Liver development is restored by blastocyst complementation of HHEX knockout in mice and pigs.

BACKGROUND: There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. METHODS: The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. RESULTS: Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. CONCLUSIONS: This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF-κB activation.

This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to ascertain the relevance of MEK5/ERK5 signalling in colon cancer. Expression of MEK5 and ERK5 was evaluated in 323 human colon cancer samples. To evaluate the role of MEK5/ERK5 signalling in colon cancer, we developed a stable cell line model with differential MEK5/ERK5 activation. Impact of differential MEK5/ERK5 signalling was evaluated on cell cycle progression by flow cytometry and cell migration was evaluated by wound healing and transwell migration assays. Finally, we used an orthotopic xenograft mouse model of colon cancer to assess tumour growth and progression. Our results demonstrated that MEK5 and ERK5 are overexpressed in human adenomas (P<0.01) and adenocarcinomas (P<0.05), where increased ERK5 expression correlated with the acquisition of more invasive and metastatic potential (P<0.05). Interestingly, we observed a significant correlation between ERK5 expression and NF-κB activation in human adenocarcinomas (P<0.001). We also showed that ERK5 overactivation significantly accelerated cell cycle progression (P<0.05) and increased cell migration (P<0.01). Furthermore, cells with overactivated ERK5 displayed increased NF-κB nuclear translocation and transcriptional activity (P<0.05), together with increased expression of the mesenchymal marker vimentin (P<0.05). We further demonstrated that increased NF-κB activation was associated with increased IκB phosphorylation and degradation (P<0.05). Finally, in the mouse model, lymph node metastasis was exclusively seen in orthotopically implanted tumours with overactivated MEK5/ERK5, and not in tumours with inhibited MEK5/ERK5. Our results suggested that MEK5/ERK5/NF-κB signalling pathway is important for tumour onset, progression and metastasis, possibly representing a novel relevant therapeutic target in colon cancer treatment.

Modulation of hepatocyte apoptosis: cross-talk between bile acids and nuclear steroid receptors.

The efficient removal of unwanted cells, such as senescent, damaged, mutated or infected cells is crucial for the maintenance of normal liver function. In fact, apoptosis has emerged as a potential contributor to the pathogenesis of a number of hepatic disorders, such as viral hepatitis, autoimmune diseases, ethanol-induced injury, cholestasis, and hepatocellular carcinoma. In contrast to the effect of cytotoxic bile acids in the liver, ursodeoxycholic acid (UDCA) has increasingly been used for the treatment of various liver disorders. The clinical efficacy of this hydrophilic bile acid was first recognized by its use in traditional Asian medicine. However, many studies have subsequently confirmed that UDCA improves liver function by three major mechanisms of action, including protection of cholangiocytes against the cytotoxicity of hydrophobic bile acids, stimulation of hepatobiliary secretion, and inhibition of liver cell apoptosis. UDCA acts as a potent inhibitor of the classical mitochondrial pathway of apoptosis, but also interferes with alternate and upstream molecular targets such as the E2F-1/p53 pathway. Together, there is growing evidence that this hydrophilic bile acid may modulate gene expression to prevent cell death. Curiously, as a cholesterol-derived molecule, UDCA interacts with nuclear steroid receptors, such as the glucocorticoid receptor. Nuclear steroid receptors play crucial roles in mediating steroid hormone signaling involved in many biological processes, including apoptosis. Here, we review the anti-apoptotic mechanisms of UDCA in hepatic cells, and discuss a potential involvement of nuclear steroid receptors in mediating the survival effects of UDCA.

p53 dephosphorylation and p21(Cip1/Waf1) translocation correlate with caspase-3 activation in TGF-beta1-induced apoptosis of HuH-7 cells.

