Publications by authors named "Elizabeth M Wilson"

85 Publications

Negative Regulation of Human Hepatic Constitutive Androstane Receptor by Cholesterol Synthesis Inhibition: Role of Sterol Regulatory Element Binding Proteins.

Drug Metab Dispos 2021 Aug 19;49(8):706-717. Epub 2021 May 19.

Institute of Environmental Health Sciences (L.C., Z.D.-D., E.A.R., A.P., T.A.K.) and Department of Chemistry (N.J.P., J.A.W.), Wayne State University, Detroit, Michigan; Division of Pharmacoengineering and Molecular Pharmaceutics, University of North Carolina, Chapel Hill, North Carolina (J.K.F., P.C.S.); and Yecuris Corporation, Tualatin, Oregon (E.M.W.)

The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.
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http://dx.doi.org/10.1124/dmd.120.000341DOI Listing
August 2021

Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans.

Hepatology 2020 08 23;72(2):656-670. Epub 2020 Mar 23.

Department of Laboratory Medicine, Division of Clinical Chemistry, Karolinska Institute, Stockholm, Sweden.

Background And Aims: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences.

Approach And Results: Fah , Rag2 , and Il2rg knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level.

Conclusions: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.
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http://dx.doi.org/10.1002/hep.31052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496592PMC
August 2020

ZFX Mediates Non-canonical Oncogenic Functions of the Androgen Receptor Splice Variant 7 in Castrate-Resistant Prostate Cancer.

Mol Cell 2018 10 27;72(2):341-354.e6. Epub 2018 Sep 27.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA. Electronic address:

Androgen receptor splice variant 7 (AR-V7) is crucial for prostate cancer progression and therapeutic resistance. We show that, independent of ligand, AR-V7 binds both androgen-responsive elements (AREs) and non-canonical sites distinct from full-length AR (AR-FL) targets. Consequently, AR-V7 not only recapitulates AR-FL's partial functions but also regulates an additional gene expression program uniquely via binding to gene promoters rather than ARE enhancers. AR-V7 binding and AR-V7-mediated activation at these unique targets do not require FOXA1 but rely on ZFX and BRD4. Knockdown of ZFX or select unique targets of AR-V7/ZFX, or BRD4 inhibition, suppresses growth of castration-resistant prostate cancer cells. We also define an AR-V7 direct target gene signature that correlates with AR-V7 expression in primary tumors, differentiates metastatic prostate cancer from normal, and predicts poor prognosis. Thus, AR-V7 has both ARE/FOXA1 canonical and ZFX-directed non-canonical regulatory functions in the evolution of anti-androgen therapeutic resistance, providing information to guide effective therapeutic strategies.
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http://dx.doi.org/10.1016/j.molcel.2018.08.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214474PMC
October 2018

Chimeric liver transplantation reveals interspecific graft remodelling.

J Hepatol 2018 Nov 20;69(5):1025-1036. Epub 2018 Jul 20.

Division of Abdominal Surgery, Department of Surgery, Faculty of Medicine, University of Geneva, Geneva, Switzerland; Hepato-Pancreato-Biliary Centre, Geneva University Hospitals, Geneva, Switzerland. Electronic address:

Background & Aims: A major limitation in the field of liver transplantation is the shortage of transplantable organs. Chimeric animals carrying human tissue have the potential to solve this problem. However, currently available chimeric organs retain a high level of xenogeneic cells, and the transplantation of impure organs needs to be tested.

Methods: We created chimeric livers by injecting Lewis rat hepatocytes into C57Bl/6 mice, and further transplanted them into newly weaned Lewis rats (45 ± 3 g) with or without suboptimal immunosuppression (tacrolimus 0.6 mg/kg/day for 56 or 112 days). Control donors included wild-type C57Bl/6 mice (xenogeneic) and Lewis rats (syngeneic).

Results: Without immunosuppression, recipients of chimeric livers experienced acute rejection, and died within 8 to 11 days. With immunosuppression, they all survived for >112 days with normal weight gain compared to syngeneic controls, while all xenogeneic controls died within 98 days due to rejection with Banff scores >6 (p = 0.0014). The chimeric grafts underwent post-transplant remodelling, growing by 670% on average. Rat hepatocytes fully replaced mouse hepatocytes starting from day 56 (absence of detectable mouse serum albumin, histological clearance of mouse hepatocytes). In addition, rat albumin levels reached those of syngeneic recipients. Four months after transplantation of chimeric livers, we observed the development of diffuse mature rat bile ducts through transdifferentiation of hepatocytes (up to 72% of cholangiocytes), and patchy areas of portal endothelium originating from the host (seen in one out of five recipients).

Conclusions: Taken together, these data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant recipient-oriented graft remodelling. Validation in a large animal model is still needed.

