Inhibition of MYC sensitizes cells to doxorubicin in RR cells. (a) The left panel showed that RR ... more Inhibition of MYC sensitizes cells to doxorubicin in RR cells. (a) The left panel showed that RR cells originated from SupM2 and Karpas 299 were treated with varying dosages of MYC inhibitor 10074-G5 for 48 h, and then the cell growth was assessed by the MTS assay. The dosage of 5 μM 10074-G5 was chosen for the following drug combination study. The right panel showed that RR cells were treated with varying dosages of doxorubicin for 48 h, and then the cell growth was assessed by the MTS assay. Fifty and 20 ng/mL doxorubicin were chosen in RR derived from Karpas 299 and SupM2, respectively, for the following drug combination study. (b, c) The cell cycle analysis was performed to assess the Sub G0/1 fraction in RR cells derived from SupM2 induced by 20 ng/mL doxorubicin, 5 μM 10074-G5, or combination of doxorubicin and 10074-G5 for 48 h; Cells with DMSO treatment were included as a control. (d) The PI staining assay was also performed in the experiment described above. (e) RU and RR cells were treated with varying doses of 10074-G5 for 72 h, then followed by the MTS assay to assess the cell growth. (PDF 341 kb)
in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREB... more in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREBPα and-β was determined. As with the animal models, glucose induced ChREBPβ expression while ChREBPα was decreased. Taken together, ChREBPβ is more responsive to changes in dietary CHO availability than the-α isoform. Diet-induced obesity increases basal expression of ChREBPβ, which may increase the risk of developing hepatic steatosis, and fructose-induced activation is independent of gluconeogenesis.
Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. ... more Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV. Methods: One million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked-urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non-chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post-HCMV inoculation, or human liver natural killer (NK) cells (20 ¥ 10 6 cells/mouse, i.v.) 1 day prior to HCMV inoculation. Results: Chimeric mice that received HCMV showed high plasma titers of HCMV DNA on days 1 and 6 that became undetectable by day 11 post-inoculation. In contrast, nontransplanted mice had only residual plasma inoculum detection at day 1 and no detectable viremia thereafter. The levels of HCMV DNA were reduced by ganciclovir treatment or by human liver NK cell adoptive transfer, while HCMV-infected chimeric mice that were not treated sustained viremia during the follow up. Conclusion: Human liver chimeric mice provide an in vivo model for the study of acute HCMV infection of hepatocytes.
Background: We have previously described the existence of two phenotypically distinct cell subset... more 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/β-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/β-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/β-catenin/MYC/Sox2 axis and increased tumorigenecity. Correlating with these findings, there was a significant correlation between the expression of active β-catenin and MYC in ALK + ALCL primary tumor cells.
Human hepatocyte transplantation is an alternative treatment for acute liver failure and liver di... more Human hepatocyte transplantation is an alternative treatment for acute liver failure and liver diseases involving enzyme deficiencies. Although it has been successfully applied in selected recipients, both isolation and transplantation outcomes have the potential to be improved by better donor selection. This study assessed the impact of various donor variables on isolation outcomes (yield and viability) and posttransplant engraftment, using the SCID/Alb-uPA (severe combined immunodeficient/urokinase type plasminogen activator under the control of an albumin promoter) human liver chimeric mouse model. Human hepatocytes were obtained from 90 human liver donor specimens and were transplanted into 3942 mice. Multivariate analysis revealed improved viability with younger donors (P ¼ 0.038) as well as with shorter warm ischemic time (P ¼ 0.012). Hepatocyte engraftment, assessed by the posttransplant level of serum human a1-antitrypsin, was improved with shorter warm ischemia time. Hepatocytes isolated from older donors (!60 years) had lower viability and posttransplant engraftment (P 0.01). In conclusion, the selection of young donors (<60 years) and rapid liver specimen retrieval, allowing for shorter warm ischemia time, are key determinants for the success of both the isolation of high viability human hepatocytes and their subsequent posttransplantation capacity for engraftment and expansion.
The severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID/Al... more 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 125 I-iodo-2¢-deoxyuridine incorporation assay. Low serum human alpha-1 antitrypsin (hAAT, <10 lg/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 lg/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)/) cc)/) /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.
