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We recently reported the isolation and characterization of a novel population of progenitor cells called liver-derived progenitor cells (LDPCs), which could differentiate into functional hepatocytes in vitro. However, our original studies resulted in relatively low and variable hepatic differentiation efficiency without validation of in vivo potential of LDPCs. Here, we report an efficient and robust hepatic differentiation of LDPCs under well-defined culture conditions and in vivo differentiation of LDPCs to mature hepatocytes. In addition to morphological studies, we performed reverse-transcription polymerase chain reaction (RT-PCR) and microRNA analyses of the in vitro hepatic differentiation of LDPCs to substantiate the efficiency of the differentiation process. The histological studies on the differentiated LDPCs showed that more than 50% of the cells were positive for albumin, cytokeratin 18, and hepatocyte nuclear factor 1 alpha and contained glycogen particles, all consistent with differentiation to functional hepatocytes. We also demonstrated by RT-PCR that upon differentiation, they expressed several markers found in mature hepatocytes and the microRNA profile of LDPCs became similar to the profile of fresh hepatocytes, confirming our morphological findings. Finally, the transplantation of LDPCs in a dipeptidyl peptidase IV-deficient (DPPIV(-/-)) rat model showed that LDPCs were able to engraft and form mature hepatocytes in the livers of the DPPIV(-/-) rats. In summary, LDPCs are a unique population of liver progenitor cells capable of hepatic differentiation both in vitro and in vivo, which makes them a potentially valuable resource for important applications such as pharmacological studies and cell therapies for a variety of liver disorders.

Background MicroRNAs (miRs or miRNAs) regulate several biological processes in the cell. However, evidence for miRNAs that control the differentiation program of specific neural cell types has been elusive. Recently, we have shown that apoptosis-associated factors, such as p53 and caspases participate in the differentiation process of mouse neural stem (NS) cells. To identify apoptosis-associated miRNAs that might play a role in neuronal development, we performed global miRNA expression profiling experiments in NS cells. Next, we characterized the expression of proapoptotic miRNAs, including miR-16, let-7a and miR-34a in distinct models of neural differentiation, including mouse embryonic stem cells, PC12 and NT2N cells. In addition, the expression of antiapoptotic miR-19a and 20a was also evaluated. Results The expression of miR-16, let-7a and miR-34a was consistently upregulated in neural differentiation models. In contrast, expression of miR-19a and miR-20a was downregulated in mouse NS cell differentiation. Importantly, differential expression of specific apoptosis-related miRNAs was not associated with increased cell death. Overexpression of miR-34a increased the proportion of postmitotic neurons of mouse NS cells. Conclusions In conclusion, the identification of miR-16, let-7a and miR-34a, whose expression patterns are conserved in mouse, rat and human neural differentiation, implicates these specific miRNAs in mammalian neuronal development. The results provide new insights into the regulation of neuronal differentiation by apoptosis-associated miRNAs.

New gene regulation study tools such as microRNA (miRNA or miR) analysis may provide unique insights into the remarkable ability of the liver to regenerate. In addition, we have previously shown that ursodeoxycholic acid (UDCA) modulates mRNA levels during liver regeneration. Bile acids are also homeotrophic sensors of functional hepatic capacity. The present study was designed to determine whether miRNAs are modulated in rats following 70% partial hepatectomy (PH) and elucidate the role of UDCA in regulating miRNA expression during liver regeneration (LR). Total RNA was isolated from livers harvested at 3–72 h following 70% PH or sham operations, from both 0.4% (wt/wt) UDCA and control diet-fed animals. By using a custom microarray platform we found that several miRNAs are significantly altered after PH by >1.5-fold, including some previously described as modulators of cell proliferation, differentiation, and death. In particular, expression of miR-21 was increased after PH. Functional modulation of miR-21 in primary rat hepatocytes increased cell proliferation and viability. Importantly, UDCA was a strong inducer of miR-21 both during LR and in cultured HepG2 cells. In fact, UDCA feeding appeared to induce a sustained increase of proliferative miRNAs observed at early time points after PH. In conclusion, miRNAs, in particular miR-21, may play a significant role in modulating proliferation and cell cycle progression genes after PH. miR-21 is additionally induced by UDCA in both regenerating rat liver and in vitro, which may represent a new mechanism behind UDCA biological functions.

The dynamic and multiple functions of p53, together with its involvement in the most common non-infectious diseases, underscore the need to elucidate the complexity of the p53 regulatory networks. Pathological conditions such as cancer, neurodegeneration, ischemia, cholestasis, and atherosclerosis are all strongly associated with deregulated levels of apoptosis in which p53 dysfunction has a prominent role. We will highlight recent developments of p53-induced apoptosis in human diseases, with a focus on modulation of liver cell apoptosis. In addition, we will discuss controversies arising from widespread p53 activation as a therapeutic approach to cancer. Recent studies have provided relevant and unprecedented information about mechanistic antiapoptotic functions of the endogenous bile acid, ursodeoxycholic acid (UDCA), suggesting that the finely tuned, complex control of p53 by Mdm-2 (mouse double minute-2, an oncoprotein) is a key step in UDCA modulation of p53-triggered apoptosis. We will also review recent therapeutic strategies and clinical applications of targeted agents, their safety, and efficacy, with particular emphasis on potential benefits of UDCA.

Apoptosis is a well-orchestrated cellular mechanism that balances the effects of cell proliferation and cell death. MicroRNAs (miRNAs) have been shown to control cell growth, differentiation, and apoptosis; and can be significantly deregulated in many cancers types. In fact, the ability to evade apoptosis is a hallmark of tumorigenesis. Although the role of miRNAs in the regulation of apoptosis is not fully understood, the recent influx of data strongly suggests that miRNAs play a significant role in regulating programmed cell death, or apoptosis. The genes involved in apoptotic pathways can be broadly classified as pro-apoptotic and anti-apoptotic. Many of these apoptotic genes, irrespective of their positive or negative functional role in apoptosis, are regulated by miRNAs. In this review, we discuss the emerging role of miRNA-mediated gene networks in the control of apoptosis.

