Diversity of hepatocellular carcinoma clones bearing hematopoietic malignancies-related chromosomal translocation.

Interpatient heterogeneity of hepatocellular carcinoma has been in-depth addressed. Intrapatient heterogeneity is less known. Four clones were freshly isolated from an Edmondson grade I HCV-associated hepatocellular carcinoma. Biochemical approaches, functional assays and cytogenetics were used. Albumin inducibility was uncoupled from canonical cytokeratin profiles, suggesting pathological combinations of hepatospecific and biliary markers. Poor differentiation and TGFß's proproliferative effect on all clones were observed. TGFß, Interferon α and doxorubicin sensitivity levels were found highly heterogeneous. Progenitor and stem cells markers OV6 and EpCAM were mutually exclusively expressed. All clones were CD44+, while none expressed CD90, CD133, or CD117. Three clones displayed a liver progenitor OV6+ phenotype, and were susceptible to hepatocytic differentiation, among which one fibroblastoid clone displayed intrahepatic parenchymal engraftment capability. A fourth clone, the less motile, displayed a cancer stem cell EpCAM+ phenotype, was essentially ß-catenin negative, and was as expected devoid of hepatocytic differentiation capability, yet the most sensitive to doxorubicin treatment. Cytogenetics evidenced in all clones a t(12;22)(p11;q11) translocation found in several myelodysplastic syndromes. All clones, that probably derive from EpCAM+ tumor cells, display aberrant E-cadherin cytosolic localization. Because of their diverse pathophysiolocal features, these freshly isolated, low population doubling-defined, HCC clones may provide novel opportunities to tackle HCC heterogeneity in a single patient background for therapy improvement purposes, especially regarding recently developed targeted strategies.

An immortalized human liver endothelial sinusoidal cell line for the study of the pathobiology of the liver endothelium.

BACKGROUND: The endothelium lines blood and lymph vessels and protects underlying tissues against external agents such as viruses, bacteria and parasites. Yet, microbes and particularly viruses have developed sophisticated ways to bypass the endothelium in order to gain access to inner organs. De novo infection of the liver parenchyma by many viruses and notably hepatitis viruses, is thought to occur through recruitment of virions on the sinusoidal endothelial surface and subsequent transfer to the epithelium. Furthermore, the liver endothelium undergoes profound changes with age and in inflammation or infection. However, primary human liver sinusoidal endothelial cells (LSECs) are difficult to obtain due to scarcity of liver resections. Relevant derived cell lines are needed in order to analyze in a standardized fashion the transfer of pathogens across the liver endothelium. By lentiviral transduction with hTERT only, we have immortalized human LSECs isolated from a hereditary hemorrhagic telangiectasia (HHT) patient and established the non-transformed cell line TRP3. TRP3 express mesenchymal, endothelial and liver sinusoidal markers. Functional assessment of TRP3 cells demonstrated a high capacity of endocytosis, tube formation and reactivity to immune stimulation. However, TRP3 displayed few fenestrae and expressed C-type lectins intracellularly. All these findings were confirmed in the original primary LSECs from which TRP3 were derived suggesting that these features were already present in the liver donor. We consider TRP3 as a model to investigate the functionality of the liver endothelium in hepatic inflammation in infection.

Plastics and carcinogenesis: The example of vinyl chloride.

The manufacture, use and disposal of various plastics can pose numerous health risks, including the risk of cancer. A model example of carcinogenic risk from plastics is provided by polyvinyl chloride, since it is composed of the known human carcinogen vinyl chloride (VC). In recent years, much has been learned about the molecular biological pathways of VC carcinogenesis. This has led to molecular epidemiologic studies of VC carcinogenesis in exposed human populations which have identified useful biomarkers of exposure, effect and susceptibility for VC. These studies have in turn provided the basis for new molecular approaches for the prevention and treatment of VC cancers. This model could have much wider applicability for many other carcinogenic exposures and many other human cancers.

Effect of the XRCC1 codon 399 polymorphism on the repair of vinyl chloride metabolite-induced DNA damage.