Inhibition of MYC sensitizes cells to doxorubicin in RR cells. (a) The left panel showed that RR ... more Inhibition of MYC sensitizes cells to doxorubicin in RR cells. (a) The left panel showed that RR cells originated from SupM2 and Karpas 299 were treated with varying dosages of MYC inhibitor 10074-G5 for 48 h, and then the cell growth was assessed by the MTS assay. The dosage of 5 μM 10074-G5 was chosen for the following drug combination study. The right panel showed that RR cells were treated with varying dosages of doxorubicin for 48 h, and then the cell growth was assessed by the MTS assay. Fifty and 20 ng/mL doxorubicin were chosen in RR derived from Karpas 299 and SupM2, respectively, for the following drug combination study. (b, c) The cell cycle analysis was performed to assess the Sub G0/1 fraction in RR cells derived from SupM2 induced by 20 ng/mL doxorubicin, 5 μM 10074-G5, or combination of doxorubicin and 10074-G5 for 48 h; Cells with DMSO treatment were included as a control. (d) The PI staining assay was also performed in the experiment described above. (e) RU and RR c...
The Wnt/β-catenin pathway is more active in RR cells than RU cells derived from SupM2. The Wnt pa... more The Wnt/β-catenin pathway is more active in RR cells than RU cells derived from SupM2. The Wnt pathway-specific oligonucleotide PCR array was performed in RU and RR cells derived from SupM2 cells. The data suggested that 24 out of 87 genes related with the Wnt pathway were more highly expressed in mRNA level (>1.4-fold) in RR cells than in RU cells. Note that one time experiment was performed in this study. (PDF 102 kb)
RU cells co-cultured with diluted (10:1) RR cells or parental SupM2 cells did not show significan... more RU cells co-cultured with diluted (10:1) RR cells or parental SupM2 cells did not show significantly increased SRR2 luciferase activity or upregulated MYC expression. SupM2-RU cells (50,000 cells in total seeded in the upper chamber) and various ratios (RU/RR = 1:1, 2:1, 5:1, 10:1) of SupM2-RR cells (seeded in the lower chamber) were co-cultured in 6-well transwell plate for 72 h. SupM2-RU cells co-cultured with the same number of SupM2-RU cells or parental SupM2 cells were included in this experiment. MYC protein expression was also assessed in this experiment. The results suggested that RU cells co-cultured with the same number of RR cells showed significantly increased SRR2 luciferase activity and a robust increased MYC expression, as compared to negative control. In contrast, RU cells co-cultured with diluted RR cells (e.g., 10:1, containing 5000 of RR cells and 45,000 of RU cells) or parental SupM2 cells did not exhibit significantly increased SRR2 luciferase activity and only ...
NPM-ALK/STAT3 is not differentially activated or expressed between RU and RR cells. (a) The prote... more NPM-ALK/STAT3 is not differentially activated or expressed between RU and RR cells. (a) The protein levels of pALKY1601, ALK, pSTAT3Y705, and STAT3 in RU and RR cells derived from SupM2 and Karpas 299. (b, c) RR and RU cells were treated with either DMSO, or 100 nM ALK inhibitor crizotinib, or 10 nM STAT3 inhibitor stattic for 24 h. The western blots were employed to assess the expression/activation levels of NPM-ALK, STAT3, MYC, and Sox2 in both RU and RR cells. The SRR2 luciferase activity in RU and RR cells was also evaluated by the luciferase assay. (PDF.488KB) (PDF 458 kb)
We have previously described the existence of two phenotypically distinct cell subsets in ALK-pos... more 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. 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. Immunofluorescenc...
We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the diffe... more We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the differential responsiveness to a Sox2 reporter (SRR2), with cells responsive to SRR2 (RR) being more stem-like than unresponsive cells (RU). Here, we report that RR cells derived from MCF7 and ZR751 displayed a higher tolerance to oxidative stress than their RU counterparts, supporting the concept that the RR phenotype correlates with cancer stemness. Sox2 is directly implicated in this differential H2O2 tolerance, since siRNA knockdown of Sox2 in RR cells leveled this difference. Interestingly, H2O2 converted a proportion of RU cells into RR cells, as evidenced by their expression of luciferase and GFP, markers of SRR2 activity. Compared to RU cells, converted RR cells showed a significant increase in mammosphere formation and tolerance to H2O2. Converted RR cells also adopted the biochemical features of RR cells, as evidenced by their substantial increase in Sox2-SRR2 binding and the expres...
