Roland Wolf

Efficient Differentiation of Hepatocytes from Human Embryonic Stem Cells Exhibiting Markers Recapitulating Liver Development In Vivo

The potential to differentiate human embryonic stem cells (hESCs) in vitro to provide an unlimited source of human hepatocytes for use in biomedical research, drug discovery, and the treatment of liver diseases holds great promise. Here we describe a three‐stage process for the efficient and reproducible differentiation of hESCs to hepatocytes by priming hESCs towards definitive endoderm with activin A and sodium butyrate prior to further differentiation to hepatocytes with dimethyl sulfoxide, followed by maturation with hepatocyte growth factor and oncostatin M. We have demonstrated that differentiation of hESCs in this process recapitulates liver development in vivo: following initial differentiation, hESCs transiently express characteristic markers of the primitive streak mesendoderm before turning to the markers of the definitive endoderm; with further differentiation, expression of hepatocyte progenitor cell markers and mature hepatocyte markers emerged sequentially. Furthermore, we have provided evidence that the hESC‐derived hepatocytes are able to carry out a range of hepatocyte functions: storage of glycogen, and generation and secretion of plasma proteins. More importantly, the hESC‐derived hepatocytes express several members of cytochrome P450 isozymes, and these P450 isozymes are capable of converting the substrates to metabolites and respond to the chemical stimulation. Our results have provided evidence that hESCs can be differentiated efficiently in vitro to functional hepatocytes, which may be useful as an in vitro system for toxicity screening in drug discovery.

GSTπ expression mediates dopaminergic neuron sensitivity in experimental parkinsonism

The cause of 95% of Parkinsons disease (PD) cases is unknown. It is hypothesized that PD arises from an interaction of free-radical-generating agents with an underlying genetic susceptibility to these compounds. Here we use the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism to examine the role of a dual function protein, GSTπ, in dopaminergic neuron death. GSTπ is the only GST family member expressed in substantia nigra neurons. GSTπ reduction by pharmacological blockade, RNA inhibition, and gene targeting increases sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, suggesting that differential expression of GSTπ contributes to the sensitivity to xenobiotics in the substantia nigra and may influence the pathogenesis of reactive oxygen species-induced neurological disorders including PD.

Identification of Dlk1-Dio3 imprinted gene cluster noncoding RNAs as novel candidate biomarkers for liver tumor promotion.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Polymorphisms in xenobiotic metabolizing enzymes and diet influence colorectal adenoma risk.

Objectives We have earlier shown that diet and xenobiotic metabolizing enzyme genotypes influence colorectal cancer risk, and now investigate whether similar associations are seen in patients with premalignant colorectal adenomas (CRA), recruited during the pilot phase of the Scottish Bowel Screening Programme. Methods Nineteen polymorphisms in 13 genes [cytochrome P450 (P450), glutathione S-transferase (GST), N-acetyl transferase, quinone reductase (NQ01) and microsomal epoxide hydrolase (EPHX1) genes] were genotyped using multiplex PCR or Taqman-based allelic discrimination assays and analyzed in conjunction with diet, assessed by food frequency questionnaire, in a case–control study [317 CRA cases (308 cases genotyped), 296 controls]. Findings significant at a nominal 5% level are reported. Results CRA risk was inversely associated with fruit (P=0.02, test for trend) and vegetable (P=0.001, test for trend) consumption. P450 CYP2C9*3 heterozygotes had reduced CRA risk compared with homozygotes for the reference allele [odds ratio (OR): 0.60; 95% confidence interval (CI): 0.36–0.99], whereas CYP2D6*4 homozygotes (OR: 2.72; 95% CI: 1.18–6.27) and GSTM1 ‘null’ individuals (OR: 1.43; 95% CI: 1.04–1.98) were at increased risk. The protective effect of fruit consumption was confined to GSTP1 (Ala114Val) reference allele homozygotes (OR: 0.49; 95% CI: 0.34–0.71, P=0.03 for interaction). CRA risk was not associated with meat consumption, although a significant interaction between red meat consumption and EPHX1 (His139Arg) genotype was noted (P=0.02 for interaction). Conclusion We report the novel associations between P450 genotype and CRA risk, and highlight the risk association with GSTM1 genotype, common to our CRA and cancer case–control series. In addition, we report a novel modifying influence of GSTP1 genotype on dietary chemoprevention. These novel findings require independent confirmation.

INTERACTION OF NITROGEN MONOXIDE WITH CYTOCHROME P-450 MONITORED BY SURFACE-ENHANCED RESONANCE RAMAN SCATTERING

The reaction of mammalian cytochrome P-450 2B4 with nitrogen monoxide and oxygen has been studied by surface-enhanced resonance Raman scattering (SERRS) to obtain sharp and definitive information in situ on the nature of the changes in the active site pocket. The initial reaction produces a six co-ordinate low spin haem-nitrogen monoxide adduct. A slower reaction leads to the irreversible formation of a five co-ordinate high-spin iron (III) haem with no nitrogen monoxide bound to it and to the nitration of an aromatic side chain, probably a tyrosine, in the proximity of the active site. In the presence of excess nitrogen monoxide, the second reaction is controlled by oxygen concentration. The sequence of events corresponds to the biphasic inhibition induced by NO in other cytochromes P-450 and peroxidases and is postulated to occur by the formation of a nitrating agent at the haem followed by diffusion to the tyrosine. The nitrated amino acid and the oxidation and spin state of the haem are observed easily by SERRS with low concentrations of protein making it a particularly suitable method for the investigation of reactions of NO in complex biological matrices.

Methylene Tetrahydrofolate Reductase Genotype Modifies the Chemopreventive Effect of Folate in Colorectal Adenoma, but not Colorectal Cancer

Epidemiologic evidence suggests a role for folate, a critical component of the 1-carbon cycle, in colorectal adenoma and cancer pathogenesis. Low folate levels, along with genetic polymorphisms in key enzymes such as methylene tetrahydrofolate reductase (MTHFR), can cause DNA hypomethylation and aberrant CpG methylation, which have been associated with colorectal tumor development. We investigated self-reported folate and alcohol intake alongside possible modifying effects of MTHFR 677 C>T and 1298 A>C polymorphisms in UK case-control studies of colorectal adenoma (317 cases, 296 controls) and cancer (500 cases, 742 controls). A significant association between MTHFR 1298 and colorectal cancer risk was observed [odds ratio, 1.57; 95% confidence interval (95% CI), 1.05-2.37], which was more pronounced in males (odds ratio, 3.02; 95% CI, 1.63-5.62). Although we found no association between MTHFR 677 and colorectal cancer, when data were stratified by sex, an increased risk was seen in females (odds ratio, 1.96; 95% CI, 1.11-3.46) but not in males. High folate intake was associated with a decreased risk for colorectal adenoma (odds ratio, 0.47; 95% CI, 0.30-0.73; Ptrend, <0.001), which was modified by MTHFR 1298 genotype (Pinteraction = 0.006). However, we found no evidence to support the hypothesis that a high-folate diet protects against colorectal cancer development. Consistent with previous studies, high alcohol intake (≥14 U/wk) was associated with a significantly increased cancer risk (odds ratio, 2.57; 95% CI, 1.81-3.64). Our data suggest that dietary folate intake may be an important determinant for premalignant colorectal disease development but not colorectal cancer, an association that is modified by MTHFR genotype. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2421–30)

Expression of xenobiotic-metabolizing enzymes by primary and secondary hepatic tumors in man

PURPOSE To determine the immunohistochemical expression of xenobiotic-metabolising enzymes (XME) in normal livers, primary hepatocellular carcinomas (hepatomas) and secondary hepatic tumors from colonic primary tumors. METHODS AND MATERIALS The expression of XME in primary (n = 16) and secondary (n = 21) hepatic tumors and patients with no malignancies (n = 20) were investigated using polyclonal antibodies raised against the following rat enzymes CYP1A1, CYP2B1, CYP2C6, CYP3A1, CYP4A1, cytochrome P-450 reductase, epoxide hydrolase and testosterone UDP-glucuronyl transferase. The rat cytochrome P-450 antibodies recognize various human isoenzymes within the same gene family. Immunohistochemistry was undertaken using the immunoperoxidase and alkaline phosphatase anti-alkaline phosphatase techniques. RESULTS There was a reduction in the overall expression of all XME by tumor tissue compared to adjacent nonneoplastic liver cells (p = 0.008), more in livers with secondary tumors (p < 0.0001) and reduced expression of XME by hepatomas and secondary liver tumors compared to livers with no malignancy. A tendency for higher expression of all XME by nonneoplastic liver cells from patients with hepatomas relative to nonmalignant livers was observed, with significantly higher expression of CYP3A4/5 and testosterone UDP-GT enzymes (odds ratio 3.12; CI 1.59-6.10). CONCLUSION The expression of XME by tumor tissue is reduced in primary and secondary hepatic malignancies. The expression of XME by nonneoplastic liver cells is higher in patients with hepatomas than patients with no hepatic malignancies. These alterations in XME activities may have important therapeutic implications in the response and toxicity to systemic anti-cancer therapy, due to altered pharmacokinetics. In addition, differential expression of these enzymes by normal and malignant cells may be important for the rational design of selective anti-tumor drugs.

Recurrent Coding Sequence Variation Explains only A Small Fraction of the Genetic Architecture of Colorectal Cancer

Whilst common genetic variation in many non-coding genomic regulatory regions are known to impart risk of colorectal cancer (CRC), much of the heritability of CRC remains unexplained. To examine the role of recurrent coding sequence variation in CRC aetiology, we genotyped 12,638 CRCs cases and 29,045 controls from six European populations. Single-variant analysis identified a coding variant (rs3184504) in SH2B3 (12q24) associated with CRC risk (OR = 1.08, P = 3.9 × 10−7), and novel damaging coding variants in 3 genes previously tagged by GWAS efforts; rs16888728 (8q24) in UTP23 (OR = 1.15, P = 1.4 × 10−7); rs6580742 and rs12303082 (12q13) in FAM186A (OR = 1.11, P = 1.2 × 10−7 and OR = 1.09, P = 7.4 × 10−8); rs1129406 (12q13) in ATF1 (OR = 1.11, P = 8.3 × 10−9), all reaching exome-wide significance levels. Gene based tests identified associations between CRC and PCDHGA genes (P < 2.90 × 10−6). We found an excess of rare, damaging variants in base-excision (P = 2.4 × 10−4) and DNA mismatch repair genes (P = 6.1 × 10−4) consistent with a recessive mode of inheritance. This study comprehensively explores the contribution of coding sequence variation to CRC risk, identifying associations with coding variation in 4 genes and PCDHG gene cluster and several candidate recessive alleles. However, these findings suggest that recurrent, low-frequency coding variants account for a minority of the unexplained heritability of CRC.

Unraveling functional networks: does gene clustering have a meaning?

Background Prediction of the role of one protein in a network of regulatory and metabolic interactions involves analysis of many complex data sets in their interference, as no experimental source along is completely and generally reliable. To discriminate the most promising pathways related to a protein of interest from background noise we integrate multiple bioinformatics data by means of a graphical designer/database of protein-protein interactions supported by data mining and logical modeling of resulted networks.

Dr. Thomas Friedberg (January 24, 1951–April 22, 2009)

It is with great sadness that we report here on the sudden and tragic death of Dr. Thomas Friedberg. He was a member of the editorial board of this journal and we are grateful that the editors invited us to contribute an obituary. Thomas was born on January 24, 1951, in Mainz, Germany. In 1978, he attained his degree (‘‘diploma’’) in chemistry, receiving the highest possible distinction, from the University of Mainz. His work from this period included pioneering studies on pesticides in tumor susceptible versus resistant mice strains. For one of the publications resulting from his work of this period he received the American Society of Pharmacology and Experimental Therapeutics ‘‘Best Paper Award’’, undoubtedly a great honor for a non-American working outside the United States. In 1981, he completed his Ph.D. on characterizing the biochemical and toxicological properties of a key detoxication system of mammalian organisms including humans, the glutathione S-transferases under the supervision of one of us (Franz Oesch). During his Ph.D. studies, Thomas also performed pioneering work on the detoxification of metabolites in polycyclic aromatic hydrocarbons together with Hansruedi Glatt. In 1982, he moved to New York as a postdoctoral fellow where he joined the laboratory of Professor Gert Kreibich at the New York University (NYU) Medical Center’s Department of Cell Biology. This sojourn in the United States was highly successful for a number of reasons. Thomas was the first person to isolate cytochrome P450 cDNAs from the CYP2C gene family, the clones at the time being called TF1 and TF2 for his initials. As part of his research in NYU, he also carried out extraordinary studies on the factors defining the homoand hetero-dimerization of glutathione S-transferase proteins. In 1984, Thomas returned to Franz Oesch’s Institute of Toxicology in Mainz. During that period Thomas made a number of significant discoveries: the detection of drug-metabolizing enzymes in several liver cell types other than hepatocytes; the development of heterologous systems for the expression of drugmetabolizing enzymes in mammalian cells and their subsequent use to establish the role of specific enzymes in the metabolic activation versus detoxication of carcinogens; the isolation of several hepatic proteins related to microsomal epoxide hydrolase (published in this journal); the identification of a single membrane anchor sequence of microsomal epoxide hydrolase and its implication for drug metabolism pathways; the repression of the crucial detoxication system, glutathione S-transferases by foreign compounds; the production of site-specific antibodies to distinguish the function of structurally very closely (more than 98% homology) related types of cytochrome P450; as well as the discovery of a novel cytochrome P450 which is expressed in the sebaceous glands but not in the liver. In 1993, Thomas took up a position as senior lecturer in the Biomedical Research Centre at the University of Dundee. One of his numerous successful activities was the development of a collaborative research program with the pharmaceutical industry on the heterologous expression of human cytochrome P450s in E. coli, where he developed reagents that are now widely used in the pharmaceutical R. Wolf (&) Biomedical Research Institute, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK e-mail: c.r.wolf@dundee.ac.uk