Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which ...must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice.
In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32-87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH.
Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.
AimTo investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions. MethodsChimeric mice with humanized liver ...were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC-MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography. ResultsCYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition. ConclusionIn the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation.
Background & Aims
Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10–20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be ...welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti‐death receptor (DR)5 monoclonal antibody (mAb) on HCC.
Methods
We first assessed the side effects of anti‐DR5 mAb administration in vivo by giving various doses of anti‐DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1‐6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti‐DR5 mAb or a combination. Next, one million Hepa1‐6 cells were transplanted into C.B17‐SCID‐beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti‐DR5 mAb alone, (3) sirolimus alone and (4) anti‐DR5 mAb + sirolimus.
Results
Anti‐DR5 mAb (200 and 300 μg/day) induced liver dysfunction with partial necrosis of the liver, but 100 μg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti‐DR5 mAb lysed Hepa1‐6 and Huh7 cells in a dose‐dependent manner, and combinations of sirolimus and anti‐DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups.
Conclusions
Combining sirolimus and low‐dose anti‐DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC.
Summary
The severe combined immunodeficiency/albumin linked‐urokinase type plasminogen activator (SCID/Alb‐uPA) human liver chimeric mouse model has added a new dimension to studies of liver based ...human diseases and has important potential for study of human hepatic drug metabolism. However, it remains unclear if natural killer (NK) cell in SCID/Alb‐uPA mice has an important negative impact on engraftment and expansion of human hepatocytes after transplantation. Here, we explore the role of mouse NK cells in the rejection of transplanted human hepatocytes in SCID/Alb‐uPA mice. We assessed NK cell activity in vivo, using 125I‐iodo‐2′‐deoxyuridine incorporation assay. Low serum human alpha‐1 antitrypsin (hAAT, <10 μg/ml) recipients, representing graft failure, showed resistance to engraftment of MHC class I knockout marrow (indicating high NK cell activity), while NK cell‐depleted low hAAT recipients and high hAAT (>100 μg/ml) recipients accepted MHC class I knockout marrow, indicating a correlation between low NK cell activity, in vivo, and high level human hepatocyte engraftment. We also showed that higher level engraftment of human hepatocytes was achieved in both NK cell‐depleted SCID/Alb‐uPA mice and Rag2−/−γc−/−/Alb‐uPA (T,B and NK cell deficient) mice compared with untreated SCID/Alb‐uPA mice. These results support a critical role for mouse NK cells in the rejection of human hepatocytes xenotransplanted to immunodeficient mice.
Carbohydrate response element binding protein (ChREBP) regulates insulin-independent de novo lipogenesis. Recently, a novel ChREBPbeta isoform was identified. The purpose of the current study was to ...define the effect of dietary carbohydrates (CHO) and obesity on the transcriptional activity of ChREBP isoforms and their respective target genes. Mice were subjected to fasting-refeeding of high-CHO diets. In all three CHO-refeeding groups, mice failed to induce ChREBPalpha, yet ChREBPbeta increased 10- to 20-fold. High-fat fed mice increased hepatic ChREBPbeta mRNA expression compared to chow-fed along with increased protein expression. To better assess the independent effect of fructose on ChREBPalpha/beta activity, HepG2 cells were treated with fructose ± a fructose-1,6-bisphosphatase inhibitor to suppress gluconeogenesis. Fructose treatment in the absence of gluconeogenesis resulted in increased ChREBP activity. To confirm the existence of ChREBPbeta in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREBPalpha and -beta was determined. As with the animal models, glucose induced ChREBPbeta expression while ChREBPalpha was decreased. Taken together, ChREBPbeta is more responsive to changes in dietary CHO availability than the -alpha isoform. Diet-induced obesity increases basal expression of ChREBPbeta, which may increase the risk of developing hepatic steatosis, and fructose-induced activation is independent of gluconeogenesis.
Background We have previously described the existence of two phenotypically distinct cell subsets in ALK-positive anaplastic large cell lymphoma (ALK + ALCL) based on their differential ...responsiveness to a Sox2 reporter (SRR2), with reporter-responsive (RR) cells being more tumorigenic and chemoresistant than reporter-unresponsive (RU) cells. However, the regulator(s) of RU/RR dichotomy are not identified. In this study, we aim to delineate the key regulator(s) of RU/RR dichotomy. Methods JASPER motif match analysis was used to identify the putative factors binding to SRR2 sequence. SRR2 probe pull-down assay and quantitate real-time PCR were performed to analyze the regulation of Sox2 transcriptional activity by MYC. Methylcellulose colony formation assay, chemoresistance to doxorubicin and mouse xenograft study were performed to investigate the biological functions of MYC. PCR array and western blotting were executed to study related signaling pathways that regulate MYC expression. Immunofluorescence and immunohistochemistry assay were initiated to evaluate the expression of MYC and its correlation with its regulator by chi-square test analysis in human primary tumor cells. Results We identified MYC as a potential regulator of RU/RR dichotomy. In support of its role, MYC was highly expressed in RR cells compared to RU cells, and inhibition of MYC substantially decreased the Sox2/SRR2 binding, Sox2 transcriptional activity, chemoresistance, and methylcellulose colony formation. In contrast, enforced expression of MYC in RU cells conferred the RR phenotype. The Wnt/beta-catenin pathway, a positive regulator of MYC, was highly active in RR but not RU cells. While inhibition of this pathway in RR cells substantially decreased MYC expression and SRR2 reporter activity, experimental activation of this pathway led to the opposite effects in RU cells. Collectively, our results support a model in which a positive feedback loop involving Wnt/beta-catenin/MYC and Sox2 contributes to the RR phenotype. In a mouse xenograft model, RU cells stably transfected with MYC showed upregulation of the Wnt/beta-catenin/MYC/Sox2 axis and increased tumorigenecity. Correlating with these findings, there was a significant correlation between the expression of active beta-catenin and MYC in ALK + ALCL primary tumor cells. Conclusions A positive feedback loop involving the Wnt/beta-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subset in ALK + ALCL. Keywords: Intra-tumoral heterogeneity, MYC, Sox2, Wnt/beta-catenin, Cancer stemness, ALK-positive anaplastic large cell lymphoma
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