Cytopathic effects are currently believed to contribute to HCV-induced liver injury and are readi... more Cytopathic effects are currently believed to contribute to HCV-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species (ROS), which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase (AMPK). The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate ROS-mediated activation of AMPK in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated and β-oxidation is enhanced in these cells. A striking increase in nuclear PPARα, which plays a dominant role in the expression of β-oxidation genes after ligand-induced activation, was also observed and we provide evidence that PPARα is const...
Carbohydrate response element binding protein (ChREBP) regulates insulin‐independent de novo lipo... more Carbohydrate response element binding protein (ChREBP) regulates insulin‐independent de novo lipogenesis. Recently, a novel ChREBPβ 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 ChREBPα, yet ChREBPβ increased 10‐ to 20‐fold. High‐fat fed mice increased hepatic ChREBPβ mRNA expression compared to chow‐fed along with increased protein expression. To better assess the independent effect of fructose on ChREBPα/β 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 ChREBPβ in human tissue, primary hepatocytes were incubated wit...
The tumorigenicity of most cases of ALK-positive anaplastic large cell lymphoma (ALK+ALCL) is dri... more The tumorigenicity of most cases of ALK-positive anaplastic large cell lymphoma (ALK+ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Since it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ALCL, thereby leaving the STAT3 signaling unchecked. Compared to normal T-cells, ALK+ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ALCL cells, as it effectively up-regulated γ-interferon, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 sign...
Cyclosporine A (CSA) has potent effects against hepatitis C virus (HCV) in vitro, but its clinica... more Cyclosporine A (CSA) has potent effects against hepatitis C virus (HCV) in vitro, but its clinical efficacy after liver transplantation (LT) is uncertain. We evaluated the impact of CSA and tacrolimus (TAC) with or without concomitant interferon (IFN) therapy on serum HCV titers in a chimeric mouse model of HCV infection. Six groups of HCV-infected mice received only the vehicle, IFN, CSA, CSA and IFN, TAC, or TAC and IFN for 4 weeks. The quantitative HCV polymerase chain reaction levels were determined after 1, 2, and 4 weeks of drug administration. There were no significant differences in the HCV titers after 4 weeks of treatment between the non-IFN-treated groups (log HCV titers: 3.5 6 0.3 for the vehicle group, 4.4 6 0.6 for the CSA group, and 4.3 6 0.4 for the TAC group, P ¼ 0.3). Although IFN had a consistent effect of reducing HCV titers across the groups, there was no significant impact of CSA on HCV levels when it was used alone or in combination with IFN at any time point. The 4-week HCV titers were as follows: 3.2 6 0.3 for the IFN group, 4.7 6 0.4 for the CSA/IFN group, and 4.0 6 0.5 for the TAC/IFN group (P ¼ 0.07). The CSA/IFN and TAC/IFN groups did not differ significantly (P ¼ 0.6). Six of the 7 animals in the IFN group (85.7%) had an HCV titer decline ! 1 log, whereas in the test groups (CSA/IFN and TAC/IFN), 6 of 9 animals (66.7%) achieved a 1-log decline in the HCV titer (P ¼ 1). Using this animal model, we could find no evidence supporting the routine use of CSA after LT in HCV-infected patients.
Background & AimsDespite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10–2... more Background & AimsDespite 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.MethodsWe 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.ResultsAnti‐DR5 mAb (200 and 300 μg/day) induced liver dysfunction with partial necrosi...
AimCytomegalovirus is a common viral pathogen that influences the outcome of organ transplantatio... more AimCytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV.MethodsOne million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked‐urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non‐chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post‐HCMV inoculation, or human liver natural killer (NK) cells (20 × 106 cells/mouse, i.v.) 1 day prior to HCMV inoculation.ResultsChimeric mice that received HCMV showed high ...
Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with in... more Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC 50) values of 0.01 to 0.14 M for genotype 1, with half maximal effective concentration (EC 50 s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 ؎ 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P ؍ 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV-796 patients treated for over 8 weeks, resulting in suspension of further trial activity. Conclusion: The RNA-dependent RNA polymerase inhibitor HCV796 demonstrated potent anti-HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose-related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti-HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment.