BACKGROUND: Recent epidemiologic evidence suggests that the common polymorphism at amino acid residue 399 of the x-ray cross complementing-1 (XRCC1) protein, a key component of the base excision repair (BER) pathway for DNA damage, plays a significant role in the genetic variability of individuals in terms of the mutagenic damage they experience following exposure to the carcinogen vinyl chloride (VC). The aim of this study was to provide support for the biological plausibility of these epidemiologic observations with experimental data derived from cell lines in culture from individuals who were either homozygous wild-type or homozygous variant for this XRCC1 polymorphism following exposure to chloroethylene oxide (CEO), the active metabolite of VC, with measurement of the induced etheno-DNA adducts before and after repair. MATERIALS AND METHODS: Immortalized lymphoblast cell lines from seven VC workers (four homozygous wild-type and three homozygous variant for the 399 XRCC1 polymorphism) were exposed to CEO, and etheno-adenosine (epsilonA) adduct levels were determined by enzyme-linked immunosorbent assay (ELISA) pre-exposure and at 0, 4, 8 and 24 h following exposure. RESULTS: The average epsilonA adduct levels were statistically significantly higher in the variant cells compared to the wild-type cells at 8 and 24 h following exposure (P Conclusion: These results are consistent with the epidemiologic findings of the types of VC-induced biomarkers observed in exposed individuals and the mutational spectra found in the resultant tumors as well as the key role that BER, especially XRCC1, plays in this carcinogenic pathway.

Gene-environment interactions between DNA repair polymorphisms and exposure to the carcinogen vinyl chloride.

We have recently suggested that polymorphisms in metabolism and repair pathways may play a role in modulating the effects of exposure to the carcinogen vinyl chloride in the production of biomarkers of its mutagenic damage. The aim of the present study was to extend these observations by examining gene-environment interactions between several common polymorphisms in the DNA repair genes XRCC1 and ERCC2/XPD and vinyl chloride exposure on the production of vinyl chloride-induced biomarkers of mutation. A cohort of 546 French vinyl chloride workers were genotyped for the XRCC1 codon 194 (Arg>Trp; rs1799782), 280 (Arg>His; rs25489) and 399 (Arg>Gln; rs25487) polymorphisms and the ERCC2/XPD codon 312 (Asp>Asn; rs1799793) and 751 (Lys>Gln; rs13181) polymorphisms. The results demonstrated a statistically significant allele dosage effect of the XRCC1 399 variant on the production of the vinyl chloride-induced mutant p53 biomarker, even after controlling for confounders including cumulative vinyl chloride exposure (p = 0.03), with a potentially supramultiplicative gene-environment interaction. In addition, the results demonstrate statistically significant allele dosage effects of the ERCC2/XPD 312 and 751 variants on the production of the vinyl chloride-induced mutant ras-p21 biomarker, even after controlling for confounders including cumulative vinyl chloride exposure (p < 0.0001 and p = 0.0006, respectively), with a potentially supramultiplicative gene-environment interaction for the codon 751 allele. Finally, the results suggest potential supramultiplicative gene-gene interactions between CYP2E1 (c2 allele; rs3813867) and ERCC2/XPD polymorphisms that are consistent with the proposed carcinogenic pathway for vinyl chloride, which requires metabolic activation by CYP2E1 to reactive intermediates that form DNA adducts that, if not removed by DNA repair mechanisms, result in oncogenic mutations.

