acteoside was involved in the hypocholesterolemic activity of CTE.

Pharmacogenomics of cardiovascular pharmacology: development of an informatics system for analysis of DNA microarray data with a focus on lipid metabolism.

Genome-wide gene-expression data from DNA-microarray technology and molecular-network data from computational text-mining have led to a paradigm shift in biological research. However, interpretation of the huge amount of data is a bottleneck. We have developed an informatics system, which we refer to as bioSpace Explorer, that can extract pathways and molecules of interest from genome-wide data and show the mutual relationships among these pathways and molecules. Differentiation of 3T3-L1 cells into adipocytes and the action of a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist or alpha-linolenic acid on this process was analyzed with bioSpace Explorer. The results suggested a biological basis for adipocyte differentiation and a strategy to enhance lipid oxidation in adipocytes. Clustered changes of molecules were apparent in the insulin, Wnt, and PPARgamma signaling pathways and in the lipogenesis, lipid oxidation, and lipid transport pathways during cell differentiation. A PPARgamma agonist enhanced lipid oxidation in adipocytes and alpha-linolenic acid gave similar results to the PPARgamma agonist. An analysis of sex hormone and thyroid hormone, in addition to PPARgamma signaling, suggested that these molecules are important for enhancement of lipid oxidation in adipocytes. The results indicate the utility of bioSpace Explorer for biological research on genome-wide molecular networks.

Administration of perilla oil coated with Calshell increases glucagon-like peptide secretion.

Recently, we found that unsaturated long-chain fatty acids (such as alpha-linolenic acid) promote the secretion of glucagon-like peptide-1 (GLP-1) via G protein-coupled receptor GPR120, which is expressed predominantly in the colon. In order to ensure that the triglycerides or free fatty acids, such as alpha-linolenic acid, reach the distal intestinal tract effectively, we developed a Calshell technique. Following single treatment of Calshell perilla oil powder, the GLP-1 secretion level was significantly higher than following vehicle treatment, 120 min after treatment. Next, we examined the effects of long-term Calshell perilla oil powder treatment on GLP-1 secretion. Plasma GLP-1 level of Calshell perilla oil powder treatment was significantly higher than of vehicle treatment for 1, 14, 28 and 56 d. We thereby demonstrated for the first time the utility of Calshell oil powder treatment for effective and sustainable GLP-1 secretion. The Calshell technique is apparently useful as a drug delivery system, since Calshell unsaturated oil powder is protected from gastric acid, reaches enteroendocrine cells in the gastrointestinal tract, and then induces effective incretin secretion.

Serial changes in expression of functionally clustered genes in progression of liver fibrosis in hepatitis C patients.

AIM: To investigate the relationship of changes in expression of marker genes in functional categories or molecular networks comprising one functional category or multiple categories in progression of hepatic fibrosis in hepatitis C (HCV) patients. METHODS: Marker genes were initially identified using DNA microarray data from a rat liver fibrosis model. The expression level of each fibrosis associated marker gene was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) in clinical biopsy specimens from HCV-positive patients (n = 61). Analysis of changes in expression patterns and interactions of marker genes in functional categories was used to assess the biological mechanism of fibrosis. RESULTS: The profile data showed several biological changes associated with progression of hepatic fibrosis. Clustered genes in functional categories showed sequential changes in expression. Several sets of clustered genes, including those related to the extracellular matrix (ECM), inflammation, lipid metabolism, steroid metabolism, and some transcription factors important for hepatic biology showed expression changes in the immediate early phase (F1/F2) of fibrosis. Genes associated with aromatic amino acid (AA) metabolism, sulfur-containing AA metabolism and insulin/ Wnt signaling showed expression changes in the middle phase (F2/F3), and some genes related to glucose metabolism showed altered expression in the late phase of fibrosis (F3/F4). Therefore, molecular networks showing serial changes in gene expression are present in liver fibrosis progression in hepatitis C patients. CONCLUSION: Analysis of gene expression profiles from a perspective of functional categories or molecular networks provides an understanding of disease and suggests new diagnostic methods. Selected marker genes have potential utility for biological identification of advanced fibrosis.

Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis.