Inhibition of MYC sensitizes cells to doxorubicin in RR cells. (a) The left panel showed that RR ... more Inhibition of MYC sensitizes cells to doxorubicin in RR cells. (a) The left panel showed that RR cells originated from SupM2 and Karpas 299 were treated with varying dosages of MYC inhibitor 10074-G5 for 48 h, and then the cell growth was assessed by the MTS assay. The dosage of 5 μM 10074-G5 was chosen for the following drug combination study. The right panel showed that RR cells were treated with varying dosages of doxorubicin for 48 h, and then the cell growth was assessed by the MTS assay. Fifty and 20 ng/mL doxorubicin were chosen in RR derived from Karpas 299 and SupM2, respectively, for the following drug combination study. (b, c) The cell cycle analysis was performed to assess the Sub G0/1 fraction in RR cells derived from SupM2 induced by 20 ng/mL doxorubicin, 5 μM 10074-G5, or combination of doxorubicin and 10074-G5 for 48 h; Cells with DMSO treatment were included as a control. (d) The PI staining assay was also performed in the experiment described above. (e) RU and RR cells were treated with varying doses of 10074-G5 for 72 h, then followed by the MTS assay to assess the cell growth. (PDF 341 kb)
in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREB... more in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREBPα and-β was determined. As with the animal models, glucose induced ChREBPβ expression while ChREBPα was decreased. Taken together, ChREBPβ is more responsive to changes in dietary CHO availability than the-α isoform. Diet-induced obesity increases basal expression of ChREBPβ, which may increase the risk of developing hepatic steatosis, and fructose-induced activation is independent of gluconeogenesis.
Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. ... more Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV. Methods: One million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked-urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non-chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post-HCMV inoculation, or human liver natural killer (NK) cells (20 ¥ 10 6 cells/mouse, i.v.) 1 day prior to HCMV inoculation. Results: Chimeric mice that received HCMV showed high plasma titers of HCMV DNA on days 1 and 6 that became undetectable by day 11 post-inoculation. In contrast, nontransplanted mice had only residual plasma inoculum detection at day 1 and no detectable viremia thereafter. The levels of HCMV DNA were reduced by ganciclovir treatment or by human liver NK cell adoptive transfer, while HCMV-infected chimeric mice that were not treated sustained viremia during the follow up. Conclusion: Human liver chimeric mice provide an in vivo model for the study of acute HCMV infection of hepatocytes.
Background: We have previously described the existence of two phenotypically distinct cell subset... more 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/β-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/β-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/β-catenin/MYC/Sox2 axis and increased tumorigenecity. Correlating with these findings, there was a significant correlation between the expression of active β-catenin and MYC in ALK + ALCL primary tumor cells.
Human hepatocyte transplantation is an alternative treatment for acute liver failure and liver di... more Human hepatocyte transplantation is an alternative treatment for acute liver failure and liver diseases involving enzyme deficiencies. Although it has been successfully applied in selected recipients, both isolation and transplantation outcomes have the potential to be improved by better donor selection. This study assessed the impact of various donor variables on isolation outcomes (yield and viability) and posttransplant engraftment, using the SCID/Alb-uPA (severe combined immunodeficient/urokinase type plasminogen activator under the control of an albumin promoter) human liver chimeric mouse model. Human hepatocytes were obtained from 90 human liver donor specimens and were transplanted into 3942 mice. Multivariate analysis revealed improved viability with younger donors (P ¼ 0.038) as well as with shorter warm ischemic time (P ¼ 0.012). Hepatocyte engraftment, assessed by the posttransplant level of serum human a1-antitrypsin, was improved with shorter warm ischemia time. Hepatocytes isolated from older donors (!60 years) had lower viability and posttransplant engraftment (P 0.01). In conclusion, the selection of young donors (<60 years) and rapid liver specimen retrieval, allowing for shorter warm ischemia time, are key determinants for the success of both the isolation of high viability human hepatocytes and their subsequent posttransplantation capacity for engraftment and expansion.
The severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID/Al... more 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 125 I-iodo-2¢-deoxyuridine incorporation assay. Low serum human alpha-1 antitrypsin (hAAT, <10 lg/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 lg/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)/) cc)/) /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.
