Purpose: Expression of organic anion transporting polypeptides (OATPs) 1B1/1B3 in hepatocellular carcinoma (HCC) induces a paradoxical enhancement of gadoxetic acid on liver magnetic resonance imaging (MRI). We examined the expression profile of OATPs with regard to tumor differentiation in a genetically modified H-Ras 12V mouse model of spontaneous HCC that undergoes multistep hepatocarcinogenesis with minimal inter-individual variation. Materials and Methods: Tumor nodules were harvested from transgenic H-Ras 12V mice. Hematoxylin and eosin-stained slides were examined for tumor differentiation and high-grade pathological components (tumor necrosis, thickened trabeculae, or vascular invasion). Immunohistochemistry of OATP 1B1/1B3 was performed, and OATP expression was assessed. Results: We examined well-differentiated HCCs (n=59) in which high-grade pathological components were absent (n=49) or present (n=10). Among the well-differentiated HCCs without high-grade pathological components (n=49), OATP expression was negative, weak positive, and moderate positive in 23, 17, and nine cases, respectively. Among the well-differentiated HCCs with highgrade pathological components (n=10), OATP expression was negative, weak positive, and moderate positive in one, two, and seven cases, respectively. The ratio of positive OATP 1B1/1B3 expressing tumors was higher in HCCs with high-grade pathological components than in those without high-grade pathological components (p=0.004). Conclusion Our findings support those of previous clinical studies that have reported the frequent appearance of gadoxetic acidenhanced MRI in moderately differentiated HCC.

Purpose: Expression of organic anion transporting polypeptides (OATPs) 1B1/1B3 in hepatocellular carcinoma (HCC) induces a paradoxical enhancement of gadoxetic acid on liver magnetic resonance imaging (MRI). We examined the expression profile of OATPs with regard to tumor differentiation in a genetically modified H-Ras 12V mouse model of spontaneous HCC that undergoes multistep hepatocarcinogenesis with minimal inter-individual variation. Materials and Methods: Tumor nodules were harvested from transgenic H-Ras 12V mice. Hematoxylin and eosin-stained slides were examined for tumor differentiation and high-grade pathological components (tumor necrosis, thickened trabeculae, or vascular invasion). Immunohistochemistry of OATP 1B1/1B3 was performed, and OATP expression was assessed. Results: We examined well-differentiated HCCs (n=59) in which high-grade pathological components were absent (n=49) or present (n=10). Among the well-differentiated HCCs without high-grade pathological components (n=49), OATP expression was negative, weak positive, and moderate positive in 23, 17, and nine cases, respectively. Among the well-differentiated HCCs with highgrade pathological components (n=10), OATP expression was negative, weak positive, and moderate positive in one, two, and seven cases, respectively. The ratio of positive OATP 1B1/1B3 expressing tumors was higher in HCCs with high-grade pathological components than in those without high-grade pathological components (p=0.004). Conclusion Our findings support those of previous clinical studies that have reported the frequent appearance of gadoxetic acidenhanced MRI in moderately differentiated HCC.

Genome editing has recently emerged as a powerful tool for generating mutant mice. Small deletions of nucleotides in the target genes are frequently found in CRISPR/Cas9 mediated mutant mice. However, there are very few reports analyzing the phenotypes in small deleted mutant mice generated by CRISPR/Cas9. In this study, we generated a mutant by microinjecting sgRNAs targeting the IL2 receptor γ gene and Cas9 protein, into the cytoplasm of IVF-derived NOD.CB17/Prkdcscid/JKrb (NOD/SCID) mice embryos, and further investigated whether a 2 bp deletion of the IL2 receptor γ gene affects severe deficiency of immune cells as seen in NOD/LtSz-scid IL2 receptor γ−/− (NSG) mice. Our results show that the thymus weight of mutant mice is significantly less than that of NOD/SCID mice, whereas the spleen weight was marginally less. T and B cells in the mutant mice were severely deficient, and NK cells were almost absent. In addition, tumor growth was exceedingly increased in the mutant mice transplanted with HepG2, Raji and A549 cells, but not in nude and NOD/SCID mice. These results suggest that the NOD/SCID mice with deletion of 2 bp in the IL2 receptor γ gene shows same phenotype as NSG mice. Taken together, our data indicates that small deletions by genome editing is sufficient to generate null mutant mice.