Distribution of ENG and ACVRL1 (ALK1) mutations in French HHT patients.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease characterized by arteriovenous malformations and resulting from mutations in two major genes: ENG and ACVRL1. The aim of the present study was to estimate the prevalence of the mutations of ENG and ACVRL1 in HHT, based on the largest series of patients reported so far, recruited through a national network. We previously reported the first mutation screening of both genes, in French HHT patients, using heteroduplex analysis. This previous study, bringing 60 novel mutations, provided a significant improvement to the knowledge of molecular pathology in HHT. However, 32% (n=48) of the patients with a confirmed clinical diagnosis remained without mutation. In these patients, we performed an extensive molecular analysis that included the sequencing of the whole coding sequence, the search for large rearrangements, and screening of the potential 5' regulatory regions. Additionally, due to the lack of large pedigrees suitable for linkage analysis, and since SMAD4 germline mutations have been reported in families with combined HHT and juvenile polyposis, we screened this gene and five other genes involved in the TGF-beta/BMP pathway in the patients without mutation of ENG or ACVRL1. Only a novel SMAD1 non-conservative substitution was found in one patient, changing a poorly conserved methionine to an isoleucin. Twenty-three mutations were found in ACVRL1 and 8 in ENG (including a duplication of exons 4 to 8 and deletions of exons 1 to 3 and 9 to 14). Our results, combined with our previous data, increase the mutation rate to 88% (n=119/136) in French patients with a confirmed clinical diagnosis. Our results also emphasize the higher prevalence of large insertions/deletions in ENG and the predominance of ACVRL1 over ENG mutations.

The role of XRCC1 polymorphisms in base excision repair of etheno-DNA adducts in French vinyl chloride workers.

OBJECTIVES: The purpose of this study was to examine whether polymorphisms in the XRCC1 DNA-repair protein can affect the base excision repair capacity to remove etheno-DNA adducts induced by vinyl chloride exposure that account for the occurrence of mutant biomarkers of effect seen in exposed workers. MATERIALS AND METHODS: Using polymerase chain reaction-restriction fragment length polymorphism and fluorescence polarization techniques, we examined the effect of three x-ray cross complementing-1 protein polymorphisms, at codons 194, 280 and 399, on the occurrence of mutant biomarkers in ras-p21 and p53 induced by vinyl chloride exposure in a cohort of 211 French vinyl chloride workers to correlate differences in genotype with differences in the presence of these biomarkers. Also, cell cultures of lymphoblast lines from a pair of individuals, one homozygous wild-type and one homozygous variant for the codon 399 polymorphism, were exposed to the reactive intermediate of vinyl chloride, and, using an enzyme-linked immunosorbent assay, levels of etheno-DNA adducts generated and repaired were measured and compared. RESULTS: After adjusting for age, smoking, alcohol drinking and cumulative vinyl chloride exposure, compared to workers who were homozygous wild-type for all alleles, the odds ratio for the presence of either biomarker increased to 2.0 (95% CI: 1.0-3.9) for workers with any one variant allele and to 2.4 (95% CI: 1.1-5.2) for workers with more than one variant allele. Data from the cell culture experiments indicating that repair of etheno-DNA adducts is considerably better in wild-type cells compared to polymorphic cells were supportive of the epidemiologic results. CONCLUSIONS: This study provides further evidence that polymorphisms in XRCC1 can be an important biomarker of susceptibility in populations exposed to agents that produce damage removed by base excision repair.

Polymorphisms of microsomal epoxide hydrolase in French vinyl chloride workers.

OBJECTIVES: The purpose of this study was to determine if polymorphisms in microsomal epoxide hydrolase, an enzyme involved in the metabolism of reactive intermediates of vinyl chloride (VC), contribute to the variable susceptibility to the mutagenic effects of vinyl chloride among exposed workers. MATERIALS AND METHODS: Polymorphisms at codons 113 and 139 were determined in DNA samples from 211 French vinyl chloride workers. Genotypes were stratified into low, medium and high activity groups and odds ratios and 95% confidence intervals were determined for the presence of one or both of two VC-induced mutant biomarkers (mutant ras-p21 and mutant p53) by logistic regression adjusting for age, smoking, drinking and cumulative VC exposure. RESULTS: Compared to the low-activity microsomal epoxide hydrolase genotype stratum, the odds ratio for the presence of the VC-induced mutant biomarkers increased to 1.16 (95% CI: 0.64-2.10) in the medium-activity genotype stratum and to 1.35 (95% CI: 0.66-2.77) in the high-activity genotype stratum. The test for trend was not statistically significant and was in the opposite direction from that expected based on increasing removal of reactive intermediates with increasing activity. CONCLUSIONS: The results suggest that polymorphisms in microsomal epoxide hydrolase do not play a significant role in susceptibility to the mutagenic effects of vinyl chloride.