Inhibition of MYC sensitizes cells to doxorubicin in RR cells. (a) The left panel showed that RR ... more Inhibition of MYC sensitizes cells to doxorubicin in RR cells. (a) The left panel showed that RR cells originated from SupM2 and Karpas 299 were treated with varying dosages of MYC inhibitor 10074-G5 for 48 h, and then the cell growth was assessed by the MTS assay. The dosage of 5 μM 10074-G5 was chosen for the following drug combination study. The right panel showed that RR cells were treated with varying dosages of doxorubicin for 48 h, and then the cell growth was assessed by the MTS assay. Fifty and 20 ng/mL doxorubicin were chosen in RR derived from Karpas 299 and SupM2, respectively, for the following drug combination study. (b, c) The cell cycle analysis was performed to assess the Sub G0/1 fraction in RR cells derived from SupM2 induced by 20 ng/mL doxorubicin, 5 μM 10074-G5, or combination of doxorubicin and 10074-G5 for 48 h; Cells with DMSO treatment were included as a control. (d) The PI staining assay was also performed in the experiment described above. (e) RU and RR c...
The Wnt/β-catenin pathway is more active in RR cells than RU cells derived from SupM2. The Wnt pa... more The Wnt/β-catenin pathway is more active in RR cells than RU cells derived from SupM2. The Wnt pathway-specific oligonucleotide PCR array was performed in RU and RR cells derived from SupM2 cells. The data suggested that 24 out of 87 genes related with the Wnt pathway were more highly expressed in mRNA level (>1.4-fold) in RR cells than in RU cells. Note that one time experiment was performed in this study. (PDF 102 kb)
RU cells co-cultured with diluted (10:1) RR cells or parental SupM2 cells did not show significan... more RU cells co-cultured with diluted (10:1) RR cells or parental SupM2 cells did not show significantly increased SRR2 luciferase activity or upregulated MYC expression. SupM2-RU cells (50,000 cells in total seeded in the upper chamber) and various ratios (RU/RR = 1:1, 2:1, 5:1, 10:1) of SupM2-RR cells (seeded in the lower chamber) were co-cultured in 6-well transwell plate for 72 h. SupM2-RU cells co-cultured with the same number of SupM2-RU cells or parental SupM2 cells were included in this experiment. MYC protein expression was also assessed in this experiment. The results suggested that RU cells co-cultured with the same number of RR cells showed significantly increased SRR2 luciferase activity and a robust increased MYC expression, as compared to negative control. In contrast, RU cells co-cultured with diluted RR cells (e.g., 10:1, containing 5000 of RR cells and 45,000 of RU cells) or parental SupM2 cells did not exhibit significantly increased SRR2 luciferase activity and only ...
NPM-ALK/STAT3 is not differentially activated or expressed between RU and RR cells. (a) The prote... more NPM-ALK/STAT3 is not differentially activated or expressed between RU and RR cells. (a) The protein levels of pALKY1601, ALK, pSTAT3Y705, and STAT3 in RU and RR cells derived from SupM2 and Karpas 299. (b, c) RR and RU cells were treated with either DMSO, or 100 nM ALK inhibitor crizotinib, or 10 nM STAT3 inhibitor stattic for 24 h. The western blots were employed to assess the expression/activation levels of NPM-ALK, STAT3, MYC, and Sox2 in both RU and RR cells. The SRR2 luciferase activity in RU and RR cells was also evaluated by the luciferase assay. (PDF.488KB) (PDF 458 kb)
We have previously described the existence of two phenotypically distinct cell subsets in ALK-pos... more 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. 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. Immunofluorescenc...
We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the diffe... more We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the differential responsiveness to a Sox2 reporter (SRR2), with cells responsive to SRR2 (RR) being more stem-like than unresponsive cells (RU). Here, we report that RR cells derived from MCF7 and ZR751 displayed a higher tolerance to oxidative stress than their RU counterparts, supporting the concept that the RR phenotype correlates with cancer stemness. Sox2 is directly implicated in this differential H2O2 tolerance, since siRNA knockdown of Sox2 in RR cells leveled this difference. Interestingly, H2O2 converted a proportion of RU cells into RR cells, as evidenced by their expression of luciferase and GFP, markers of SRR2 activity. Compared to RU cells, converted RR cells showed a significant increase in mammosphere formation and tolerance to H2O2. Converted RR cells also adopted the biochemical features of RR cells, as evidenced by their substantial increase in Sox2-SRR2 binding and the expres...