The hepatitis B virus (HBV) infects approximately 240 million people worldwide, causing chronic liver disease (CLD) and liver cancer. Although numerous studies have been performed to date, unfortunately there is no conclusive drug or treatment for HBV induced liver disease. The hepatitis B virus X (HBx) is considered a key player in inducing CLD and hepatocellular carcinoma (HCC). We generated transgenic (Tg) mice expressing HBx protein, inducing HCC at the age of 11–18 months. The incidence of histological phenotype, including liver tumor, differed depending on the genetic background of HBx Tg mice. Fatty change and tumor generation were observed much earlier in livers of HBx Tg hybrid (C57BL/6 and CBA) (HBx-Tg hybrid) mice than in HBx Tg C57BL/6 (HBx-Tg B6) mice. Inflammation was also enhanced in the HBx-Tg B6 mice as compared to HBx-Tg hybrid mice. HBx may be involved in inducing and promoting hepatic steatosis, glycemia, hepatic fibrosis, and liver cancer. Reactive oxygen species (ROS) generation was remarkably increased in livers of HBx Tg young mice compared to young wild type control mice.Previous studies on HBx Tg mice indicate that the HBx-induced ROS plays a role in inducing and promoting CLD and HCC.

The hepatitis B virus (HBV) infects approximately 240 million people worldwide, causing chronic liver disease (CLD) and liver cancer. Although numerous studies have been performed to date, unfortunately there is no conclusive drug or treatment for HBV induced liver disease. The hepatitis B virus X (HBx) is considered a key player in inducing CLD and hepatocellular carcinoma (HCC). We generated transgenic (Tg) mice expressing HBx protein, inducing HCC at the age of 11–18 months. The incidence of histological phenotype, including liver tumor, differed depending on the genetic background of HBx Tg mice. Fatty change and tumor generation were observed much earlier in livers of HBx Tg hybrid (C57BL/6 and CBA) (HBx-Tg hybrid) mice than in HBx Tg C57BL/6 (HBx-Tg B6) mice. Inflammation was also enhanced in the HBx-Tg B6 mice as compared to HBx-Tg hybrid mice. HBx may be involved in inducing and promoting hepatic steatosis, glycemia, hepatic fibrosis, and liver cancer. Reactive oxygen species (ROS) generation was remarkably increased in livers of HBx Tg young mice compared to young wild type control mice.Previous studies on HBx Tg mice indicate that the HBx-induced ROS plays a role in inducing and promoting CLD and HCC.

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP(C230X−/−) mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP(C230X−/−) mice displayed reduced hyperlocomotion in response to intrastriatal injection of the μ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.
Analgesia* ; Animals ; Brain ; Codon, Nonsense ; Dopamine ; Enkephalin, Ala(2)-MePhe(4)-Gly(5)- ; Ethylnitrosourea ; Fertilization in Vitro ; Gene Library ; Mass Screening ; Mice ; Morphine* ; Phosphotransferases ; Protein Tyrosine Phosphatases ; Receptors, Opioid ; Reward* ; Street Drugs ; Substance Withdrawal Syndrome*

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.
Animals ; Antineoplastic Agents/*therapeutic use ; Apoptosis/drug effects ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/metabolism/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Humans ; Lung/drug effects/metabolism/pathology ; Lung Neoplasms/*drug therapy/genetics/metabolism/pathology ; Mice, Inbred BALB C ; Mice, Nude ; Oxidative Stress/drug effects ; Peroxiredoxins/*genetics/metabolism ; Quinazolines/*therapeutic use ; Reactive Oxygen Species/metabolism