The last two decades have shown significant advances in our understanding of age- and disease-related alterations in the regulation of gene expression and the underlying endogenous gene repair pathways. As a result, there have been some important strides in the development of engineered technologies for repairing mutated or damaged DNA via the development, delivery, and integration of specific and selective modified DNA into precise locations. This chapter describes available strategies for engineered gene repair including homologous recombination, the use of ribozymes, antisense nucleotides and DNA ribonucleases, single strand replacement/ chimeraplasty, and triplex DNA. The evolution of these approaches together with RNA interference is discussed, and relevant mechanisms and pathways are described. Present demonstrations of the utility of each of these gene modification approaches for therapeutic use imply that these methods can be employed to correct inherited and aging-related mutations and their consequences in future applications.

MicroRNAs (miRNAs) have emerged as one of the major regulatory mechanisms of gene expression. A major function of miRNAs involves the post-transcriptional regulation of target mRNAs, which is reported to occur primarily in the cytoplasm. However, there is a significant amount of evidence demonstrating the existence of small non-coding RNAs, including small-interfering RNA (siRNA), miRNA, and Piwi-interacting RNA (piRNA) in the nucleus. In order to elucidate the potential subcellular localizations and functions of miRNAs, we have identified numerous miRNAs that are present in isolated nuclei from human colon cancer HCT116 cells. MicroRNA profiles were compared between cytoplasmic and nuclear fractions of the HCT116 cell line on the basis of multiple microarray analyses. MicroRNA species showing significant existence in isolated and highly purified populations of nuclei were selected and further tested with RT-PCR. The nuclear localization of the mature form of miRNAs was verified again by control RT-PCR excluding the detection of premature forms of miRNA, such as pri-miRNA or pre-miRNA. The elevated levels of representative miRNAs identified in purified nuclei were confirmed by Northern blot analysis, supporting the notion that significant numbers of mature miRNAs exist not only in the cytoplasm but also in the nucleus. These results will likely provide a basis for further studies concerning the intracellular trafficking and nuclear location of miRNAs.

Prostate cancer, a complex disease, can be relatively harmless or extremely aggressive. To identify candidate genes involved in causal pathways of aggressive prostate cancer, we implemented a systems biology approach by combining differential expression analysis and coexpression network analysis to evaluate transcriptional profiles using lymphoblastoid cell lines from 62 prostate cancer patients with aggressive phenotype (Gleason grade ≥ 8) and 63 prostate cancer patients with nonaggressive phenotype (Gleason grade ≤ 5). From 13,935 mRNA genes and 273 microRNAs (miRNA) tested, we identified significant differences in 1,100 mRNAs and 7 miRNAs with a false discovery rate (FDR) of

Despite important roles in mammalian gene regulation, the critical targeting mechanism of DNA methylation for specific DNA sequences remains unclear. Recently, small non-coding RNAs were reported to be essential for DNA methylation in plants as well as in mice, suggesting that small non-coding RNAs might interact with DNA methyltransferases (DNMTs) to provide them with target sequence information. In the present study, we attempted to detect and isolate microRNAs by immunoprecipitation (IP) that might be associated with human DNMT3a. When analyzed by gel electrophoresis after radioisotope-labeling, DNMT3a IP revealed no detectable levels of microRNA. RNA from DNMT3a IP was also analyzed with microRNA microarray and with subsequent RT-PCR. The results showed no specific enrichment of candidate microRNAs in the DNMT3a IP. DNMT3a was not directly associated with microRNAs. But, other classes of small non-coding RNA could still be implicated with DNMTs in a specific tissue such as testis where such RNA species are highly abundant.

MicroRNAs are aberrantly expressed in cancer; microRNA-143 (miR-143) is down-regulated in colon cancer. HCT116 human colorectal cancer cells were used to investigate the biological role of miR-143. Transient miR-143 overexpression resulted in an approximate 60% reduction in cell viability. In addition, stable miR-143 overexpressing cells were selected with G418 and exposed to 5-fluorouracil. Increased stable expression of miR-143 was associated with decreased viability and increased cell death after exposure to 5-fluorouracil. These changes were associated with increased nuclear fragmentation and caspase -3, -8 and -9 activities. In addition, extracellular-regulated protein kinase 5, nuclear factor-kappaB and Bcl-2 protein expression was down-regulated by miR-143, and further reduced by exposure to 5-fluorouracil. In conclusion, miR-143 modulates the expression of key proteins involved in the regulation of cell proliferation, death and chemotherapy response. In addition, miR-143 increases the sensitivity of colon cancer cells to 5-fluorouracil, probably acting through extracellular-regulated protein kinase 5/nuclear factor-kappaB regulated pathways. Collectively, the data obtained in the present study suggest anti-proliferative, chemosensitizer and putative pro-apoptotic roles for miR-143 in colon cancer.

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 μm in diameter. RA1 cells proliferated in vitro with a doubling time of 5–7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.

Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli -galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8g/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of CriglerNajjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1. Conclusion: FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application. (HEPATOLOGY 2009;50:815-824.)

Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type-specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain-deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.

Hepatocellular carcinoma (HCC) is a common malignant tumor that almost always occurs within a preexisting background of chronic liver disease and cirrhosis. Currently, medical therapy is not effective in treating most HCC, and the only hope of cure is either resection or liver transplantation. A small minority of patients is eligible for these therapies, which entail major morbidity at the very least. In spite of immense scientific advances during the past 3 decades, patient survival has improved very little. In order to reduce morbidity and mortality from HCC, improvements in early diagnosis and development of novel local and systemic therapies for advanced disease are essential, in addition to efforts geared towards primary prevention. Studies with experimental animal models that closely mimic human disease are very valuable in understanding physiological, cellular and molecular mechanisms underlying the disease. Furthermore, appropriate animal models have the potential to increase our understanding of the effects of image-guided minimally invasive therapies and thereby help to improve such therapies. In this review, we examine the evidence for stem cell origins of such tumors, critically evaluate existing models and reflect on how to develop new models for minimally invasive, image-guided treatment of HCC.

