Rebecca C. Fry

Rat liver sinusoidal endothelial cells survive without exogenous VEGF in 3D perfused co-cultures with hepatocytes

Liver sinusoidal endothelial cells (SECs) are generally refractory to extended in vitro culture. In an attempt to recreate some features of the complex set of cues arising from the liver parenchyma, we cocul‐tured adult rat liver SECs, identified by the expression of the marker SE‐1, with primary adult rat hepatocytes in a 3D culture system that provides controlled mi‐croscale perfusion through the tissue mass. The culture was established in a medium containing serum and VEGF, and these factors were then removed to assess whether cells with the SE‐1 phenotype could be supported by the local microenvironment in vitro. Rats expressing enhanced green fluorescent protein (EGFP) in all liver cells were used for isolation of the SE‐1‐positive cells added to cocultures. By the 13th day of culture, EGFP‐expressing cells had largely disappeared from 2D control cultures but exhibited moderate proliferation in 3D perfused cultures. SE‐1‐positive cells were present in 3D cocultures after 13 days, and these cultures also contained Kupffer cells, stellate cells, and CD31‐expressing endothelial cells. Global transcrip‐tional profiling did not reveal profound changes between 2D and 3D cultures in expression of most canonical angiogenic factors but suggested changes in several pathways related to endothelial cell function.—Hwa, A. J., Fry, R. C., Sivaraman, A., So, P. T., Samson, L. D., Stolz, D. B., Griffith, L. G. Rat liver sinusoidal endothelial cells survive without exogenous VEGF in 3D perfused co‐cultures with hepatocytes. FASEB J. 21, 2564–2579 (2007)

Hepatocellular Carcinoma Associated with Liver-Gender Disruption in Male Mice

Hepatocellular carcinoma (HCC) is a male-predominant cancer associated with chronic hepatitis. Like human viral hepatitis, murine Helicobacter hepaticus infection produces inflammation and HCC with a masculine bias. We used this model to identify potential mechanisms of male HCC predisposition. Male weanling A/JCr mice (n = 67) were gavaged with H. hepaticus or vehicle. At 1 year, mice were distributed into four groups: surgical castration, chemical castration, castration followed by dihydrotestosterone supplementation, or sexually intact controls. Responses to infection were compared with IFN-gamma challenge alone. At 21 months, there was no significant difference in hepatitis between groups. Neither castration nor androgen receptor agonism altered tumor incidence. Infected mice with severe, but not mild, disease exhibited a mosaic of alterations to sexually dimorphic genes and microsomal long-chain fatty acids. By microarray, tumorigenic hepatitis was strongly associated with liver-gender disruption, defined as the loss of a gender-identifying hepatic molecular signature. IFN-gamma alone produced similar changes, demonstrating a role for proinflammatory cytokines in this process. In conclusion, hepatocarcinogenesis in male mice with chronic hepatitis is maturationally imprinted and androgen-independent. Proinflammatory cytokines may promote HCC in a male-predominant fashion due to high sensitivity of the masculinized liver to loss of sex-specific transcriptional balance. Liver-gender disruption has pleiotropic implications for hepatic enzyme activity, lipid processing, nuclear receptor activation, apoptosis, and proliferation. We propose a multistep model linking chronic hepatitis to liver cancer through cytokine-mediated derangement of gender-specific cellular metabolism. This model introduces a novel mechanism of inflammation-associated carcinogenesis consistent with male-predominant HCC risk.

Arsenic-Associated Changes to the Epigenome: What Are the Functional Consequences?

Inorganic arsenic (iAs) poses a major threat to worldwide human health, and yet the molecular mechanisms underlying the toxic effects associated with iAs exposure are not well understood. There is increasing experimental evidence indicating that epigenetic modifications may play a major role in the development of diseases associated with exposure to environmental toxicants. Research in the field has firmly established that iAs exposure is associated with epigenetic alterations including changes in DNA methylation, miRNA abundance, and post-translational histone modifications. Here, we summarize recent studies that have expanded the current knowledge of these relationships. These studies have pinpointed specific regions of the genome and genes that are targets of arsenical-induced epigenetic changes, including those associated with in utero iAs exposure. The recent literature indicates that iAs biotransformation likely plays an important role in the relationship between iAs exposure and the epigenome, in addition to the sex and genetic background of individuals. The research also shows that relatively low to moderate exposure to iAs is associated with epigenetic effects. However, while it is well established that arsenicals can alter components of the epigenome, in many cases, the biological significance of these alterations remains unknown. The manner by which these and future studies may help inform the role of epigenetic modifications in the development of iAs-associated disease is evaluated and the need for functional validation emphasized.