The p53 tumor suppressor gene product plays an important role in the regulation of apoptosis. Transforming growth factor beta1 (TGF-beta1)-induced apoptosis in hepatic cells is associated with reduced expression of the retinoblastoma protein (pRb) and subsequent E2F-1-activated expression of apoptosis-related genes. In this study, we explored the potential role of p53 in TGF-beta1-induced apoptosis. HuH-7 human hepatoma cells were either synchronized in G1, S and G2/M phases, or treated with 1 nM TGF-beta1. The results indicated that greater than 90% of the TGF-beta1-treated cells were arrested in G1 phase of the cell cycle. This was associated with enhanced p53 dephosphorylation and p21(Cip1/Waf1) expression, which coincided with decreased Cdk2, Cdk4, and cyclin E expression, compared with synchronized G1 cells. In addition, p53 dephosphorylation coincided with caspase-3 activation, and translocation of p21(Cip1/Waf1) and p27(Kip1) into the cytoplasm, all of which were suppressed by caspase inhibition of TGF-beta1-induced apoptosis. Finally, phosphatase inhibition and pRb overexpression partially inhibited p53-mediated apoptosis. In conclusion, the results demonstrated that TGF-beta1-induced p53 dephosphorylation is associated with caspase-3 activation, and cytosolic translocation of p21(Cip1/Waf1) and p27(Kip1), resulting in decreased expression of Cdks and cyclins. Further, p53 appears to mediate TGF-beta1-induced apoptosis downstream of the pRb/E2F-1 pathway.

Modification of hepatic genomic DNA using RNA/DNA oligonucleotides.

The ideal gene therapy is one that repairs the precise genetic defect without additional modification of the genome. Such a strategy has been developed for correcting single nucleotide mutations by using RNA/DNA oligonucleotides, or chimeraplasts. This approach for in situ repair is based on the delivery of exogenous DNA designed to mediate genomic base conversion, insertion, or deletion, thereby, correcting the genetic mutation. Using in vivo delivery systems to hepatocytes via the asialoglycoprotein receptor, we targeted rat liver DNA and successfully modified the genomic sequence by chimeraplasty. The changes in both the hepatic genes, and their associated phenotypes remained stable for 2 years. In addition, we also examined the potential to alter sequence defects in mitochondrial DNA. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeraplast-mediated DNA changes. Using an in vitro DNA repair assay of mutagenized plasmids and an Escherichia coli readout system, we showed that extracts from highly purified rat liver mitochondria have the essential enzymatic activity to mediate precise single-nucleotide changes at a frequency similar to liver nuclear extracts. Moreover, single-stranded oligonucleotides carrying a single nucleotide mismatch with the target sequence were capable of promoting gene conversion using either mitochondrial or nuclear extracts. Several approaches now exist for the precise repair of genetic mutations using either single-stranded or RNA/DNA chimeric oligonucleotides.

A bile acid protects against motor and cognitive deficits and reduces striatal degeneration in the 3-nitropropionic acid model of Huntington's disease.

There is currently no effective treatment for Huntington's disease (HD), a progressive, fatal, neurodegenerative disorder characterized by motor and cognitive deterioration. It is well established that HD is associated with perturbation of mitochondrial energy metabolism. Tauroursodeoxycholic acid (TUDCA), a naturally occurring bile acid, can stabilize the mitochondrial membrane, inhibit the mitochondrial permeability transition, decrease free radical formation, and derail apoptotic pathways. Here we report that TUDCA significantly reduced 3-nitropropionic acid (3-NP)-mediated striatal neuronal cell death in cell culture. In addition, rats treated with TUDCA exhibited an 80% reduction in apoptosis and in lesion volumes associated with 3-NP administration. Moreover, rats which received a combination of TUDCA + 3-NP exhibited sensorimotor and cognitive task performance that was indistinguishable from that of controls, and this effect persisted at least 6 months. Bile acids have traditionally been used as therapeutic agents for certain liver diseases. This is the first demonstration, however, that a bile acid can be delivered to the brain and function as a neuroprotectant and thus may offer potential therapeutic benefit in the treatment of certain neurodegenerative diseases.

Targeted gene correction strategies.

We are now approaching the reality of success in gene therapy as our knowledge of the genetic basis of disease continues to grow, coupled with improved delivery methods for therapeutic nucleic acid molecules. It is apparent that gene therapy can be divided into two specific and very different approaches in which gene replacement, or augmentation, is differentiated from gene repair. In fact, gene augmentation is characterized by the delivery of the coding sequence of the gene of interest in an expression cassette. In contrast, gene repair differs in that the process targets for correction of the mutation responsible for the genetic disorder. The in situ repair of a gene has many advantages over conventional replacement methods. This review will concentrate on the various strategies currently available for gene repair. The potential benefits of correction versus augmentation will be addressed and possible future developments outlined.

Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair.

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitro DNA repair assay and an Escherichia coli readout system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.

Modulation of steady-state messenger RNA levels in the regenerating rat liver with bile acid feeding.

Liver regeneration after two thirds partial hepatectomy (PH) is an orchestrated hyperplastic growth process requiring coordinated expression of many genes. The synchronous progression of 95% of the remnant hepatocytes through the cell cycle provides an in vivo model for examining the influence of bile acids on the molecular regulation of hepatocyte replication and growth. In this study, we examined the effects of endogenous deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) on messenger RNA (mRNA) expression and growth rate during liver regeneration. Rats were fed diets containing no addition, 0.4% DCA, UDCA, or both for 14 days; they then underwent 70% PH and were maintained on the diets for an additional 14 days. mRNA transcript levels for a variety of cell cycle-regulated genes were examined post-PH by Northern blot analysis. Bile acid concentrations were determined in liver, isolated nuclei, and plasma by gas chromatography and mass spectrometry. The results indicated that the addition of DCA and UDCA to the diet markedly shifted the bile-acid compositions of liver and plasma. In addition, DCA dramatically altered the abundance of many transcripts post-PH, whereas coadministration of UDCA suppressed the effect. DCA feeding significantly inhibited liver growth through day 3; however, by day 8, it induced an approximately 20% increase in mass compared with controls, UDCA-fed, or combination-fed animals. UDCA was concentrated greater than 20-fold in nuclei compared with whole liver in controls and DCA-fed animals and greater than 2-fold with UDCA feeding. These data suggest that bile acids may have a key role in liver regeneration, which is significantly altered by modulation of the bile-acid pool.

Creatine-supplemented diet extends Purkinje cell survival in spinocerebellar ataxia type 1 transgenic mice but does not prevent the ataxic phenotype.

It is not known why expression of a protein with an expanded polyglutamine region is pathogenic in spinocerebellar ataxia, Huntington's disease and several other neurodegenerative diseases. Dietary supplementation with creatine improves survival and motor performance and delays neuronal atrophy in the R6/2 transgenic mouse model of Huntington's disease. These effects may be due to improved energy and calcium homeostasis, enhanced presynaptic glutamate uptake, or protection of mitochondria from the mitochondrial permeability transition. We tested the effects of a 2% creatine-supplemented diet and treatment with taurine-conjugated ursodeoxycholic acid, a bile constituent that can inhibit the mitochondrial permeability transition, on ataxia and Purkinje cell survival in a transgenic model of spinocerebellar ataxia type 1. After 24 weeks, transgenic mice on the 2% creatine diet had cerebellar phosphocreatine levels that were 72.5% of wildtype controls, compared to 26.8% in transgenic mice fed a control diet. The creatine diet resulted in maintenance of Purkinje cell numbers in these transgenic mice at levels comparable to wildtype controls, while transgenic mice fed a control diet lost over 25% of their Purkinje cell population. Nevertheless, the ataxic phenotype was neither improved nor delayed. Repeated s.c. ursodeoxycholic acid injections markedly elevated ursodeoxycholic acid levels in the brain without adverse effects, but provided no improvement in phenotype or cell survival in spinocerebellar ataxia type 1 mice. These results demonstrate that preserving neurons from degeneration is insufficient to prevent a behavioral phenotype in this transgenic model of polyglutamine disease. In addition, we suggest that the means by which creatine mitigates against the neurodegenerative effects of an ataxin-1 protein containing an expanded polyglutamine region is through mechanisms other than stabilization of mitochondrial membranes.

The therapeutic effects of ursodeoxycholic acid as an anti-apoptotic agent.