The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies.

Liver progenitor cells may play an important role in carcinogenesis in vivo and represent therefore useful cellular materials for in vitro studies. The HepaRG cell line, which is a human bipotent progenitor cell line capable to differentiate toward two different cell phenotypes (i.e., biliary-like and hepatocyte-like cells), has been established from a liver tumor associated with chronic hepatitis C. This cell line represents a valuable alternative to ex vivo cultivated primary human hepatocytes (PHH), as HepaRG cells share some features and properties with adult hepatocytes. The cell line is particularly useful to evaluate drugs and perform drug metabolism studies, as many detoxifying enzymes are expressed and functional. It is also an interesting tool to study some aspect of progenitor biology (e.g., differentiation process), carcinogenesis, and the infection by some pathogens for which the cell line is permissive (e.g., HBV infection). Overall, this chapter gives a concise overview of the biological properties and potential applications of this cell line.

The effect of genetic polymorphisms in the vinyl chloride metabolic pathway on mutagenic risk.

Vinyl chloride (VC) is a human carcinogen known to undergo metabolism by cytochrome P450 2E1 (CYP2E1) to reactive intermediates that can cause oncogene and tumor suppressor gene mutations and that are further metabolized by acetaldehyde dehydrogenase (ALDH2) and glutathione-S-transferases (GSTs) to non-mutagenic end products. These metabolic enzymes have known polymorphisms that could lead to increased levels of the VC reactive intermediates and thus an increased risk for mutations and cancer following exposure. Using restriction fragment length polymorphism (RFLP) analysis, we have examined a cohort of 597 French VC workers for polymorphisms in CYP2E1, ALDH2, GSTM1 and GSTT1 in relation to the occurrence of mutant oncogene and tumor suppressor gene biomarkers that are attributable to VC exposure. The presence of the biomarkers for mutant ras-p21 and mutant p53 was found to be highly significantly associated with cumulative VC exposure (P for trend <0.0001). The presence of the CYP2E1 variant c2 allele was found to be significantly associated with the presence of either or both mutant biomarkers even after controlling for potential confounders including cumulative VC exposure (OR = 2.3, 95% CI = 1.2-4.1), and the effects of the c2 allele and VC exposure were approximately additive. GSTT1 null status was found to have an increased, but not significant association with the presence of either or both biomarkers after controlling for confounders (OR = 1.3, 95% CI = 0.8-2.0). These results suggest the existence of a possible gene-environment interaction between polymorphisms in the VC metabolic pathway and VC exposure that could contribute to the variable susceptibility to the mutagenic effects of VC in exposed populations.

Glutathione S-transferase M1 and GST T1 genetic polymorphisms and Raynaud's phenomenon in French vinyl chloride monomer-exposed workers.

Occupational vinyl chloride monomer (VCM) exposure can induce Raynaud's phenomenon (RP). However, not all VCM workers developed RP, which suggests an underlying genetic susceptibility. Genetic polymorphisms of glutathione S-transferases (GSTs), involved in VCM metabolism, have been shown to influence certain VCM-related health effects. We have conducted a case-control study of 58 subjects with RP along with 247 subjects without RP, from a population of 305 French workers exposed or formerly exposed to VCM, to assess any association between GST M1 and GST T1 gene polymorphisms, either separately or in combination, and the presence of RP. None of the GST M1 or GST T1 genotypes were significantly associated with the presence of RP among studied VCM workers. A combination of positive genotypes for both GST M1 and GST T1 was significantly associated with RP presence, compared to the other combinations of genotypes (OR=2.1, 95% CI=1.1-3.8). OR adjusted for age, smoking status, alcohol consumption and history of treated hypertension did not reach significance (OR=2.0, 95% CI=0.9-5.2). None of the GST M1 and GST T1 genotypes seem to contribute separately to the presence of RP, suggesting that they are not, when taken alone, a major determinant of interindividual variability for VCM-induced PR. However, the combination of both positive GST M1 and GST T1 genotypes appears to contribute slightly to susceptibility to RP in VCM-exposed subjects. Nevertheless, our study-the first to examine the role of a genetic component in the occurrence of RP secondary to occupational exposure to a chemical-corroborates the previous considerations that interaction between the genetic constitution and environmental factors is of importance in determining the health-adverse effects of VCM exposure.