AIM: To determine the gene expression profile data for the whole liver during development of dimethylni-trosamine (DMN)-induced hepatic fibrosis. METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells), and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells. RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSC-specific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis, suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocyte-specific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis. CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis.

Nucleotide sequences and organization of the genes for carotovoricin (Ctv) from Erwinia carotovora indicate that Ctv evolved from the same ancestor as Salmonella typhi prophage.

Carotovoricin Er (CtvEr), which is produced by a plant soft rot disease causative agent, Erwinia carotovora subsp. carotovora Er, is a high-molecular-weight bacteriocin showing Myoviridae phage-tail-like morphology with contractile sheath and plural tail fibers. We determined the complete nucleotide sequences of CtvEr genes on the E. carotovora Er chromosome and report that CtvEr genes consist of lysis cassette, major and minor structural protein gene clusters. Four promoters were identified. The lysis gene cassette, which is composed of the genes for lysis enzyme and holin, was also identified and characterized. The nucleotide sequences and organization of the genes for CtvCGE, which is produced by E. carotovora strain CGE234-M403 with the morphology similar to CtvEr, were also determined and compared to that of CtvEr, and it was found that CtvCGE is almost identical to CtvEr except for tail fibers which are involved in the killing spectra of both bacteriocins. We also explain that the gene organization and the deduced amino acid sequences of both carotovoricins are very close to those of prophage, which is lysogenized in the chromosome on Salmonella enterica serovar Typhi CT18. These findings strongly suggest that Ctv evolved as a phage tail-like bacteriocin from a common ancestor with Salmonella typhi prophage.

Replicated studentized-deviate detection applied to the identification of differentially expressed genes in TNF-alpha-stimulated cells.

Among the applications of DNA microarray expression data, identification of differentially expressed genes is widely used to search for genes involved in specific biological processes. Previous studies have shown that replication is necessary to reliably identify differentially expressed genes. In practice, however, further guidelines are required to provide references for experiment design, that is, to fulfill the needs for determining the replication number on a desired confidence level or the detection sensitivity of the microarrays. Here, we performed simulations to build a simple statistical table of general guidelines adapted specifically for identifying differentially expressed genes using replications of a microarray experiment, and we present an accompanying nonparametric method, replicated studentized-deviate detection (RSD). The analysis results of the TNF-alpha-stimulated cells showed that RSD is simple and robust, and that the resulting statistical table is plausible.

A novel hepatic-targeting system for therapeutic cytokines that delivers to the hepatic asialoglycoprotein receptor, but avoids receptor-mediated endocytosis.

PURPOSE: To demonstrate the utilities of a synthetic low-affinity ligand ((Gal)3) for the asialoglycoprotein receptor (ASGP-R) as a hepatic targeting device for therapeutic cytokines. METHODS: The site-specific incorporation of (Gal)3 or a typical high-affinity ligand (GaINAc)3 into IL-2 was catalyzed by microbial transglutaminase. The anti-tumor activities, pharmacokinetic profiles and receptor-mediated endocytosis in hepatocytes of the ligand-IL-2 conjugates were examined in mouse. RESULTS: The (Gal)3 has approximately 50 times lower affinity to ASGP-R than (GalNAc)3. Nevertheless, the antitumor effects were in the order of (Gal)3-IL-2 > unmodified IL-2 > (GalNAc)3-IL-2. The systemic elimination and the hepatic uptake of (GalNAc)374L-2 were more rapid than (Gal)3-IL-2. The ratio of the rate constant representing dissociation from the cell-surface receptor (k(off) to that representing endocytosis of the ligand (k(int) was greater for (Gal)3-IL-2 than (GalNAc)s-IL-2, suggesting that (Gal)3-IL-2 preferably avoids internalization due to its lower affinity to the receptor. The simulation studies demonstrated that (Gal)3-L-2 was present in the hepatic extracellular space for a longer period than (GaINAc)3 IL-2. CONCLUSIONS: The (Gal)3 ligand increases the therapeutic efficacy of IL-2 by enhancing its exposure to the cell-surface. The k(off)/k(int) affects the targeting efficacy of the conjugates to ASGP-R.