The epigenetic effects of a high prenatal folate intake in male mouse fetuses exposed in utero to arsenic.

Inorganic arsenic (iAs) is a complete transplacental carcinogen in mice. Previous studies have demonstrated that in utero exposure to iAs promotes cancer in adult mouse offspring, possibly acting through epigenetic mechanisms. Humans and rodents enzymatically convert iAs to its methylated metabolites. This reaction requires S-adenosylmethionine (SAM) as methyl group donor. SAM is also required for DNA methylation. Supplementation with folate, a major dietary source of methyl groups for SAM synthesis, has been shown to modify iAs metabolism and the adverse effects of iAs exposure. However, effects of gestational folate supplementation on iAs metabolism and fetal DNA methylation have never been thoroughly examined. In the present study, pregnant CD1 mice were fed control (i.e. normal folate, or 2.2 mg/kg) or high folate diet (11 mg/kg) from gestational day (GD) 5 to 18 and drank water with 0 or 85 ppm of As (as arsenite) from GD8 to 18. The exposure to iAs significantly decreased body weight of GD18 fetuses and increased both SAM and S-adenosylhomocysteine (SAH) concentrations in fetal livers. High folate intake lowered the burden of total arsenic in maternal livers but did not prevent the effects of iAs exposure on fetal weight or hepatic SAM and SAH concentrations. In fact, combined folate-iAs exposure caused further significant body weight reduction. Notably, iAs exposure alone had little effect on DNA methylation in fetal livers. In contrast, the combined folate-iAs exposure changed the CpG island methylation in 2,931 genes, including genes known to be imprinted. Most of these genes were associated with neurodevelopment, cancer, cell cycle, and signaling networks. The canonical Wnt-signaling pathway, which regulates fetal development, was among the most affected biological pathways. Taken together, our results suggest that a combined in utero exposure to iAs and a high folate intake may adversely influence DNA methylation profiles and weight of fetuses, compromising fetal development and possibly increasing the risk for early-onset of disease in offspring.

Hepatic temporal gene expression profiling in Helicobacter hepaticus-infected A/JCr mice

Helicobacter hepaticus infection of A/JCr mice is a model of infectious liver cancer. We monitored hepatic global gene expression profiles in H. hepaticus infected and control male A/JCr mice at 3 months, 6 months, and 1 year of age using an Affymetrix-based oligonucleotide microarray platform on the premise that a specific genetic expression signature at isolated time points would be indicative of disease status. Model based expression index comparisons generated by dChip yielded consistent profiles of differential gene expression for H. hepaticus infected male mice with progressive liver disease versus uninfected control mice within each age group. Linear discriminant analysis and principal component analysis allowed segregation of mice based on combined age and lesion status, or age alone. Up-regulation of putative tumor markers correlated with advancing hepatocellular dysplasia. Transcriptionally down-regulated genes in mice with liver lesions included those related to peroxisome proliferator, fatty acid, and steroid metabolism pathways. In conclusion, transcriptional profiling of hepatic genes documented gene expression signatures in the livers of H. hepaticus infected male A/JCr mice with chronic progressive hepatitis and preneoplastic liver lesions, complemented the histopathological diagnosis, and suggested molecular targets for the monitoring and intervention of disease progression prior to the onset of hepatocellular neoplasia.

Haem-regulated eIF2α kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency

Haem‐regulated eIF2α kinase (HRI) is essential for the regulation of globin gene translation and the survival of erythroid precursors in iron/haem deficiency. This study found that that in iron deficiency, fetal definitive erythropoiesis is inhibited at the basophilic erythroblast stage with increased proliferation and elevated apoptosis. This hallmark of ineffective erythropoiesis is more severe in HRI deficiency. Microarray gene profiling analysis showed that HRI was required for adaptive gene expression in erythroid precursors during chronic iron deficiency. The number of genes with expression affected more than twofold increased, from 213 in iron deficiency and 73 in HRI deficiency, to 3135 in combined iron and HRI deficiencies. Many of these genes are regulated by Gata1 and Fog1. We demonstrate for the first time that Gata1 expression in developing erythroid precursors is decreased in iron deficiency, and is decreased further in combined iron and HRI deficiencies. Additionally, Fog1 expression is decreased in combined deficiencies, but not in iron or HRI deficiency alone. Our results indicate that HRI confers adaptive gene expression in developing erythroblasts during iron deficiency through maintaining Gata1/Fog1 expression.