Liver involvement in hereditary hemorrhagic telangiectasia: consensus recommendations.

STUDY PURPOSE: To formulate recommendations about clinical management of liver involvement in hereditary hemorrhagic telangiectasia (HHT), using a formal consensus development process. CONSENSUS PROCESS: A nominal group technique was used. A list of main clinical, diagnostic and therapeutic issues about liver involvement in HHT was generated by the organizing committee. Panel members then scored their agreement with each statement; the median score, and standard deviation for each statement were determined for each of the three successive panel rounds. These consensus statements formed the basis for recommendations graded with the strength and quality of supporting evidence. RECOMMENDATION STATEMENTS: Doppler US is sufficiently accurate and suitable for first-line imaging of the liver in the general HHT population. Liver biopsy in any patient with proven or suspected HHT should be avoided. Liver involvement in HHT is generally asymptomatic; in the minority of patients where it is symptomatic, morbidity and mortality can be substantial. The prevalence of focal nodular hyperplasia is much higher in patients with liver involvement by HHT than in the general population. Invasive therapies for liver involvement by HHT (namely liver transplantation) should be considered only in patients who have failed to respond to intensive medical therapy.

A common polymorphism in XRCC1 as a biomarker of susceptibility for chemically induced genetic damage.

We have recently demonstrated a significant dose-response relationship between vinyl chloride exposure and mutant p53 biomarkers in humans. The aim of this study was to examine a common polymorphism in the DNA repair gene XRCC1 as a potential biomarker of susceptibility modifying this relationship, consistent with the known mechanism of production of p53 mutations via vinyl chloride-induced etheno-DNA adducts, which are repaired by XRCC1. A cohort of 211 French vinyl chloride workers were genotyped for the XRCC1 codon 399 polymorphism (CGG>CAG; Arg>Gln). Among the homozygous Arg-Arg individuals, 34% were biomarker positive compared with 47% in the heterozygous Arg-Gln individuals (adjusted odds ratio 1.73, 95% CI0.93-3.22) and 66% in the homozygous Gln-Gln individuals (adjusted odds ratio 3.95, 95% CI 1.68-9.28), with a significant trend for increasing Gln allele dosage (p=0.002). These preliminary results suggest that a common polymorphism in a DNA repair gene can be an important biomarker of susceptibility for chemically induced genetic damage.

Origin and characterization of a human bipotent liver progenitor cell line.

BACKGROUND & AIMS: Liver progenitor cells may be important in carcinogenesis resulting from human chronic liver diseases. The HepaRG cell line has been established from a liver tumor associated with chronic hepatitis C. We observed that these cells showed an evident morphological heterogeneity, displaying both hepatocyte-like and biliary-like epithelial phenotypes. Our goal was to determine whether they could share some features with liver progenitor cells. METHODS: Phenotypic studies using immunofluorescence, immunoblotting, and flow cytometry were performed at different culture stages. RESULTS: HepaRG cells progressively exhibited polarized and functional hepatocytes and bile duct-like cell features under defined conditions. Cytokeratin 18 and 19 coexpression was, however, observed all along the maturation process together with oval cell-specific markers (M2-PK, OV-1, OV-6, and CD34); kinetics and expression profiles were dependent on the cell population. In addition, a strong commitment toward the hepatocytic lineage could be observed in the presence of epidermal growth factor. Immunohistochemistry on the fibrotic liver showing the atypical ductular reaction from which HepaRG cells originated displayed a comparable immunophenotype specifically restricted to bile neoductules. CONCLUSIONS: HepaRG cells constitute the first described human hepatic bipotent progenitor cell line regarding phenotype and histological origin.