The dihydroxy bile acid, ursodeoxycholic acid (UDCA), has been in widespread clinical use in the Western world since the mid 1980s, when it was initially used for gallstone dissolution [1,2] and subsequently for the treatment of chronic cholestatic liver diseases [3,4]. Many clinical trials of UDCA in a variety of cholestatic disorders established biochemical and clinical improvements, and most importantly showed a significant prolongation of transplant-free survival after four years of treatment with UDCA in patients with primary biliary cirrhosis [5]. Despite its clinical efficacy, the precise mechanism(s) by which UDCA improves liver function during cholestasis is still a matter of debate [6]. It was initially considered that the choleretic effect of UDCA, coupled with its ability to cause a marked shift in the composition of the bile acid pool towards hydrophilicity, accounted for its mechanism of action. In recent years, however, it has become evident that UDCA and its conjugated derivatives are capable of exerting direct effects at the cellular, subcellular, and molecular levels by stabilising cell membranes, affecting signal transduction pathways, and regulating immune responses. In addition, we have shown that UDCA plays a unique role in modulating the apoptotic threshold in both hepatic and non-hepatic cells [7-10]. The purpose of this article is to examine the mechanism(s) by which UDCA prevents apoptotic cell death associated with cholestasis. In addition, we will also review a potentially novel and, heretofore, unrecognised role of UDCA as a therapeutic agent in the treatment of non-liver diseases associated with increased levels of apoptosis as a pathogenesis of the disorder.

Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition.

Ursodeoxycholic acid (UDCA) has been shown to be a strong modulator of the apoptotic threshold in both hepatic and nonhepatic cells. 3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, appears to cause apoptotic neuronal cell death in the striatum, reminiscent of the neurochemical and anatomical changes associated with Huntington's disease (HD). This study was undertaken (a) to characterize further the mechanism by which 3-NP induces apoptosis in rat neuronal RN33B cells and (b) to determine if and how the taurine-conjugated UDCA, tauroursodeoxycholic acid (TUDCA), inhibits apoptosis induced by 3-NP. Our results indicate that coincubation of cells with TUDCA and 3-NP was associated with an approximately 80% reduction in apoptosis (p < 0.001), whereas neither taurine nor cyclosporin A, a potent inhibitor of the mitochondrial permeability transition (MPT), inhibited cell death. Moreover, TUDCA, as well as UDCA and its glycine-conjugated form, glycoursodeoxycholic acid, prevented mitochondrial release of cytochrome c (p < 0.001), which probably accounts for the observed inhibition of DEVD-specific caspase activity and poly(ADP-ribose) polymerase cleavage. 3-NP decreased mitochondrial transmembrane potential (p < 0.001) and increased mitochondrial-associated Bax protein levels (p < 0.001). Coincubation with TUDCA was associated with significant inhibition of these mitochondrial membrane alterations (p < 0.01). The results suggest that TUDCA inhibits 3-NP-induced apoptosis via direct inhibition of mitochondrial depolarization and outer membrane disruption, together with modulation of Bax translocation from cytosol to mitochondria. In addition, cell death by 3-NP apparently occurs through pathways that are independent of the MPT.

Genomic organization and promoter characterization of the rat cyclin B1 gene.

Cyclin B1 is a key regulatory protein involved in cellular mitosis. We have cloned 1.8kb of DNA sequence upstream of the rat cyclin B1 gene translation start site from Rattus norvegicus liver genomic DNA and a commercial rat testis genomic library. The mRNA transcription start point (tsp) was determined by primer extension and mRNA end ligation followed by RT-PCR across the ligated 3' and 5' ends. An authentic tsp was confirmed approximately 100bp upstream of the translation start site. A second potential tsp was also detected approximately 32bp downstream from the first. RT-PCR analysis of rat liver poly(A)(+) RNA using 5'-derived oligonucleotide primers indicated that the 5' end sequence was present in both the 1.6 and 2. 4kb rat liver cyclin B1 mRNA species. Like many other cyclin promoters, there was no apparent TATA box upstream of the transcription initiation sites. However, computer analysis of the promoter region identified a group of consensus transcription factor binding sites, some of which are also reported in other cyclin promoters. These include those for p53, p21, Ap-1, Ap-2, Ets-1, CAATT, E-Box and Yi. We also performed luciferase reporter assays using a set of promoter deletion constructs in human HuH-7 hepatoma and HeLa carcinoma cell lines. Our results suggest that an E-Box and/or CCAAT binding sites are important for transcription, and that there may be negative regulatory elements present between 1800 and 1100bp upstream of the translation start site.

Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation.

The hydrophilic bile salt ursodeoxycholic acid (UDCA) is a potent inhibitor of apoptosis. In this paper, we further characterize the mechanism by which UDCA inhibits apoptosis induced by deoxycholic acid, okadaic acid and transforming growth factor beta1 in primary rat hepatocytes. Our data indicate that coincubation of cells with UDCA and each of the apoptosis-inducing agents was associated with an approximately 80% inhibition of nuclear fragmentation (P<0.001). Moreover, UDCA prevented mitochondrial release of cytochrome c into the cytoplasm by 70 - 75% (P<0.001), thereby, inhibiting subsequent activation of DEVD-specific caspases and cleavage of poly(ADP-ribose) polymerase. Each of the apoptosis-inducing agents decreased mitochondrial transmembrane potential and increased mitochondrial-associated Bax protein levels. Coincubation with UDCA was associated with significant inhibition of these mitochondrial membrane alterations. The results suggest that the mechanism by which UDCA inhibits apoptosis involves an interplay of events in which both depolarization and channel-forming activity of the mitochondrial membrane are inhibited.

Correction of the UDP-glucuronosyltransferase gene defect in the gunn rat model of crigler-najjar syndrome type I with a chimeric oligonucleotide.

Crigler-Najjar syndrome type I is characterized by unconjugated hyperbilirubinemia resulting from an autosomal recessive inherited deficiency of hepatic UDP-glucuronosyltransferase (UGT) 1A1 activity. The enzyme is essential for glucuronidation and biliary excretion of bilirubin, and its absence can be fatal. The Gunn rat is an excellent animal model of this disease, exhibiting a single guanosine (G) base deletion within the UGT1A1 gene. The defect results in a frameshift and a premature stop codon, absence of enzyme activity, and hyperbilirubinemia. Here, we show permanent correction of the UGT1A1 genetic defect in Gunn rat liver with site-specific replacement of the absent G residue at nucleotide 1206 by using an RNA/DNA oligonucleotide designed to promote endogenous repair of genomic DNA. The chimeric oligonucleotide was either complexed with polyethylenimine or encapsulated in anionic liposomes, administered i.v., and targeted to the hepatocyte via the asialoglycoprotein receptor. G insertion was determined by PCR amplification, colony lift hybridizations, restriction endonuclease digestion, and DNA sequencing, and confirmed by genomic Southern blot analysis. DNA repair was specific, efficient, stable throughout the 6-month observation period, and associated with reduction of serum bilirubin levels. Our results indicate that correction of the UGT1A1 genetic lesion in the Gunn rat restores enzyme expression and bilirubin conjugating activity, with consequent improvement in the metabolic abnormality.

Gene repair using chimeric RNA/DNA oligonucleotides.

An experimental strategy has been developed for the site-specific alteration of genomic DNA. The approach is based on the observation that oligonucleotides containing complementary RNA/DNA hybrid regions are more active than duplex DNA in homologous pairing reactions in vitro. The chimeric molecules are designed with a homologous targeting sequence comprised of a DNA region flanked by blocks of 2'-O-methyl RNA residues (the chimeric strand), its complementary all-DNA strand, thymidine hairpin caps, a single-strand break, and a double-stranded clamp region. The oligonucleotide can align in perfect register with a genomic target except for the designed single base pair mismatch, which is recognized and corrected by harnessing the cell's endogenous DNA repair system. The mechanism of repair has been studied using mammalian cell-free extracts and bacterial systems and has revealed a mismatch correction pathway distinct from homologous recombination. The chimeric molecules have been demonstrated to be effective in the alteration of single nucleotides in episomal and genomic DNA in cell culture, as well as genomic DNA of cells in situ. This is a potentially powerful strategy for gene repair for the myriad hepatic genetic diseases caused by point mutations.

Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor.

Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19-24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides.