Chicken Hepatoma Cells Research Articles

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-α) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ERα antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Species-specific mechanisms for cholesterol 7α-hydroxylase (CYP7A1) regulation by drugs and bile acids

The gene encoding cholesterol 7α-hydroxylase (CYP7A1) is tightly regulated in order to control intrahepatic cholesterol and bile acid levels. Ligands of the xenobiotic-sensing pregnane X receptor inhibit CYP7A1 expression. To retrace the evolution of the molecular mechanisms underlying CYP7A1 inhibition, we used a chicken hepatoma cell system that retains the ability to be induced by phenobarbital and other drugs. Whereas bile acids regulate CYP7A1 via small heterodimer partner and liver receptor homolog-1, mRNA expression of these nuclear receptors is unchanged by xenobiotics. Instead, drugs repress chicken hepatic nuclear factor 4α (HNF4α) transcript levels concomitant with a reduction in CYP7A1 expression. Importantly, no reduction of HNF4α levels is found in mouse liver in vivo and in human primary hepatocyte cultures, respectively. Thus, besides the importance of HNF4α in CYP7A1 regulation in all species, birds and mammals use different signaling pathways to adjust CYP7A1 levels after exposure to xenobiotics.

Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration.

Integrated hepadnaviral DNA in livers and tumors of chronic hepatitis B patients has been reported for many years. In this study, we investigated whether hepatitis B virus DNA integration occurs preferentially at sites of cell DNA damage. A single I-SceI homing endonuclease recognition site was introduced into the DNA of the chicken hepatoma cell line LMH by stable DNA transfection, and double-strand breaks were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. Alteration of the target cleavage site by imprecise nonhomologous end joining occurred at a frequency of approximately 10(-3) per transfected cell. When replication of an avian hepadnavirus, duck hepatitis B virus, occurred at the time of double-strand break repair, we observed integration of viral DNA at the site of the break with a frequency of approximately 10(-4) per transfected cell. Integration depended on the production of viral double-stranded linear DNA and the expression of I-SceI, and integrated DNA was stable through at least 17 cell divisions. Integration appeared to occur through nonhomologous end joining between the viral linear DNA ends and the I-SceI-induced break, because small deletions or insertions were observed at the sites of end joining. The results suggest that integration of hepadnaviral DNA in infected livers occurs at sites of DNA damage and may indicate the presence of more widespread genetic changes beyond that caused by viral DNA integration itself [corrected].

The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals

BackgroundDrugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals.ResultsTo explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR.ConclusionOur finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species.

Transcriptional Regulation of Heme Oxygenase-1 Gene Expression by MAP Kinases of the JNK and p38 Pathways in Primary Cultures of Rat Hepatocytes

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with -754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38alpha, beta, and delta decreased and p38gamma increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (-668/-654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (-47/-42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediates HO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box.

Regulation by estrogen of synthesis and secretion of apolipoprotein A-I in the chicken hepatoma cell line, LMH-2A

The synthesis and secretion of apolipoprotein A-I (apoA-I) in response to the treatment with estrogen were investigated in the chicken hepatoma cell line, LMH-2A. Exposure of these cells to exogenous estrogen for up to 48 h results in a decrease of apoA-I production, as evident from Western blotting, immunoprecipitation, and immunofluorescence experiments. Likewise, the secretion of apoA-I is also decreased in estrogen-treated cells when compared to controls. However, under both conditions, the disappearance of the apoprotein from the cells occurs very rapidly and with similar kinetics. The bulk of apoA-I secreted from LMH-2A cells is recovered on lipoprotein particles with a buoyant density of ≥1.10 g/ml, corresponding to HDL and heavy LDL. Interestingly, apoA-I is detectable on apoB-containing lipoproteins by sequential immunoprecipitation, suggesting that the two apoproteins co-reside at least on a subfraction of the secreted particles, or that apoB- and apoA-I-containing particles interact. These interactions are more pronounced in estrogen-treated cells, most likely due to the dramatic estrogen-mediated induction of apoB synthesis and secretion.

Transcriptional Activation of Cytochrome P450 CYP2C45 by Drugs Is Mediated by the Chicken Xenobiotic Receptor (CXR) Interacting with a Phenobarbital Response Enhancer Unit

Cytochromes P450 (CYP)-2C enzymes fulfill an important role in xenobiotic metabolism and therefore have extensively been studied in rodents and humans. However, no CYP2C genes have been described in avian species to date. In this paper, we report the cloning, functional analysis, and regulation of chicken CYP2C45. The sequence shares up to 58% amino acid identity with CYP2Cs in other species. The overexpression of CYP2C45 in chicken hepatoma cells leghorn male hepatoma (LMH) led to increased scoparone metabolism. CYP2C45 regulation was studied in LMH cells at the mRNA level and in reporter gene assays using a construct containing 2.6 kb of its 5'-flanking region. Exposure of LMH cells to phenobarbital or metyrapone led to a 95- or 210-fold increase in CYP2C45 mRNA and a 140- or 290-fold increase in reporter gene expression, respectively. A phenobarbital response enhancer unit (PBRU) of 239 bp containing a DR-4 nuclear receptor binding site was identified within the 2.6-kb fragment. Site-specific mutation of the DR-4 revealed the requirement of this motif for CYP2C45 induction by drugs. The chicken xenobiotic receptor CXR interacted with the PBRU in electromobility shift and transactivation assays. Furthermore, the related nuclear receptors, mouse PXR and mouse CAR, transactivated this enhancer element, suggesting evolutionary conservation of nuclear receptor-DNA interactions in CYP2C induction.