Cytopathic effects are currently believed to contribute to HCV-induced liver injury and are readi... more Cytopathic effects are currently believed to contribute to HCV-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species (ROS), which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase (AMPK). The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate ROS-mediated activation of AMPK in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated and β-oxidation is enhanced in these cells. A striking increase in nuclear PPARα, which plays a dominant role in the expression of β-oxidation genes after ligand-induced activation, was also observed and we provide evidence that PPARα is const...
Carbohydrate response element binding protein (ChREBP) regulates insulin‐independent de novo lipo... more Carbohydrate response element binding protein (ChREBP) regulates insulin‐independent de novo lipogenesis. Recently, a novel ChREBPβ 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 ChREBPα, yet ChREBPβ increased 10‐ to 20‐fold. High‐fat fed mice increased hepatic ChREBPβ mRNA expression compared to chow‐fed along with increased protein expression. To better assess the independent effect of fructose on ChREBPα/β 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 ChREBPβ in human tissue, primary hepatocytes were incubated wit...
The tumorigenicity of most cases of ALK-positive anaplastic large cell lymphoma (ALK+ALCL) is dri... more The tumorigenicity of most cases of ALK-positive anaplastic large cell lymphoma (ALK+ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Since it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ALCL, thereby leaving the STAT3 signaling unchecked. Compared to normal T-cells, ALK+ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ALCL cells, as it effectively up-regulated γ-interferon, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 sign...
Cyclosporine A (CSA) has potent effects against hepatitis C virus (HCV) in vitro, but its clinica... more Cyclosporine A (CSA) has potent effects against hepatitis C virus (HCV) in vitro, but its clinical efficacy after liver transplantation (LT) is uncertain. We evaluated the impact of CSA and tacrolimus (TAC) with or without concomitant interferon (IFN) therapy on serum HCV titers in a chimeric mouse model of HCV infection. Six groups of HCV-infected mice received only the vehicle, IFN, CSA, CSA and IFN, TAC, or TAC and IFN for 4 weeks. The quantitative HCV polymerase chain reaction levels were determined after 1, 2, and 4 weeks of drug administration. There were no significant differences in the HCV titers after 4 weeks of treatment between the non-IFN-treated groups (log HCV titers: 3.5 6 0.3 for the vehicle group, 4.4 6 0.6 for the CSA group, and 4.3 6 0.4 for the TAC group, P ¼ 0.3). Although IFN had a consistent effect of reducing HCV titers across the groups, there was no significant impact of CSA on HCV levels when it was used alone or in combination with IFN at any time point. The 4-week HCV titers were as follows: 3.2 6 0.3 for the IFN group, 4.7 6 0.4 for the CSA/IFN group, and 4.0 6 0.5 for the TAC/IFN group (P ¼ 0.07). The CSA/IFN and TAC/IFN groups did not differ significantly (P ¼ 0.6). Six of the 7 animals in the IFN group (85.7%) had an HCV titer decline ! 1 log, whereas in the test groups (CSA/IFN and TAC/IFN), 6 of 9 animals (66.7%) achieved a 1-log decline in the HCV titer (P ¼ 1). Using this animal model, we could find no evidence supporting the routine use of CSA after LT in HCV-infected patients.
Background & AimsDespite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10–2... more Background & AimsDespite 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.MethodsWe 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.ResultsAnti‐DR5 mAb (200 and 300 μg/day) induced liver dysfunction with partial necrosi...
AimCytomegalovirus is a common viral pathogen that influences the outcome of organ transplantatio... more AimCytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV.MethodsOne million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked‐urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non‐chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post‐HCMV inoculation, or human liver natural killer (NK) cells (20 × 106 cells/mouse, i.v.) 1 day prior to HCMV inoculation.ResultsChimeric mice that received HCMV showed high ...
Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with in... more Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC 50) values of 0.01 to 0.14 M for genotype 1, with half maximal effective concentration (EC 50 s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 ؎ 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P ؍ 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV-796 patients treated for over 8 weeks, resulting in suspension of further trial activity. Conclusion: The RNA-dependent RNA polymerase inhibitor HCV796 demonstrated potent anti-HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose-related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti-HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment.
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Papers by Jamie Lewis