Neural stem cells (NSCs) differentiate into neurons and glia, and a large percentage undergoes apoptosis. The engagement and activity of apoptotic pathways may favor either cell death or differentiation. In addition, Akt represses differentiation by up-regulating the inhibitor of differentiation 1 (Id 1), through phosphorylation of its repressor FOXO3A. The aim of this study was to investigate the potential cross-talk between apoptosis and proliferation during mouse NSC differentiation. We determined the time of neurogenesis and gliogenesis using neuronal beta-III tubulin and astroglial GFAP to confirm that both processes occurred at similar to 3 and 8 days, respectively. p-Akt, p-FOXO3A, and Id 1 were significantly reduced throughout differentiation. Caspase-3 processing, p53 phosphorylation, and p53 transcriptional activation increased at 3 days of differentiation, with no evidence of apoptosis. Importantly, in cells exposed to the pancaspase inhibitor z-VAD.fmk, p-FOXO3A and Id 1 were no longer down-regulated, p53 phosphorylation and transcriptional activation were reduced, while neurogenesis and gliogenesis were significantly delayed. The effect of siRNA-mediated silencing of p53 on FOXO3A/Id 1 was similar to that of z-VAD.fmk only at 3 days of differentiation. Interestingly, caspase inhibition further increased the effect of p53 knockdown during neurogenesis. In conclusion, apoptosis-associated factors such as caspases and p53 temporally modulate FOXO3A/Id 1 signaling and differentiation of mouse NSCs. J. Cell. Biochem. 107: 748-758, 2009. (C) 2009 Wiley-Liss, Inc.. - Fundacao para a Ciencia e a Tecnologia (FCT) ; FEDER [PTDC/SAU-FCF/67912/2006]. - Fundacao para a Ciencia e a Tecnologia (FCT) and FEDER; Grant number: PTDC/SAU-FCF/67912/2006.

Background Colon cancer arises from the accumulation of multiple genetic and epigenetic alterations to normal colonic tissue. microRNAs (miRNAs) are small, non-coding regulatory RNAs that post-transcriptionally regulate gene expression. Differential miRNA expression in cancer versus normal tissue is a common event and may be pivotal for tumor onset and progression. Methods To identify miRNAs that are differentially expressed in tumors and tumor subtypes, we carried out highly sensitive expression profiling of 735 miRNAs on samples obtained from a statistically powerful set of tumors (n = 80) and normal colon tissue (n = 28) and validated a subset of this data by qRT-PCR. Results Tumor specimens showed highly significant and large fold change differential expression of the levels of 39 miRNAs including miR-135b, miR-96, miR-182, miR-183, miR-1, and miR-133a, relative to normal colon tissue. Significant differences were also seen in 6 miRNAs including miR-31 and miR-592, in the direct comparison of tumors that were deficient or proficient for mismatch repair. Examination of the genomic regions containing differentially expressed miRNAs revealed that they were also differentially methylated in colon cancer at a far greater rate than would be expected by chance. A network of interactions between these miRNAs and genes associated with colon cancer provided evidence for the role of these miRNAs as oncogenes by attenuation of tumor suppressor genes. Conclusion Colon tumors show differential expression of miRNAs depending on mismatch repair status. miRNA expression in colon tumors has an epigenetic component and altered expression that may reflect a reversion to regulatory programs characteristic of undifferentiated proliferative developmental states.

We have developed computational techniques that allow image averaging to be applied to electron micrographs of filamentous molecules that exhibit tight and variable curvature. These techniques, which involve straightening by cubic-spline interpolation, image classification, and statistical analysis of the molecules' curvature properties, have been applied to purified brain clathrin. This trimeric filamentous protein polymerizes, both in vivo and in vitro, into a wide range of polyhedral structures. Contrasted by low-angle rotary shadowing, dissociated clathrin molecules appear as distinctive three-legged structures, called "triskelions" (E. Ungewickell and D. Branton (1981) Nature 289, 420). We find triskelion legs to vary from 35 to 62 nm in total length, according to an approximately bell-shaped distribution (mu = 51.6 nm). Peaks in averaged curvature profiles mark hinges or sites of enhanced flexibility. Such profiles, calculated for each length class, show that triskelion legs are flexible over their entire lengths. However, three curvature peaks are observed in every case: their locations define a proximal segment of systematically increasing length (14.0-19.0 nm), a mid-segment of fixed length (approximately 12 nm), and a rather variable end-segment (11.6-19.5 nm), terminating in a hinge just before the globular terminal domain (approximately 7.3 nm diameter). Thus, two major factors contribute to the overall variability in leg length: (1) stretching of the proximal segment and (2) stretching of the end-segment and/or scrolling of the terminal domain. The observed elasticity of the proximal segment may reflect phosphorylation of the clathrin light chains.

Cellular processing of 125I-labeled transforming growth factor-β1 was investigated in the human hepatoma cell lines Hep G2 and Hep 3B. Binding of 125I-transforming growth factor-β1 to cell surface receptors was specific, saturable and calciumindependent. Both cell lines exhibited a single class of high-affinity (Kd = 2.2 × 10−10 mol/L) binding sites (4.5 × 103 for the Hep G2 cell; 1.5 × 103 for the Hep 3B cell) for both human and porcine transforming growth factor-β1. Binding was temperature dependent, time dependent and pH dependent. Cell-bound 125I-transforming growth factor-β1 was removed by brief exposure to acidic medium (pH

The effects of chronic ethanol consumption on enzyme systems involved in carcinogen activation and detoxification were studied in a rat model of liver regeneration. In control rats, steady-state messenger RNAs of cytochrome P450j decreased 12 to 24 hr after partial hepatectomy but were fully recovered by 48 to 72 hr. In contrast, messenger RNA levels of cytochrome P450b and P450d did not vary significantly during that period. Steady-state messenger RNA levels for the placental form of glutathione S-transferase decreased within 30 min after partial hepatectomy but fluctuated until levels returned to normal by 48 hr. Preliminary nuclear run-on analyses suggest that the regulation of cytochrome P450j and the placental form of glutathione S-transferase messenger RNA levels involves posttranscriptional control in these animals. In ethanol-fed rats, as in controls, expression of cytochrome P450j and the placental form of glutathione S-transferase decreased transiently after partial hepatectomy. However, compared with control values, messenger RNA levels for cytochrome P450j were greater in ethanol-fed rats at each time point. Similar results were noted for placental glutathione S-transferase levels from 12 to 48 hr after partial hepatectomy. Ethanol feeding had no apparent effect on steady-state messenger RNA levels of cytochrome P450d, P450b or the multidrug-resistant gene. In both ethanol and control rats, only prehepatectomy levels of cytochrome P450 transcripts correlated with levels of the respective P450 isoenzymes. These data indicate that liver regeneration selectively decreases the steady-state messenger RNA expression of certain isoenzymes of cytochrome P450 and glutathione S-transferase. Chronic ethanol consumption induces basal messenger RNA expression of cytochrome P450j and blunts regeneration-associated decreases in cytochrome P450j and glutathione S-transferase expression. These alterations may be involved in ethanol's ability to function as a co-carcinogen in liver. (HEPATOLOGY 1991;13:722–727.)

MicroRNAs (miRNAs) are a class of small approximately 22 nt noncoding (nc) RNAs that regulate gene expression post-transcriptionally by direct binding to target sites on mRNAs. They comprise more than 1,000 novel species in mammalian cells and exert their function by modulating gene expression through several different mechanisms, including translational inhibition, and/or degradation of target mRNAs. Mitochondria maintain and express their own genome, which is distinct from the nuclear transcriptional and translational apparatus. Thus, they provide a potential site for miRNA mediated post-transcriptional regulation. To determine whether they maintain a unique miRNA population, we examined the miRNA profile from highly purified and RNase treated mitochondria from adult rat liver. Fifteen miRNAs were identified by microarray analysis of which, five were confirmed by TaqMan 5'nuclease assays using rat specific probes. Functional analysis of the miRNAs indicated that they were not targeted to the mitochondrial genome nor were they complementary to nuclear RNAs encoding mitochondrial proteins. Rather, the mitochondria-associated miRNAs appear to be involved in the expression of genes associated with apoptosis, cell proliferation, and differentiation. Given the central role that mitochondria play in apoptosis, the results suggest that they might serve as reservoirs of select miRNAs that may modulate these processes in a coordinate fashion.

Hepatocellular carcinoma (HCC) typically has poor prognosis, because it is often diagnosed at an advanced stage. Heterogeneous phenotypic and genetic traits of affected individuals and a wide range of risk factors have classified it a complex disease. HCC is not amenable to standard chemotherapy and is resistant to radiotherapy. In most cases, surgical resection and liver transplantation remain the only curative treatment options. Therefore, development of novel, effective therapies is of prime importance. Extensive research over the past decade has identified a number of molecular biomarkers as well as cellular networks and signaling pathways affected in liver cancer. Recent studies using a combination of "omics" technologies, microRNA studies, combinatorial chemistry, and bioinformatics are providing new insights into the gene expression and protein profiles during various stages of the disease. In this review, we discuss the contribution of these newer approaches toward an understanding of molecular mechanisms of HCC and for the development of novel cancer therapeutics.

Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, the total HO-1 mRNA was enriched 2-fold relative to control cells, and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and was injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate the transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery, and monitoring of gene-therapy vectors.

Current techniques for the alteration of gene expression in the liver have a number of limitations, including the lack of stable somatic gene transfer and the technical challenges of germline transgenesis. Rapid and stable genetic engineering of the liver would allow systematic, in vivo testing of contributions by many genes to disease. After fumaryl acetoacetate hydrolase (Fah) gene transfer to hepatocytes, selective repopulation of the liver occurs in FAH-deficient mice. This genetic correction is readily mediated with transposons. Using this approach, we show that genes with biological utility can be linked to a selectable Fah transposon cassette. First, net conversion of Fah−/− liver tissue to transgenic tissue, and its outgrowth, was monitored by bioluminescence in vivo from a luciferase gene linked to the FAH gene. Second, coexpressed short hairpin RNAs (shRNAs) stably reduced target gene expression, indicating the potential for loss-of-function assays. Third, a mutant allele of human α1-antitrypsin (hAAT) was linked to Fah and resulted in protein inclusions within hepatocytes, which are the histopathological hallmark of hAAT deficiency disorder. Finally, oncogenes linked to Fah resulted in transformation of transduced hepatocytes. Conclusion: Coexpression with FAH is an effective technique for lifelong expression of transgenes in adult hepatocytes with applicability to a wide variety of genetic studies in the liver. (HEPATOLOGY 2008;47:1714–1724.)

Radiation inactivation and sedimentation equilibrium analysis were used to determine the functional and physical size of the chicken hepatic membrane receptor that binds N-acetylglucosamine-terminated glycoproteins. Purified plasma membranes from chicken liver were irradiated with high energy electrons and assayed for 125I-agalactoorosomucoid binding. Increasing the dose of ionizing radiation resulted in a monoexponential decay in binding activity due to a progressive loss of binding sites. The molecular mass of the chicken lectin, determined in situ by target analysis, was 69,000 +/- 9,000 Da. When the same irradiated membranes were solubilized in Brij 58 and assayed, the binding protein exhibited a target size of 62,000 +/- 4,000 Da; in Triton X-100, the functional size of the receptor was 85,000 +/- 10,000 Da. Sedimentation equilibrium measurements of the purified binding protein yielded a lower limit molecular weight of 79,000 +/- 7,000. However, the solubilized lectin was detected as a heterogeneous population of oligomers with molecular weights as high as 450,000. Addition of calcium or calcium plus N-acetylglucosamine decreased the higher molecular weight species, but the lower limit molecular weights remained invariant. Similar results were determined when the chicken lectin was solubilized in Brij 58, C12E9, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS). Results from the present study suggest that in the plasma membrane, the functional species of the chicken hepatic lectin exists as a trimer. However, in detergent solution, the purified receptor forms a heterogeneous population of irreversible oligomers that exhibit binding activity proportional to size.

The potential role of apoptosis in Alzheimer's disease (AD) has been an area of intense research in recent years. Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA) are endogenous bile acids that act as potent inhibitors of apoptosis. Their therapeutic effects have been tested in many experimental pathological conditions, including neurological disorders, such as AD. TUDCA regulates precise transcriptional and post-transcriptional events that impact mitochondrial function in neurons. TUDCA not only stabilizes the mitochondrial membrane and prevents Bax translocation, inhibiting the release of cytochrome c and the activation of caspases, but also interferes with upstream factors, including cell cycle-related proteins. In addition, TUDCA is capable of inducing survival pathways. Here, we review the role of apoptosis in AD and discuss the therapeutic potential of TUDCA in treating this disease.

Apoptosis represents a universal and exquisitely efficient cellular suicide pathway essential for a variety of normal biological processes ranging from embryonic development to ageing. In fact, tissue homeostasis is dependent on the perfect balance between positive and negative signals that determines the decision between life and death. Therefore, any imbalance can result in a wide range of pathologic disorders associated with unwanted apoptosis or cell growth. During the apoptotic process, the molecular players interact closely with each other in ways relevant to accelerate or interrupt the cellular death process. In addition, two major pathways of apoptosis activation have been recognized as the "intrinsic" mitochondrial pathway and the "extrinsic" death receptor pathway. Although these pathways act independently to initiate apoptosis, a delicate balance and cross-talk between the extrinsic and intrinsic pathways is thought to occur in many cell types. Interestingly, we have shown that ursodeoxycholic acid (UDCA), an endogenous hydrophilic bile acid, is a potent inhibitor of apoptosis by either stabilizing the mitochondrial membrane or modulating the expression of specific upstream targets. Herein, we review the main effectors involved in the death machinery, describe how they interact to regulate apoptosis, and discuss the main pathways that control cell death and survival. Further, we address multiple interesting targets as well as the potential application of UDCA as a therapeutic modality for apoptosis-related disorders.

Bear bile has been used in Asia for over 3,000 years to treat visual disorders, yet its therapeutic potential remains unexplored in Western vision research. The purpose of this study was to test whether treatment of mice undergoing retinal degeneration with tauroursodeoxycholic acid (TUDCA), a primary constituent of bear bile, alters the course of degeneration.Two retinal degeneration models were tested: the rd10 mouse, which has a point mutation in the gene encoding the beta subunit of rod phosphodiesterase, and light induced retinal damage (LIRD). For LIRD studies, albino Balb/C adult mice were subcutaneously injected with TUDCA (500 mg/kg body weight) or vehicle (0.15 M NaHCO(3)). Sixteen h later, each mouse received repeat injections. Half of each treatment group was then placed in bright light (10,000 lux) or dim light (200 lux) for seven h. At the end of exposure, animals were transferred to their regular housing. Electroretinograms (ERGs) were assessed 24 h later, mice sacrificed, eyes embedded in paraffin and sectioned, and retina sections assayed for morphology and apoptosis by TUNEL and anti-active caspase-3 immunoreactivity via fluorescent confocal microscopy. A subset of mice were sacrificed 8 and 15 days after exposure and retina sections analyzed for morphology and apoptosis. For rd10 studies, mice were injected subcutaneously with TUDCA or vehicle at postnatal (P) days 6, 9, 12, and 15. At p18, ERGs were recorded, mice were euthanized and eyes were harvested, fixed, and processed. Retinal sections were stained (toluidine blue), and retinal cell layers morphometrically analyzed by light microscopy. Consecutive sections were analyzed for apopotosis as above.By every measure, TUDCA greatly slowed retinal degeneration in LIRD and rd10 mice. ERG a-wave and b-wave amplitudes were greater in mice treated with TUDCA compared to those treated with vehicle. Retinas of TUDCA-treated mice had thicker outer nuclear layers, more photoreceptor cells, and more fully-developed photoreceptor outer segments. Finally, TUDCA treatments dramatically suppressed signs of apoptosis in both models.Systemic injection of TUDCA, a primary constituent of bear bile, profoundly suppressed apoptosis and preserved function and morphology of photoreceptor cells in two disparate mouse models of retinal degeneration. It may be that bear bile has endured so long in Asian pharmacopeias due to efficacy resulting from this anti-apoptotic and neuroprotective activity of TUDCA. These results also indicate that a systematic, clinical assessment of TUDCA may be warranted.

Black bear bile has been used in traditional Chinese medicine to treat liver and eye related illnesses for centuries. A major constituent of bile is ursodeoxycholic acid (UDCA). Recent analysis of the cellular effects of UDCA and its taurine conjugate tauroursodeoxycholic acid (TUDCA) have demonstrated their antiapoptotic properties through regulation of Bcl-2 family and survival signaling proteins (Bax, Bad, phosphatidylinositol-3-kinase). In this study, we tested the hypothesis that TUDCA administered to rats prior to a myocardial infarction (MI) would exhibit anti-apoptotic effects and improve cardiac function. Prior to ligation of the left anterior descending (LAD) coronary artery, TUDCA (50 mg/ml, 400 mg/kg, IV) or PBS was administered to rats. Animals were sacrificed 24 hours after ligation for terminal transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) and caspase-3 activity to assess apoptosis. Additional TUDCA or PBS treated rats underwent pre-operative,1 and 4 week transthoracic ultrasounds to assess heart function by quantification of shortening fraction (SF) and infarct area. TUNEL labeling of the cardiac tissue revealed a significant reduction in apoptotic cells in rats given TUDCA prior to ischemic injury ( p = 0.05). In support of reducing apoptosis, caspase-3 activity in the TUDCA treated animals also decreased ( p = 0.02). By 4 weeks, a significantly smaller infarct area was present in the TUDCA group compared to the PBS group (0.05 vs. 0.13 cm2, p = NS) and there was also an improvement in SF. The results provide evidence for TUDCA as a viable treatment for reducing apoptosis in a model of myocardial infarction. Additional studies will distinguish the functional result of improved cell survival following infarction, suggesting the potential for clinical application of this anti-apoptotic drug in treatment of acute MI.

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). Hemolysis releases free hemoglobin (Hb) and Hb-containing microparticles into the vasculature that upon oxidation to methemoglobin frees heme from the globin, which in turn can promote oxidative stress and activate toll-like receptor 4 (TLR4) signaling. Hemopexin (HPX), a plasma β1-glycoprotein, binds heme with a very high affinity (Kd < 10-12 M), and transports it to the liver for catabolism via CD91 receptor-mediated uptake. SCD patients have low serum HPX levels likely due to chronic hemolysis leading to increased HPX catabolism with insufficient compensatory increase in synthesis. We and others have shown that HPX transports heme to the liver, and inhibits heme toxicity and the activation of endothelial, leukocyte and platelet TLR4 signaling. Acute studies have shown HPX infusion prior to a heme challenge protects sickle mice from vaso-occlusion and developing acute pulmonary injury while chronic HPX infusion therapy modified heme toxicity to endothelium. We hypothesize that in SCD mice, hepatic overexpression of HPX will bind the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis (vaso-occlusion). To examine the protective role of HPX in SCD, we transplanted bone marrow from NY1DD SCD mice into HPX-/- or normal C57BL/6 mice. After 12 weeks, conversion to the HbS phenotype was confirmed by isoelectric focusing. Dorsal skin fold chambers (DSFC) were implanted in week 13 and microvascular stasis (% non-flowing venules) assessed in response to heme (3.2 µmol/kg) infusion. HPX-/- sickle mice had 34% ± 3% and 24% ± 2% at 1h and 4h post heme, significantly greater than HPX+/+ C57BL/6 sickle mice which had 21% ± 5% and 13% ± 8% at 1 and 4 h, (mean ± SD, p DSFCs were implanted 4 weeks after plasmid infusion and microvascular stasis was assessed in response to heme (3.2 µmol/kg) infusion. NY1DD and Townes-SS mice overexpressing rat HPX (SB-HPX) had significantly less stasis than LRS or SB-LUC treated SCD mice (Figure 1A and B). HPX overexpression markedly increased nuclear Nrf2 expression in the livers of sickle mice, presumably by promoting delivery of heme to the liver and activating the Keap1-Nrf2 axis. In addition, hepatic HO-1 activity and protein and CD91 protein were increased in sickle mice overexpressing HPX and NF-ĸB activation was markedly decreased as assessed by nuclear phospho-p65-NF-ĸB expression on western blots demonstrating the anti-inflammatory properties of HPX in sickle mice. In conclusion, supplementing HPX levels in transgenic sickle mice via gene therapy activates the Nrf2 anti-oxidant axis and ameliorates inflammation and vaso-occlusion. We speculate that plasma HPX supplementation may be beneficial in SCD especially during hemolytic crises or acute chest syndrome. Figure 1. Figure 1. Disclosures Vercellotti: Cydan: Research Funding; CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding. Belcher:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding.

Introduction: Intercellular communication is critical for cancer growth and progression. Research to date has focused on gap junctions, micro-vesicles/exosomes, and diffusible factors. There is limited understanding of how efficient cell-to-cell communication occurs between distant cells in the complex tumor microenvironment. Tunneling nanotubes (TNTs) are thin, non-adherent actin filament-based, open conduits that facilitate direct sharing of various cellular cargos between cells. TNTs can be 50-800 nanometers in diameter and up to several hundred micrometers in length in vitro. Our lab was the first to provide in vivo evidence of TNTs in human solid tumors. We hypothesized that TNT formation is significantly upregulated in human cancers compared to their non-malignant counterparts. In this study, we examined the rate of TNT formation in cell lines from multiple human cancers. Methods: Cell lines from four cancers (colon, mesothelioma, pancreatic, and ovarian cancer) were plated at similar density in 6-well tissue culture plates. The number of TNTs and cells in 10 randomly chosen fields of each well were quantified every 24 hrs for up to 96 hrs and representative images were taken. Experiments were performed in duplicate for each cell line. Results: The number of TNTs per cell (TNTs/cell), which we have titled “TNT index,” was calculated in order to control for cell proliferation. The ratio of TNTs formed between malignant colorectal cells compared benign adenoma ranged from 20:1 to 100:1 by 96 hrs. For mesothelioma, malignant:mesothelial TNTs/cell ratio ranged from 8:1 to 78:1 over 72 hrs. The mesothelioma cells were too confluent at 96 hrs to quantify TNTs. The pancreatic malignant:stellate human pancreatic ductal epithelial TNTs/cell ratio ranged between 2:1 to 63:1. Interestingly, we found that non-malignant ovarian epithelial (IOSE) cells had a significantly higher rate of TNT formation than malignant ovarian cells. Conclusions: We have demonstrated that malignant cells have significantly higher rates of TNT formation compared to stromal or premalignant cells of their corresponding tumor. TNTs potentially provide malignant cells a direct route for transmitting influential messages across long distances in the complex tumor microenvironment. We believe that upregulation of TNTs in cancer may amplify cellular cross-talk and thus increase rates of tumor recurrence and invasion. Citation Format: Snider Desir, Subbaya Subramanian, Clifford Steer, Emil Lou. Tunneling nanotube formation is significantly upregulated in invasive cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3189. doi:10.1158/1538-7445.AM2015-3189

© 2015 Macmillan Publishers Limited All rights reserved. Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0, 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, This study was supported by Fundação para a Ciência e Tecnologia (HMSP-ICT/0018/2011, SFRH/BD/96517/2013, SFRH/BD/88619/2012 and SFRH/BD/79356/2011).

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.

Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA), play a unique role in modulating the apoptotic threshold in cells. The mechanism is thought to involve, in part, inhibition of translocation for Bax from the cytosol to mitochondria. Here, we attempted to use the native fluorescence of the tryptophan residues of Bax to determine whether bile acids bind directly to recombinant Bax protein. The results showed that UDCA had no effect on the tryptophan fluorescence of Bax. Similarly, there was no evidence of direct binding between Bax protein and the more hydrophobic bile acid, deoxycholic acid (DCA). In contrast, the fluorescence change detected for Bax solution titrated against TUDCA in dimethylsulfoxide was greater than that observed with solvent alone. In conclusion, data from fluorescence spectroscopy does not support a direct interaction of UDCA or DCA with Bax protein, whereas it suggests that there may be some potential interaction with TUDCA.

Early onset familial Alzheimer's disease (FAD) is linked to autosomal dominant mutations in the amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2) genes. These are critical mediators of total amyloid beta-peptide (Abeta) production, inducing cell death through uncertain mechanisms. Tauroursodeoxycholic acid (TUDCA) modulates exogenous Abeta-induced apoptosis by interfering with E2F-1/p53/Bax. Here, we used mouse neuroblastoma cells that express either wild-type APP, APP with the Swedish mutation (APPswe), or double-mutated human APP and PS1 (APPswe/DeltaE9), all exhibiting increased Abeta production and aggregation. Cell viability was decreased in APPswe and APPswe/DeltaE9 but was partially reversed by z-VAD.fmk. Nuclear fragmentation and caspase 2, 6 and 8 activation were also readily detected. TUDCA reduced nuclear fragmentation as well as caspase 2 and 6, but not caspase 8 activities. p53 activity, and Bcl-2 and Bax changes, were also modulated by TUDCA. Overexpression of p53, but not mutant p53, in wild-type and mutant neuroblastoma cells was sufficient to induce apoptosis, which, in turn, was reduced by TUDCA. In addition, inhibition of the phosphatidylinositide 3'-OH kinase pathway reduced TUDCA protection against p53-induced apoptosis. In conclusion, FAD mutations are associated with the activation of classical apoptotic pathways. TUDCA reduces p53-induced apoptosis and modulates expression of Bcl-2 family.

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The Sleeping Beauty (SB) transposon (Tn) system is a nonviral gene delivery tool that has widespread application for transfer of therapeutic genes into the mammalian genome. To determine its utility as a gene delivery system, it was important to assess the epigenetic modifications associated with SB insertion into the genome and potential inactivation of the transgene. This study investigated the DNA methylation pattern of an SB Tn as well as the flanking genomic region at insertion sites in the mouse genome. The ubiquitous ROSA26 promoter and an initial part of the eGFP coding sequence in the SB Tn exhibited high levels of CpG methylation in transgenic mouse lines, irrespective of the chromosomal loci of the insertion sites. In contrast, no detectable CpG methylation in the endogenous mouse ROSA26 counterpart was observed in the same animals. Furthermore, significant hypomethylation was detected in neighboring chromosomal sequences of two unique SB Tn insertions compared to wild-type patterns. Taken together, these results suggest that SB Tn insertions into the mouse genome can be discriminated by DNA methylation machinery from an identical endogenous DNA sequence and can profoundly alter the DNA methylation status of the transgene cargo as well as flanking host genomic regions.

To date, no other studies have examined the seasonal changes in circulating levels of various bile acids in the plasma of wild North American black bears, Ursus americanus. Using gas chromatography, bile acid concentrations were measured in plasma samples obtained during either early or late hibernation, and during summer active periods. Thus, specific compositional changes from individual animals were examined through a given year. Total bile acid concentrations in the plasma of these normal animals were found to range between 0.2 and 3.1 micromol/L (0.9 +/- 0.2 micromol/L, mean +/- SEM). Cholic, ursodeoxycholic and chenodeoxycholic acids were the major bile acid species identified. Ursodeoxycholic acid represented 28.0 +/- 2.6% of the total bile acid pool. Deoxycholic and lithocholic acids were found only in small amounts. In addition, total bile acid concentrations were lower in plasma samples obtained during hibernation compared with those obtained during summer active periods (0.6 +/- 0.1 and 1.2 +/- 0.4 micromol/L, respectively; p0.05). However, the relative proportion of ursodeoxycholic acid, was significantly greater in winter than in summer (31.5 +/- 3.2% and 22.2 +/- 4.5%, p0.05). Finally, taurine-conjugated bile acids were the predominant species in bear plasma, accounting for67% of the total bile acids. These data demonstrate that ursodeoxycholic acid is a major bile acid in black bear plasma, mostly conjugated with taurine. Further, the finding of seasonal variation in plasma bile acid composition provides evidence to support the possible role that ursodeoxycholic acid may play in cellular protection in hibernating black bears.

How genetic and environmental factors interact in Parkinson disease is poorly understood. We have now compared the patterns of vulnerability and rescue of Caenorhabditis elegans with genetic modifications of three different genetic factors implicated in Parkinson disease (PD). We observed that expressing alpha-synuclein, deleting parkin (K08E3.7), or knocking down DJ-1 (B0432.2) or parkin produces similar patterns of pharmacological vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than nontransgenic nematodes to mitochondrial complex I inhibitors, including rotenone, fenperoximate, pyridaben, or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity to paraquat, sodium azide, divalent metal ions (Fe(II) or Cu(II)), or etoposide compared with the nontransgenic nematodes. Each of the PD-related lines was also partially rescued by the antioxidant probucol, the mitochondrial complex II activator, D-beta-hydroxybutyrate, or the anti-apoptotic bile acid tauroursodeoxycholic acid. Complete protection in all lines was achieved by combining d-beta-hydroxybutyrate with tauroursodeoxycholic acid but not with probucol. These results show that diverse PD-related genetic modifications disrupt the mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD.

Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis-related genes from transforming growth factor β1 (TGF-β1)–induced hepatocyte apoptosis by a nuclear steroid receptor (NSR)–dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the antiapoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-β1–induced apoptosis of primary rat hepatocytes through a GR-dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy-terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF-β1–induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-β1. Finally, using a flourescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild-type or mutant forms of GR showed that nuclear trafficking occurs through a GR-dependent mechanism. In conclusion, these results further clarify the antiapoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis. (HEPATOLOGY 2005;42:925–934.)

SSOs (single-stranded oligonucleotides) can mediate site-specific alteration of base-pairs in episomal and chromosomal target genes in mammalian cells. The TNE (targeted nucleotide exchange) can result in either repair or mutation of a gene sequence and is mediated through endogenous DNA repair pathway(s). Thus the approach provides a technique for the treatment of monogenic disorders associated with specific point mutations such as SCD (sickle cell disease). We studied the potential application of SSOs for SCD by introducing either an A to T substitution at the sixth codon of the human beta-globin gene (sickle locus) or a C to G mutation at -202 of the Ggamma-globin gene promoter region. The latter TNE is an alternative strategy to ameliorate the clinical manifestations of sickle cell anaemia by re-activating fetal haemoglobin gene expression in adult erythrocytes. A sensitive and valid PCR assay system was developed, which allows detection of point mutations as low as 0.01% at these sites. Using this system, TNE between 0.01 and 0.1% at the sickle locus or gamma-globin gene promoter region was detected after transfection with SSOs in cultured human cell lines. TNE in the Ggamma-globin promoter region exhibited varying degrees of strand bias that was dependent on SSO design and the cell's DNA mismatch repair activity. The results suggest that the endogenous DNA repair machinery may permit SSO correction of the sickle defect by modification of the beta- and/or gamma-globin genes.