Lay Summary: Chimeric animals are composed of cells from different species. Chimeric animals carrying human tissue have the potential to increase the availability of transplantable organs. We transplanted rat-to-mouse liver grafts into newly weaned rats. The chimeric grafts underwent post-transplant remodelling with rat hepatocytes replacing all mouse hepatocytes within 56 days. In addition, we observed the post-transplant development of diffuse mature rat bile ducts through the transformation of hepatocytes, and patchy areas of portal endothelium originating from the host. These data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant graft remodelling.
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http://dx.doi.org/10.1016/j.jhep.2018.07.008DOI Listing
November 2018

Liver Stage Infection and Transition to Stable Blood Stage Infection in Liver-Humanized and Blood-Humanized FRGN KO Mice Enables Testing of Blood Stage Inhibitory Antibodies (Reticulocyte-Binding Protein Homolog 5) .

Front Immunol 2018 14;9:524. Epub 2018 Mar 14.

Center for Infectious Disease Research, Seattle, WA, United States.

The invention of liver-humanized mouse models has made it possible to directly study the preerythrocytic stages of . In contrast, the current models to directly study blood stage infection are extremely limited. Humanization of the mouse blood stream is achievable by frequent injections of human red blood cells (hRBCs) and is currently the only system with which to study human malaria blood stage infections in a small animal model. Infections have been primarily achieved by direct injection of -infected RBCs but as such, this modality of infection does not model the natural route of infection by mosquito bite and lacks the transition of parasites from liver stage infection to blood stage infection. Including these life cycle transition points in a small animal model is of relevance for testing therapeutic interventions. To this end, we used FRGN KO mice that were engrafted with human hepatocytes and performed a blood exchange under immune modulation to engraft the animals with more than 50% hRBCs. These mice were infected by mosquito bite with sporozoite stages of a luciferase-expressing parasite, resulting in noninvasively measurable liver stage burden by bioluminescent imaging (IVIS) at days 5-7 postinfection. Transition to blood stage infection was observed by IVIS from day 8 onward and then blood stage parasitemia increased with a kinetic similar to that observed in controlled human malaria infection. To assess the utility of this model, we tested whether a monoclonal antibody targeting the erythrocyte invasion ligand reticulocyte-binding protein homolog 5 (with known growth inhibitory activity ) was capable of blocking blood stage infection when parasites emerge from the liver and found it highly effective. Together, these results show that a combined liver-humanized and blood-humanized FRGN mouse model infected with luciferase-expressing will be a useful tool to study preerythrocytic and erythrocytic stages and enables the testing of interventions that target either one or both stages of parasite infection.
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http://dx.doi.org/10.3389/fimmu.2018.00524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861195PMC
May 2019

Evolution of Melanoma Antigen-A11 (MAGEA11) During Primate Phylogeny.

J Mol Evol 2018 04 24;86(3-4):240-253. Epub 2018 Mar 24.

Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, 27599-7500, USA.

Melanoma antigen-A11 (MAGE-A11) is an X-linked and primate-specific steroid hormone receptor transcriptional coregulator and proto-oncogenic protein whose increased expression promotes the growth of prostate cancer. The MAGEA11 gene is expressed at low levels in normal human testis, ovary, and endometrium, and at highest levels in castration-resistant prostate cancer. Annotated genome predictions throughout the surviving primate lineage show that MAGEA11 acquired three 5' coding exons unique within the MAGEA subfamily during the evolution of New World monkeys (NWM), Old World monkeys (OWM), and apes. MAGE-A11 in all primates has a conserved FXXIF coactivator-binding motif that suggests interaction with p160 coactivators contributed to its early evolution as a transcriptional coregulator. An ancestral form of MAGE-A11 in the more distantly related lemur has significant amino acid sequence identity with human MAGE-A11, but lacks coregulator activity based on the absence of the three 5' coding exons that include a nuclear localization signal (NLS). NWM MAGE-A11 has greater amino acid sequence identity than lemur to human MAGE-A11, but inframe premature stop codons suggest that MAGEA11 is a pseudogene in NWM. MAGE-A11 in OWM and apes has nearly identical 5' coding exon amino acid sequence and conserved interaction sites for p300 acetyltransferase and cyclin A. We conclude that the evolution of MAGEA11 within the lineage leading to OWM and apes resulted in steroid hormone receptor transcriptional coregulator activity through the acquisition of three 5' coding exons that include a NLS sequence and nonsynonymous substitutions required to interact with cell cycle regulatory proteins and transcription factors.
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http://dx.doi.org/10.1007/s00239-018-9838-8DOI Listing
April 2018

Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade.

Oncotarget 2018 Feb 10;9(13):11227-11242. Epub 2018 Jan 10.

Department of Urology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.

Androgen deprivation therapy (ADT) is palliative and prostate cancer (CaP) recurs as lethal castration-recurrent/resistant CaP (CRPC). One mechanism that provides CaP resistance to ADT is primary backdoor androgen metabolism, which uses up to four 3α-oxidoreductases to convert 5α-androstane-3α,17β-diol (DIOL) to dihydrotestosterone (DHT). The goal was to determine whether inhibition of 3α-oxidoreductase activity decreased conversion of DIOL to DHT. Protein sequence analysis showed that the four 3α-oxidoreductases have identical catalytic amino acid residues. Mass spectrometry data showed combined treatment using catalytically inactive 3α-oxidoreductase mutants and the 5α-reductase inhibitor, dutasteride, decreased DHT levels in CaP cells better than dutasteride alone. Combined blockade of frontdoor and backdoor pathways of DHT synthesis provides a therapeutic strategy to inhibit CRPC development and growth.
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http://dx.doi.org/10.18632/oncotarget.24107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834294PMC
February 2018

Assessing drug efficacy against Plasmodium falciparum liver stages in vivo.