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.
Animals ; Antineoplastic Agents/*therapeutic use ; Apoptosis/drug effects ; Carcinoma, Non-Small-Cell Lung/*drug therapy/genetics/metabolism/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Humans ; Lung/drug effects/metabolism/pathology ; Lung Neoplasms/*drug therapy/genetics/metabolism/pathology ; Mice, Inbred BALB C ; Mice, Nude ; Oxidative Stress/drug effects ; Peroxiredoxins/*genetics/metabolism ; Quinazolines/*therapeutic use ; Reactive Oxygen Species/metabolism

Peroxiredoxins (Prxs) are a family of antioxidant proteins that reduce peroxide levels by using reducing agents such as thioredoxin. These proteins were characterized to have a number of cellular functions, including cell proliferation and differentiation and protection of specific proteins from oxidative damage. Thus, it is important to clarify the physiological role of Prxs by generating mouse models deficient in each Prx to better understand the in vivo function of Prxs. We have generated and characterized mice deficient in Prx I and II that are abundantly expressed in almost all types of cells. The Prx II-/- mice were healthy in appearance and fertile, however showed several pathophysiological disorders. Using the mice, we found that Prx II is an essential antioxidant enzyme that prevents oxidative stress in erythropoiesis, protects against endotoxin-induced lethal shock, regulates platelet-derived growth factor signaling and angiogenesis, inhibits cellular senescence, preserves cognitive function against age-linked hippocampal oxidative damage and exacerbates tumorigenesis in a liver cancer mouse model. The Prx I-/- mice were also healthy in appearance and fertile like Prx II-/- mice. With the mice, we found that Prx I suppresses K-ras-driven lung tumorigenesis by opposing the redox-sensitive extracellular-signal-regulated kinase/cyclin D1 pathway and plays concerted action with sulfiredoxin in preventing against alcohol-induced oxidative injury in the mouse liver. The results obtained suggest that Prx I and II are essential antioxidant enzymes for maintaining redox homeostasis in mice.
Animals ; Antioxidants ; Cell Aging ; Cell Proliferation ; Cell Transformation, Neoplastic ; Erythropoiesis ; Homeostasis ; Humans ; Liver ; Liver Neoplasms ; Lung ; Mice ; Mice, Knockout ; Oxidation-Reduction ; Oxidative Stress ; Peroxiredoxins ; Platelet-Derived Growth Factor ; Proteins ; Reducing Agents ; Shock ; Thioredoxins

Retinoids regulate not only various cell functions including proliferation and differentiation but also glucose and lipid metabolism. After we observed a marked up-regulation of cellular retinol-binding protein-I (CRBP-I) in the liver of hepatitis B virus x antigen (HBx)-transgenic (HBx Tg) mice which are prone to hepatocellular carcinoma (HCC) and fatty liver, we aimed to evaluate retinoid pathway, including genes for the retinoid physiology, CRBP-I protein expression, and retinoid levels, in the liver of HBx Tg mice. We also assessed the effect of chronic metformin treatment on HCC development in the mice. Many genes involved in hepatic retinoid physiology, including CRBP-I, were altered and the tissue levels of retinol and all-trans retinoic acid (ATRA) were elevated in the liver of HBx Tg mice compared to those of wild type (WT) control mice. CRBP-I protein expression in liver, but not in white adipose tissue, of HBx Tg mice was significantly elevated compared to WT control mice while CRBP-I protein expressions in the liver and WAT of high-fat fed obese and db/db mice were comparable to WT control mice. Chronic treatment of HBx Tg mice with metformin did not affect the incidence of HCC, but slightly increased hepatic CRBP-I level. In conclusion, hepatic CRBP-I level was markedly up-regulated in HCC-prone HBx Tg mice and neither hepatic CRBP-I nor the development of HCC was suppressed by metformin treatment.
Adipose Tissue, White ; Animals ; Carcinoma, Hepatocellular* ; Fatty Liver ; Hepatitis B virus ; Incidence ; Lipid Metabolism ; Liver* ; Metformin* ; Mice ; Mice, Transgenic* ; Retinoids ; Retinol-Binding Proteins, Cellular* ; Trans-Activators ; Tretinoin ; Up-Regulation ; Vitamin A