Huntington disease (HD) is a devastating neurologic disorder that is characterized by abnormal expansion of a CAG nt repeat in the first exon of the huntingtin (htt) gene, producing a mutant protein with an elongated polyglutamine stretch. The presence of this mutant protein is correlated with the characteristic loss of striatal neurons and the clinical manifestation of HD. Currently there is no effective treatment for the associated cell death. The aim of this study was to evaluate an innovative strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon system to down-regulate Htt expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human htt gene. Real-time RT-PCR and Western blot analysis were used to quantify Htt mRNA and protein levels, respectively, in human cell lines. The results indicated that selected siRNA constructs significantly decreased Htt mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Htt by approximately 90%. The combination of siRNA, the SB transposon, and an accurate transgenic mouse model may permit evaluation of this approach in preventing the pathogenesis associated with expression of mutant Htt.

RNA interference is an evolutionarily conserved mechanism of post-transcriptional gene silencing. Small interfering RNAs (siRNA) of 21-23 nucleotides generated from processing double-stranded RNA (dsRNA) by ribonuclease III, Dicer, are widely used for selective sequence-specific gene silencing in a broad range of organisms. In plants, siRNA is associated with de novo RNA-directed DNA methylation (RdDM) at the homologous target genomic region. To examine RdDM in somatic cells, human glioblastoma cell lines were treated with siRNAs homologous to the human huntingtin gene responsible for Huntington's disease. Methylation of CpG dinucleotides in the plasmid vectors expressing the dsRNAs and homologous genomic region was investigated by bisulfite-mediated genomic sequencing. Target regions of the siRNA in the huntingtin gene showed no significant change in the pattern of DNA methylation, and no CpG methylation was observed on the plasmid vectors. These results indicate that siRNA is not directly linked to DNA methylation at the target huntingtin genomic locus in human cells.

We have recently shown that both ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) prevent transforming growth factor beta1 (TGF-beta1)-induced hepatocyte apoptosis by modulating the E2F-1/p53/Bax pathway. In addition, activation of glucocorticoid (GR) and mineralocorticoid receptors (MR) inhibits apoptosis in various systems. UDCA induces a ligand-independent activation of the GR, thus potentially regulating a number of targets. In this study, we investigated the role of GR and MR during TGF-beta1-induced hepatocyte apoptosis, and identified additional antiapoptotic targets for UDCA. Our results showed that in primary hepatocytes, TGF-beta1 induced 40-50% decreases in gr and mr mRNA expression (p < 0.01), together with up to 10-fold reductions in their protein levels (p < 0.01). Notably, pretreatment with UDCA resulted in a significant upregulation of nuclear steroid receptors (p < 0.05), which coincided with 2- and 3-fold increases in the level of GR and MR nuclear translocation, respectively, when compared with that of TGF-beta1 alone (p < 0.05). Similarly, TUDCA induced GR and MR nuclear translocations (p < 0.05) and markedly prevented MR protein changes associated with TGF-beta1 (p < 0.05) without affecting GR protein levels. Moreover, when interference RNA was used to inhibit GR and MR, UDCA no longer protected hepatocytes against TGF-beta1-induced apoptosis. In fact, the protective effect of UDCA in TGF-beta1-associated caspase activation decreased from 65 to

Publisher Summary This chapter discusses the role of bile acids in the modulation of apoptosis. Bile acids are inherently toxic molecules, which can have a profound influence on cellular function and structure. The proposed mechanisms of bile acid-induced hepatocyte damage range from a simple detergent effect on binding to plasma membranes to the induction of cell death. Bile acids induce apoptosis via ligand-independent, death receptor pathways, involving the Fas receptor and also through classic mitochondrial pathways. In contrast, the oral administration of ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) has been used clinically for years to treat cholestasis and a number of other liver diseases. There is now strong evidence that the cytoprotective mechanism of UDCA and its conjugates results from their ability to inhibit apoptosis in hepatic cells. Interestingly, the anti-apoptotic properties of these bile acids are independent of the cell type and death signal, suggesting a common anti-apoptotic mechanism(s). Specifically, UDCA prevents apoptosis by modulating mitochondrial membrane perturbation, opening of the permeability transition pore, Bax translocation, cytochrome c release, and subsequent caspase activation and substrate cleavage. Apoptosis can not only be inhibited by blocking pro-apoptotic pathways but also by activating survival signals for example through the cAMP, Akt, NF-κB, MAPK and PI3K-mediated pathways.

Transforming growth factor beta1 (TGF-beta1)-induced hepatocyte apoptosis is associated with activation of E2F transcription factors and p53 stabilization through Mdm-2, thus potentially modulating a number of target genes. In previous studies, we have shown that ursodeoxycholic acid (UDCA) prevents TGF-beta1-induced hepatocyte apoptosis by inhibiting the mitochondrial pathway of cell death. In this study we examined the role of p53 in the induction of apoptosis by TGF-beta1, and identified additional antiapoptosis targets for UDCA. Our data show a significant transcriptional activation of E2F-1 in primary rat hepatocytes incubated with TGF-beta1, as well as a 5-fold increase in p53 and a 2-fold decrease in its inhibitor, Mdm-2 (p0.05). In addition, bax mRNA expression was significantly induced at 36 h (p0.01), resulting in increased levels of Bax protein. In contrast, Bcl-2 transcript and protein levels were decreased at all time points (p0.01). Notably, UDCA inhibited E2F-1 transcriptional activation, p53 stabilization and Bcl-2 family expression (p0.05), in part, through a caspase-independent mechanism. Moreover, in the absence of TGF-beta1, UDCA prevented induction of p53 and Bax by overexpression of E2F-1 and p53, respectively (p0.05). In addition, UDCA inhibited TGF-beta1-induced degradation of nuclear factor kappaB (NF-kappaB) and its inhibitor IkappaB (p0.05). In conclusion, these results demonstrate that UDCA inhibits E2F-1 transcriptional activation of hepatocyte apoptosis, thus modulating p53 stabilization, NF-kappaB degradation, and expression of Bcl-2 family members.