JCI Insight 2018 01 11;3(1). Epub 2018 Jan 11.

Center for Infectious Disease Research, Seattle, Washington, USA.

Malaria eradication necessitates new tools to fight the evolving and complex Plasmodium pathogens. These tools include prophylactic drugs that eliminate Plasmodium liver stages and consequently prevent clinical disease, decrease transmission, and reduce the propensity for resistance development. Currently, the identification of these drugs relies on in vitro P. falciparum liver stage assays or in vivo causal prophylaxis assays using rodent malaria parasites; there is no method to directly test in vivo liver stage activity of candidate antimalarials against the human malaria-causing parasite P. falciparum. Here, we use a liver-chimeric humanized mouse (FRG huHep) to demonstrate in vivo P. falciparum liver stage development and describe the efficacy of clinically used and candidate antimalarials with prophylactic activity. We show that daily administration of atovaquone-proguanil (ATQ-PG; ATQ, 30 mg/kg, and PG, 10 mg/kg) protects 5 of 5 mice from liver stage infection, consistent with the use in humans as a causal prophylactic drug. Single-dose primaquine (60 mg/kg) has similar activity to that observed in humans, demonstrating the activity of this drug (and its active metabolites) in FRG huHep mice. We also show that DSM265, a selective Plasmodial dihydroorotate dehydrogenase inhibitor with causal prophylactic activity in humans, reduces liver stage burden in FRG huHep mice. Finally, we measured liver stage-to-blood stage transition of the parasite, the ultimate readout of prophylactic activity and measurement of infective capacity of parasites in the liver, to show that ATQ-PG reduces blood stage patency to below the limit of quantitation by quantitative PCR (qPCR). The FRG huHep model, thus, provides a platform for preclinical evaluation of drug candidates for liver stage causal prophylactic activity, pharmacokinetic/pharmacodynamics studies, and biological studies to investigate the mechanism of action of liver stage active antimalarials.
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http://dx.doi.org/10.1172/jci.insight.92587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821200PMC
January 2018

Murine model of hepatic breast cancer.

Biochem Biophys Rep 2016 Dec 4;8:1-5. Epub 2016 Aug 4.

Children's Cancer Therapy Development Institute, Fort Collins, CO 80524, USA.

Background And Aims: Breast cancer is the most common cancer in women and the second leading cause of cancer-related deaths in this population. Breast cancer related deaths have declined due to screening and adjuvant therapies, yet a driving clinical need exists to better understand the cause of the deadliest aspect of breast cancer, metastatic disease. Breast cancer metastasizes to several distant organs, the liver being the third most common site. To date, very few murine models of hepatic breast cancer exist.

Methods: In this study, a novel murine model of liver breast cancer using the MDA-MB-231 cell line is introduced as an experimental (preclinical) model.

Results: Histological typing revealed consistent hepatic breast cancer tumor foci. Common features of the murine model were vascular invasion, lung metastasis and peritoneal seeding.

Conclusions: The novel murine model of hepatic breast cancer established in this study provides a tool to be used to investigate mechanisms of hepatic metastasis and to test potential therapeutic interventions.
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http://dx.doi.org/10.1016/j.bbrep.2016.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613703PMC
December 2016

Rotavirus Infection in the Auckland Region After the Implementation of Universal Infant Rotavirus Vaccination: Impact on Hospitalizations and Laboratory Implications.

Pediatr Infect Dis J 2018 Jan;37(1):e1-e5

From the *Microbiology Department, Auckland City Hospital, †Microbiology Department, Middlemore Hospital, ‡Microbiology Department, North Shore Hospital, and §Department of Paediatric Infectious Diseases, Starship Children's Hospital, Auckland, New Zealand.

Background: In July 2014, New Zealand introduced universal infant vaccination with RotaTeq (Merk & Co.) administered as 3 doses at 6 weeks, 3 and 5 months of age. We sought to assess the impact of rotavirus vaccination on gastroenteritis (GE) hospitalizations in the greater Auckland region and analyze changes in rotavirus testing in the period around vaccine introduction.

Methods: Hospitalizations, laboratory testing rates and methods were compared between the pre-vaccine period (2009-2013), post-vaccine period (January 2015 to December 2015) and year of vaccine introduction (2014).

Results: There was a 68% decline in rotavirus hospitalizations of children <5 years of age after vaccine introduction (from 258/100,000 to 83/100,000) and a 17% decline in all-cause gastroenteritis admissions (from 1815/100,000 to 1293/100,000). Reductions were also seen in pediatric groups too old to have received vaccine. Despite these changes, rotavirus testing rates in our region remained static in the year after vaccine introduction compared with the 2 prior years, and after vaccine introduction, we observed a high rate of false positives 19/58 (33%) in patients with reactive rotavirus tests.