The Cycad Genotoxin MAM Modulates Brain Cellular Pathways Involved in Neurodegenerative Disease and Cancer in a DNA Damage-Linked Manner

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O 6-methyldeoxyguanosine lesions, O 6-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O 6-mG DNA methyltransferase (MGMT) showed elevated O 6-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimers disease.

Genetic Susceptibility to Chronic Hepatitis Is Inherited Codominantly in Helicobacter hepaticus-Infected AB6F1 and B6AF1 Hybrid Male Mice, and Progression to Hepatocellular Carcinoma Is Linked to Hepatic Expression of Lipogenic Genes and Immune Function-Associated Networks

ABSTRACT Helicobacter hepaticus causes hepatitis in susceptible strains of mice. Previous studies indicated that A/JCr mice are susceptible and C57BL/6NCr mice are resistant to H. hepaticus-induced hepatitis. We used F1 hybrid mice derived from A/J and C57BL/6 matings to investigate their phenotype and determine their hepatic gene expression profile in response to H. hepaticus infection. F1 hybrid mice, as well as parental A/J and C57BL/6 mice, were divided equally into control and H. hepaticus-infected groups and euthanized at 18 months postinoculation. Hepatic lesions were evaluated histologically and the differential hepatic gene expression in F1 mice was determined by microarray-based global gene expression profiling analysis. H. hepaticus-infected parental strains including A/J and C57BL/6 mice, as well as F1 mice, developed significant hepatitis. Overall, hepatocellular carcinomas or dysplastic liver lesions were observed in 69% of H. hepaticus-infected F1 male mice and H. hepaticus was isolated from hepatic tissues of all F1 mice with liver tumors. Liver tumors, characterized by hepatic steatosis, developed in livers with high hepatitis scores. To identify gene expression specific to H. hepaticus-induced hepatitis and progression to hepatocellular carcinoma in F1 mice, a method using comparative group transcriptome analysis was utilized. The canonical pathway most significantly enriched was immunological disease. Fatty acid synthase and steaoryl-coenzyme A desaturase, the two rate-limiting enzymes in lipogenesis, were upregulated in neoplastic relative to dysplastic livers. This study suggests a synergistic interaction between hepatic steatosis and infectious hepatitis leading to hepatocellular carcinoma. The use of AB6F1 and B6AF1 mice, as well as genetically engineered mice, on a C57BL/6 background will allow studies investigating the role of chronic microbial hepatitis and steatohepatitis in the pathogenesis of liver cancer.

RNA steady‐state defects in myotonic dystrophy are linked to nuclear exclusion of SHARP

We describe a new mechanism by which CTG tract expansion affects myotonic dystrophy (DM1). Changes to the levels of a panel of RNAs involved in muscle development and function that are downregulated in DM1 are due to aberrant localization of the transcription factor SHARP (SMART/HDAC1‐associated repressor protein). Mislocalization of SHARP in DM1 is consistent with increased CRM1‐mediated export of SHARP to the cytoplasm. A direct link between CTG repeat expression and SHARP mislocalization is demonstrated as expression of expanded CTG repeats in normal cells recapitulates cytoplasmic SHARP localization. These results demonstrate a role for the inactivation of SHARP transcription in DM1 biology.

DNA damage and stress transcripts in Saccharomyces cerevisiae Mutant sgs1

The human aging diseases Werner and Bloom syndromes are a result of mutation of the WRN and BLM genes, respectively. The SGS1 gene of Saccharomyces cerevisiae is homologous to the human WRN and BLM genes of the RecQ DNA helicase family. Deletion of SGS1 results in accelerated yeast aging and a reduction in life span as well as cell cycle arrest. We demonstrate that SGS1 deletion, DNA damage, and stress show similar transcriptional responses in yeast. Our comparative analysis of the genome-wide expression response of SGS1 deletion, stress and DNA damage indicates parallel transcriptional responses to cellular insult and aging in yeast.