Conclusions: Rotavirus vaccine has had a significant early impact on gastroenteritis hospitalizations for children in the Auckland region. However, continued rotavirus testing at pre-vaccine rates risks generating false positive results. Laboratories and clinicians should consider reviewing their testing algorithms before vaccine introduction.
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http://dx.doi.org/10.1097/INF.0000000000001706DOI Listing
January 2018

Androgen receptor regulation by histone methyltransferase Suppressor of variegation 3-9 homolog 2 and Melanoma antigen-A11.

Mol Cell Endocrinol 2017 03 29;443:42-51. Epub 2016 Dec 29.

Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, United States. Electronic address:

Androgen receptor (AR) transcriptional activity depends on interactions between the AR NH-terminal region and transcriptional coregulators. A yeast two-hybrid screen of a human testis library using predicted α-helical NH-terminal fragment AR-(370-420) as bait identified suppressor of variegation 3-9 homolog 2 (SUV39H2) histone methyltransferase as an AR interacting protein. SUV39H2 interaction with AR and the AR coregulator, melanoma antigen-A11 (MAGE-A11), was verified in two-hybrid, in vitro glutathione S-transferase affinity matrix and coimmunoprecipitation assays. Fluorescent immunocytochemistry colocalized SUV39H2 and AR in the cytoplasm without androgen, in the nucleus with androgen, and with MAGE-A11 in the nucleus independent of androgen. Chromatin immunoprecipitation using antibodies raised against SUV39H2 demonstrated androgen-dependent recruitment of AR and SUV39H2 to the androgen-responsive upstream enhancer of the prostate-specific antigen gene. SUV39H2 functioned cooperatively with MAGE-A11 to increase androgen-dependent AR transcriptional activity. SUV39H2 histone methyltransferase is an AR coactivator that increases androgen-dependent transcriptional activity through interactions with AR and MAGE-A11.
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http://dx.doi.org/10.1016/j.mce.2016.12.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303141PMC
March 2017

Up-Regulation of Follistatin-Like 1 By the Androgen Receptor and Melanoma Antigen-A11 in Prostate Cancer.

Prostate 2017 04 14;77(5):505-516. Epub 2016 Dec 14.

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina.

Background: High affinity androgen binding to the androgen receptor (AR) activates genes required for male sex differentiation and promotes the development and progression of prostate cancer. Human AR transcriptional activity involves interactions with coregulatory proteins that include primate-specific melanoma antigen-A11 (MAGE-A11), a coactivator that increases AR transcriptional activity during prostate cancer progression to castration-resistant/recurrent prostate cancer (CRPC).

Methods: Microarray analysis and quantitative RT-PCR were performed to identify androgen-regulated MAGE-A11-dependent genes in LAPC-4 prostate cancer cells after lentivirus shRNA knockdown of MAGE-A11. Chromatin immunoprecipitation was used to assess androgen-dependent AR recruitment, and immunocytochemistry to localize an androgen-dependent protein in prostate cancer cells and tissue and in the CWR22 human prostate cancer xenograft.

Results: Microarray analysis of androgen-treated LAPC-4 prostate cancer cells indicated follistatin-like 1 (FSTL1) is up-regulated by MAGE-A11. Androgen-dependent up-regulation of FSTL1 was inhibited in LAPC-4 cells by lentivirus shRNA knockdown of AR or MAGE-A11. Chromatin immunoprecipitation demonstrated AR recruitment to intron 10 of the FSTL1 gene that contains a classical consensus androgen response element. Increased levels of FSTL1 protein in LAPC-4 cells correlated with higher levels of MAGE-A11 relative to other prostate cancer cells. FSTL1 mRNA levels increased in CRPC and castration-recurrent CWR22 xenografts in association with predominantly nuclear FSTL1. Increased nuclear localization of FSTL1 in prostate cancer was suggested by predominantly cytoplasmic FSTL1 in benign prostate epithelial cells and predominantly nuclear FSTL1 in epithelial cells in CRPC tissue and the castration-recurrent CWR22 xenograft. AR expression studies showed nuclear colocalization of AR and endogenous FSTL1 in response to androgen.

Conclusion: AR and MAGE-A11 cooperate in the up-regulation of FSTL1 to promote growth and progression of CRPC. Prostate 77:505-516, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/pros.23288DOI Listing
April 2017

Successful Engraftment of Human Hepatocytes in uPA-SCID and FRG KO Mice.

Methods Mol Biol 2017 ;1506:117-130

Departments of Clinical Chemistry, Microbiology and Immunology, Ghent University, University Hospital Ghent, MRB2, De Pintelaan 185, Ghent, 9000, Belgium.

Mice with humanized chimeric liver are promising in vivo tools to evaluate the efficacy of novel compounds or vaccine induced antibodies directed against pathogens that infect the human liver. In addition they can be used to study the human-type metabolism of medicinal compounds and hepatotoxicity.
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http://dx.doi.org/10.1007/978-1-4939-6506-9_8DOI Listing
January 2018

Melanoma antigen-A11 regulates substrate-specificity of Skp2-mediated protein degradation.

Mol Cell Endocrinol 2017 01 6;439:1-9. Epub 2016 Oct 6.

Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA. Electronic address:

Melanoma antigen-A11 (MAGE-A11) is a proto-oncogene involved in androgen receptor signaling and androgen-dependent cell growth. In this report we provide evidence that MAGE-A11 interacts with Skp2 (S phase kinase-associated protein), the substrate recognition protein of the Skp1-Cullin1-F-box E3 ubiquitin ligase, and with Skp2 binding protein, cyclin A. A similar cyclin A binding motif in MAGE-A11 and Skp2 was consistent with a competitive relationship between MAGE-A11 and Skp2 in binding cyclin A. Skp2 inhibited MAGE-A11 interaction with cyclin A. Differential effects of MAGE-A11 on Skp2-mediated protein degradation were also revealed. MAGE-A11 increased Skp2-mediated degradation of cyclin A and retinoblastoma-related protein p130. In contrast, MAGE-A11 decreased Skp2-mediated degradation of E2F1 and Skp2 self-ubiquitination. Stabilization of E2F1 by MAGE-A11 was associated with sequestration and inactivation of Skp2 through the formation of an E2F1-MAGE-A11-Skp2 complex. We conclude that direct interactions of MAGE-A11 with Skp2 and cyclin A regulate the substrate-specificity of Skp2-mediated protein degradation.
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http://dx.doi.org/10.1016/j.mce.2016.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123923PMC
January 2017

Characterization of fibroblast-free CWR-R1ca castration-recurrent prostate cancer cell line.

Prostate 2016 09 8;76(12):1067-77. Epub 2016 Jun 8.

Department of Cancer Genetics, Center for Genetics and Pharmacology, Roswell Park Cancer Institute, Buffalo, New York.

Background: The previously established CWR-R1 cell line has been used as an in vitro model representing castration-recurrent prostate cancer. Microscopic observation of subconfluent cells demonstrated two distinct cellular morphologies: polygonal closely aggregated epithelial cells surrounded by bipolar fibroblastic cells with long processes. This study sought to establish and characterize a fibroblast-free derivative of the CWR-R1 cell line.

Methods: The CWR-R1ca cell line was established from CWR-R1 cells by removing fibroblasts using multiple cycles of short-term trypsinization, cloning, and pooling single-cell colonies. Authentication of fibroblast-free CWR-R1ca cells was demonstrated by analyzing the expression of cytodifferentiation and prostate-associated markers, DNA and cytogenetic profiling, and growth pattern in the absence or presence of androgen.

Results: CWR-R1ca is an androgen-sensitive cell line that expresses the androgen receptor (AR) and its splice variant 7 and the luminal epithelia markers, CK-8, CK-18, and c-Met. CWR-R1fb fibroblasts isolated from CWR-R1 cells express AR, hepatocyte growth factor-α, and mouse β-actin but not AR-V7 or epithelial markers. Cytogenetic analysis of CWR-R1ca cells revealed a hyperdiploid male with numerical gains in chromosomes 1, 7, 8, 10, 11, and 12, deletion of one chromosome 2 allele, structural abnormalities that include der(1)t(1:4), der(4)t(2:4), der(10)t(4:10), and an unbalanced reciprocal translocation between chromosome 6 and 14. DNA-profiling revealed that CWR-R1ca cells had significant short-tandem repeat marker homology with CWR22Pc and CWR22Rv1 cell lines, which indicated lineage derivation from CWR22 prostate cancer xenografts. CWR-R1ca cells were responsive to the growth stimulatory effects of dihydrotestosterone (DHT) in the femtomolar range.

Conclusion: This study establishes CWR-R1ca cells as a fibroblast-free derivative of the castration-recurrent CWR-R1 cell line. Prostate 76:1067-1077, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/pros.23190DOI Listing
September 2016

Sex bias in CNS autoimmune disease mediated by androgen control of autoimmune regulator.

Nat Commun 2016 Apr 13;7:11350. Epub 2016 Apr 13.

Division of Endocrinology, Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

Male gender is protective against multiple sclerosis and other T-cell-mediated autoimmune diseases. This protection may be due, in part, to higher androgen levels in males. Androgen binds to the androgen receptor (AR) to regulate gene expression, but how androgen protects against autoimmunity is not well understood. Autoimmune regulator (Aire) prevents autoimmunity by promoting self-antigen expression in medullary thymic epithelial cells, such that developing T cells that recognize these self-antigens within the thymus undergo clonal deletion. Here we show that androgen upregulates Aire-mediated thymic tolerance to protect against autoimmunity. Androgen recruits AR to Aire promoter regions, with consequent enhancement of Aire transcription. In mice and humans, thymic Aire expression is higher in males compared with females. Androgen administration and male gender protect against autoimmunity in a multiple sclerosis mouse model in an Aire-dependent manner. Thus, androgen control of an intrathymic Aire-mediated tolerance mechanism contributes to gender differences in autoimmunity.
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http://dx.doi.org/10.1038/ncomms11350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512610PMC
April 2016

Post-translational Down-regulation of Melanoma Antigen-A11 (MAGE-A11) by Human p14-ARF Tumor Suppressor.

J Biol Chem 2015 Oct 1;290(41):25174-87. Epub 2015 Sep 1.

From the Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599

X-linked primate-specific melanoma antigen-A11 (MAGE-A11) is a human androgen receptor (AR) coactivator and proto-oncogene expressed at low levels in normal human reproductive tract tissues and at higher levels in castration-resistant prostate cancer where it is required for androgen-dependent cell growth. In this report, we show that MAGE-A11 is targeted for degradation by human p14-ARF, a tumor suppressor expressed from an alternative reading frame of the p16 cyclin-dependent kinase inhibitor INK4a/ARF gene. MAGE-A11 degradation by the proteasome was mediated by an interaction with p14-ARF and was independent of lysine ubiquitination. A dose-dependent inverse relationship between MAGE-A11 and p14-ARF correlated with p14-ARF inhibition of the MAGE-A11-induced increase in androgen-dependent AR transcriptional activity and constitutive activity of a splice variant-like AR. Reciprocal stabilization between MAGE-A11 and AR did not protect against degradation promoted by p14-ARF. p14-ARF prevented MAGE-A11 interaction with the E2F1 oncoprotein and inhibited the MAGE-A11-induced increase in E2F1 transcriptional activity. Post-translational down-regulation of MAGE-A11 promoted by p14-ARF was independent of HDM2, the human homologue of mouse double minute 2, an E3 ubiquitin ligase inhibited by p14-ARF. However, MAGE-A11 had a stabilizing effect on HDM2 in the absence or presence of p14-ARF and cooperated with HDM2 to increase E2F1 transcriptional activity in the absence of p14-ARF. We conclude that degradation of MAGE-A11 promoted by the human p14-ARF tumor suppressor contributes to low levels of MAGE-A11 in nontransformed cells and that higher levels of MAGE-A11 associated with low p14-ARF increase AR and E2F1 transcriptional activity and promote the development of castration-resistant prostate cancer.
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http://dx.doi.org/10.1074/jbc.M115.663641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599020PMC
October 2015

Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.

Cell Stem Cell 2014 Nov 9;15(5):605-18. Epub 2014 Oct 9.

Department of Pediatrics, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.

Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass.
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http://dx.doi.org/10.1016/j.stem.2014.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254170PMC
November 2014

Extensive double humanization of both liver and hematopoiesis in FRGN mice.

Stem Cell Res 2014 Nov 6;13(3 Pt A):404-12. Epub 2014 Sep 6.

Yecuris Corp., Tigard, OR, USA.

Preclinical research in animals often fails to adequately predict the outcomes observed in human patients. Chimeric animals bearing individual human tissues have been developed to provide improved models of human-specific cellular processes. Mice transplanted with human hematopoietic stem cells can be used to study human immune responses, infections of blood cells and processes of hematopoiesis. Animals with humanized livers are useful for modeling hepatotropic infections as well as drug metabolism and hepatotoxicity. However, many pathophysiologic processes involve both the liver and the hematolymphoid system. Examples include hepatitis C/HIV co-infection, immune mediated liver diseases, liver injuries with inflammation such as steatohepatitis and alcoholic liver disease. We developed a robust protocol enabling the concurrent double-humanization of mice with mature hepatocytes and human blood. Immune-deficient, fumarylacetoacetate hydrolase (Fah(-/-)), Rag2(-/-) and Il2rg(-/-) deficient animals on the NOD-strain background (FRGN) were simultaneously co-transplanted with adult human hepatocytes and hematopoietic stem cells after busulfan and Ad:uPA pre-conditioning. Four months after transplantation the average human liver repopulation exceeded 80% and hematopoietic chimerism also was high (40-80% in bone marrow). Importantly, human macrophages (Kupffer cells) were present in the chimeric livers. Double-chimeric FRGN mice will serve as a new model for disease processes that involve interactions between hepatocytes and hematolymphoid cells.
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http://dx.doi.org/10.1016/j.scr.2014.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275629PMC
November 2014

Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.

Nature 2014 Feb 25;506(7488):382-6. Epub 2013 Dec 25.

Stanford University, School of Medicine, Departments of Pediatrics and Genetics, 269 Campus Drive, Stanford, California 94305, USA.

Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly-approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.
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http://dx.doi.org/10.1038/nature12875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939040PMC
February 2014

More evidence intratumoral DHT synthesis drives castration-resistant prostate cancer.

Asian J Androl 2014 Jan-Feb;16(1):99-100

Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA.

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http://dx.doi.org/10.4103/1008-682X.122200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901888PMC
September 2014

Mice with chimeric livers are an improved model for human lipoprotein metabolism.

PLoS One 2013 4;8(11):e78550. Epub 2013 Nov 4.

Department of Clinical Science, Intervention and Technology (CLINTEC) Division of Transplantation Surgery, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden.

Objective: Rodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism.

Design: FRG [ F ah(-/-) R ag2(-/-)Il2r g (-/-)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR.

Results: Chimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal.

Conclusion: Humanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078550PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817217PMC
August 2014

Mechanism of androgen receptor corepression by CKβBP2/CRIF1, a multifunctional transcription factor coregulator expressed in prostate cancer.

Mol Cell Endocrinol 2014 Jan 5;382(1):302-313. Epub 2013 Oct 5.

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, School of Medicine, Chapel Hill, NC, United States; Lineberger Comprehensive Cancer Center, University of North Carolina, School of Medicine, Chapel Hill, NC, United States. Electronic address:

The transcription factor coregulator Casein kinase IIβ-binding protein 2 or CR6-interacting factor 1 (CKβBP2/CRIF1) binds the androgen receptor (AR) in prostate cancer cells and in response to dihydrotestosterone localizes with AR on the prostate-specific antigen gene enhancer, but does not bind DNA suggesting CKβBP2/CRIF1 localization in chromatin is determined by AR. In this study we show also that CKβBP2/CRIF1 inhibits wild-type AR and AR N-terminal transcriptional activity, binds to the AR C-terminal region, inhibits interaction of the AR N- and C-terminal domains (N/C interaction) and competes with p160 coactivator binding to the AR C-terminal domain, suggesting CKβBP2/CRIF1 interferes with AR activation functions 1 and 2. CKβBP2/CRIF1 is expressed mainly in stromal cells of benign prostatic hyperplasia and in stroma and epithelium of prostate cancer. CKβBP2/CRIF1 protein is increased in epithelium of androgen-dependent prostate cancer compared to benign prostatic hyperplasia and decreased slightly in castration recurrent epithelium compared to androgen-dependent prostate cancer. The multifunctional CKβBP2/CRIF1 is a STAT3 interacting protein and reported to be a coactivator of STAT3. CKβBP2/CRIF1 is expressed with STAT3 in prostate cancer where STAT3 may help to offset the AR repressor effect of CKβBP2/CRIF1 and allow AR regulation of prostate cancer growth.
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http://dx.doi.org/10.1016/j.mce.2013.09.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880566PMC
January 2014

Proto-oncogene activity of melanoma antigen-A11 (MAGE-A11) regulates retinoblastoma-related p107 and E2F1 proteins.

J Biol Chem 2013 Aug 12;288(34):24809-24. Epub 2013 Jul 12.

Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

Melanoma antigen-A11 (MAGE-A11) is a low-abundance, primate-specific steroid receptor coregulator in normal tissues of the human reproductive tract that is expressed at higher levels in prostate cancer. Increased expression of MAGE-A11 enhances androgen receptor transcriptional activity and promotes prostate cancer cell growth. Further investigation into the mechanisms of MAGE-A11 function in prostate cancer demonstrated interactions with the retinoblastoma-related protein p107 and Rb tumor suppressor but no interaction with p130 of the Rb family. MAGE-A11 interaction with p107 was associated with transcriptional repression in cells with low MAGE-A11 and transcriptional activation in cells with higher MAGE-A11. Selective interaction of MAGE-A11 with retinoblastoma family members suggested the regulation of E2F transcription factors. MAGE-A11 stabilized p107 by inhibition of ubiquitination and linked p107 to hypophosphorylated E2F1 in association with the stabilization and activation of E2F1. The androgen receptor and MAGE-A11 modulated endogenous expression of the E2F1-regulated cyclin-dependent kinase inhibitor p27(Kip1). The ability of MAGE-A11 to increase E2F1 transcriptional activity was similar to the activity of adenovirus early oncoprotein E1A and depended on MAGE-A11 interactions with p107 and p300. The immunoreactivity of p107 and MAGE-A11 was greater in advanced prostate cancer than in benign prostate, and knockdown with small inhibitory RNA showed that p107 is a transcriptional activator in prostate cancer cells. These results suggest that MAGE-A11 is a proto-oncogene whose increased expression in prostate cancer reverses retinoblastoma-related protein p107 from a transcriptional repressor to a transcriptional activator of the androgen receptor and E2F1.
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http://dx.doi.org/10.1074/jbc.M113.468579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750176PMC
August 2013

DNA methylation and nucleosome occupancy regulate the cancer germline antigen gene MAGEA11.

Epigenetics 2013 Aug 9;8(8):849-63. Epub 2013 Jul 9.

Department of Pharmacology and Therapeutics; Roswell Park Cancer Institute; Buffalo, NY USA.

MAGEA11 is a cancer germline (CG) antigen and androgen receptor co-activator. Its expression in cancers other than prostate, and its mechanism of activation, has not been reported. In silico analyses reveal that MAGEA11 is frequently expressed in human cancers, is increased during tumor progression, and correlates with poor prognosis and survival. In prostate and epithelial ovarian cancers (EOC), MAGEA11 expression was associated with promoter and global DNA hypomethylation, and with activation of other CG genes. Pharmacological or genetic inhibition of DNA methyltransferases (DNMTs) and/or histone deacetylases (HDACs) activated MAGEA11 in a cell line specific manner. MAGEA11 promoter activity was directly repressed by DNA methylation, and partially depended on Sp1, as pharmacological or genetic targeting of Sp1 reduced MAGEA11 promoter activity and endogenous gene expression. Importantly, DNA methylation regulated nucleosome occupancy specifically at the -1 positioned nucleosome of MAGEA11. Methylation of a single Ets site near the transcriptional start site (TSS) correlated with -1 nucleosome occupancy and, by itself, strongly repressed MAGEA11 promoter activity. Thus, DNA methylation regulates nucleosome occupancy at MAGEA11, and this appears to function cooperatively with sequence-specific transcription factors to regulate gene expression. MAGEA11 regulation is highly instructive for understanding mechanisms regulating CG antigen genes in human cancer.
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http://dx.doi.org/10.4161/epi.25500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3883788PMC
August 2013

New potential cell source for hepatocyte transplantation: discarded livers from metabolic disease liver transplants.

Stem Cell Res 2013 Jul 27;11(1):563-73. Epub 2013 Mar 27.

Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden.

Unlabelled: Domino liver transplantation is a method used to increase the number of liver grafts available for orthotopic liver transplantation (OLT). Reports indicate that livers from patients with metabolic liver disease can be safely transplanted into select recipients if the donor's defect and the recipient's metabolic needs are carefully considered. The liver of patients with many types of metabolic liver disease is morphologically and biochemically normal, except for the mutation that characterizes that disease. Other biochemical functions normally performed by the liver are present and presumably "normal" in these hepatocytes. Hepatocytes were isolated from the liver of 35 organ donors and 35 liver tissues taken at OLT from patients with liver disease were analyzed for 9 different measures of viability and function. The data indicate that cells isolated from some diseased livers performed as well or better than those isolated from organ donors with respect to viability, cell yield, plating efficiency and in assays of liver function, including drug metabolism, conjugation reactions and ammonia metabolism. Cells from metabolic diseased livers rapidly and efficiently repopulated a mouse liver upon transplantation.

Conclusions: As with domino liver transplantation, domino cell transplantation deserves consideration as method to extend the pool of available organs and cells for transplantation.
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http://dx.doi.org/10.1016/j.scr.2013.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262521PMC
July 2013

Melanoma antigen-A11 (MAGE-A11) enhances transcriptional activity by linking androgen receptor dimers.

J Biol Chem 2013 Jan 21;288(3):1939-52. Epub 2012 Nov 21.

Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill, NC 27599-7500, USA.

Prostate cancer growth and progression depend on androgen receptor (AR) signaling through transcriptional mechanisms that require interactions with coregulatory proteins, one of which is the primate-specific steroid receptor coregulator melanoma antigen-A11 (MAGE-A11). In this report, we provide evidence how increased expression of MAGE-A11 during prostate cancer progression enhances AR signaling and prostate cancer growth. MAGE-A11 protein levels were highest in castration-recurrent prostate cancer. The cyclic AMP-induced increase in androgen-dependent and androgen-independent AR transcriptional activity correlated with an increase in MAGE-A11 and was inhibited by silencing MAGE-A11 expression. MAGE-A11 mediated synergistic AR transcriptional activity in LAPC-4 prostate cancer cells. The ability of MAGE-A11 to rescue transcriptional activity of complementary inactive AR mutants and promote coimmunoprecipitation between unlike forms of AR suggests that MAGE-A11 links transcriptionally active AR dimers. A model for the AR·MAGE-A11 multidimeric complex is proposed in which one AR FXXLF motif of the AR dimer engages in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction, whereas the second FXXLF motif region of the AR dimer interacts with dimeric MAGE-A11. The AR·MAGE-A11 multidimeric complex accounts for the dual functions of the AR FXXLF motif in the androgen-dependent AR NH(2)- and carboxyl-terminal interaction and binding MAGE-A11 and for synergy between reported AR splice variants and full-length AR. We conclude that the increased expression of MAGE-A11 in castration-recurrent prostate cancer, which is enhanced by cyclic AMP signaling, increases AR-dependent growth of prostate cancer by MAGE-A11 forming a molecular bridge between transcriptionally active AR dimers.
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http://dx.doi.org/10.1074/jbc.M112.428409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548502PMC
January 2013

Complete Plasmodium falciparum liver-stage development in liver-chimeric mice.

J Clin Invest 2012 Oct 10;122(10):3618-28. Epub 2012 Sep 10.

Seattle Biomedical Research Institute, Seattle, Washington 98109, USA.

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase-deficient mouse (Fah-/-, Rag2-/-, Il2rg-/-, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS-to-blood-stage transition of a human malaria parasite.
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http://dx.doi.org/10.1172/JCI62684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461911PMC
October 2012

Primate-specific melanoma antigen-A11 regulates isoform-specific human progesterone receptor-B transactivation.

J Biol Chem 2012 Oct 13;287(41):34809-24. Epub 2012 Aug 13.

Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH(2)-terminal (110)LLXXVLXXLL(119) motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium.
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http://dx.doi.org/10.1074/jbc.M112.372797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464583PMC
October 2012

A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes.

J Biol Chem 2012 Jul 15;287(28):23368-80. Epub 2012 May 15.

Department of Biochemistry,University of Illinois, Urbana, Illinois 61801-3602, USA.

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.
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http://dx.doi.org/10.1074/jbc.M112.344671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390614PMC
July 2012
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