Mapping of the Chick Heme Oxygenase-1 Proximal Promoter for Responsiveness to Metalloporphyrins

Heme oxygenase (HO) catalyzes the rate-controlling step of physiologic heme catabolism, namely, the oxidation of the α-methene bridge of the macrocycle with formation of CO, Fe, and biliverdin. HO-1, the first isoform of HO to be identified, is highly inducible by a large number of physical and chemical factors. Many of these factors cause oxidative or other stresses to cells. In this work, we have studied the regulation of the chick HO-1 gene, using selected promoter–reporter constructs of the gene transiently or stably transfected into primary cultures of chick embryo liver cells or into the LMH line of chicken hepatoma cells. By use of deletional and mutational analyses, DNase protection, and electromobility shift DNA-binding assays, we identified a heretofore undefined regulatory region in the 5′-UTR of the chick HO-1 gene which confers up-regulation of reporter gene (luciferase) expression in the presence of heme and other selected metalloporphyrins. This new metalloporphyrin-responsive element (MPRE) was localized to a 200-bp region 3.8 to 3.6 kb upstream of the transcription starting point of the chick HO-1 gene. It responded particularly to heme and cobalt protoporphyrin with maximal inductions at 10–15 μM concentrations and 15–18 h of exposure. In contrast, sodium arsenite, a prototypical stress-type inducer of HO-1, led to down-regulation of the reporter gene down stream of MPRE. DNase analysis identified an 18-mer oligonucleotide that was required for the metalloporphyrin response (5′-−3711TATTGCAGCTGTGTGGGG-3′). Mutations at any of four sites within this oligonucleotide abrogated the metalloporphyrin-dependent up-regulation of reporter gene expression. Nuclear protein extracts of cells treated with heme or cobalt protoporphyrin showed specific enhanced binding to this 18-mer. We conclude that the chick HO-1 promoter region contains a unique sequence that subserves up-regulation of the gene by metalloporphyrins and propose the name “metalloporphyrin-responsive element” for this sequence.

A Link between Cholesterol Levels and Phenobarbital Induction of Cytochromes P450

Squalestatin1 (SQ1), a potent inhibitor of squalene synthase produced a dose-dependent induction of cytochromes P450 CYP2H1 and CYP3A37 mRNAs in chicken hepatoma cells. The effect of SQ1 was completely reversed by 25-hydroxycholesterol. Bile acids elicited an induction of CYP3A37 and CYP2H1 mRNA. Bile acids also reduced the phenobarbital induction of CYP2H1 but not of CYP3A37 mRNA. The effects of SQ1 and its reversal by 25-hydroxycholesterol and the effects of bile acids were reproduced in reporter gene assays with a phenobarbital-responsive enhancer unit of CYP2H1. These data suggest that an endogenous molecule related to cholesterol homeostasis regulates induction of drug-inducible CYPs.

Conservation of signaling pathways of xenobiotic-sensing orphan nuclear receptors, chicken xenobiotic receptor, constitutive androstane receptor, and pregnane X receptor, from birds to humans.

Chicken xenobiotic receptor, pregnane X receptor, and constitutive androstane receptor are orphan nuclear receptors that have recently been discovered to regulate drug- and steroid-mediated induction of hepatic cytochromes P450 (CYP). This induction is part of an adaptive response involving numerous genes to exposure to drugs and chemicals and has major clinical and toxicological implications. Here we report experiments in the chicken hepatoma cell line LMH that suggest evolutionary conservation of the signaling pathways triggered by pregnane X receptor, constitutive androstane receptor, and chicken xenobiotic receptor. Thus, the phenobarbital-inducible enhancer units of the mouse Cyp2b10, rat CYP2B2, and human CYP2B6 genes were activated in reporter gene assays by the same compounds that activate the chicken CYP2H1 phenobarbital-inducible enhancer units. Chicken xenobiotic receptor, pregnane X receptor, and constitutive androstane receptor all bound to the CYP2H1 phenobarbital-inducible enhancer units in gel-shift experiments. In CV-1 cell transactivation assays, mammalian pregnane X receptors activate the chicken phenobarbital-inducible enhancer units to the same extent as does chicken xenobiotic receptor, each receptor maintaining its species-specific ligand spectrum. To assess the reported role of protein phosphorylation in drug-mediated induction, we treated LMH cells with okadaic acid and observed increased mRNA of delta-aminolevulinate synthase and CYP2H1 whereas expression of CYP3A37 was decreased. The effects of okadaic acid and other modifiers of protein phosphorylation in LMH cells are comparable to those seen on CYP2Bs and CYP3As in mammalian primary hepatocyte cultures. These results indicate that closely related nuclear receptors, transcription factors, and signaling pathways are mediating the transcriptional activation of multiple genes by xenobiotics in chicken, rodents, and man.

Characterization and expression of three forms of cDNA encoding chicken platelet-derived growth factor-A chain

Platelet-derived growth factor (PDGF) affects cell proliferation and differentiation during mammalian embryogenesis. In a number of avian species, PDGF-α receptors and PDGF-A chain (PDGF-A) are present during chicken limb and lens development. However, little is understood about the chicken PDGF-A gene. The present study identified short form type 1 (S1), long form (L) and short form type 2 (S2) cDNA clones encoding chicken PDGF-A chain (PDGF-A). These clones were isolated from a chicken hepatoma cell line (LMH) mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA library cloning. Genomic sequencing and Southern blotting revealed that these forms were generated by alternative splicing. The mRNAs of S1 and L contained two transcription start sites on one exon. At the amino acid level, the mature protein encoded by the L clone showed 90 and 85% homology with the processed coding regions of the long form of human and Xenopus PDGF-A, respectively. The putative mature peptides of all forms of chicken PDGF-A encompassed the eight cysteine residues conserved in all known forms of PDGF. We examined the expression of the three forms in chicken tissues and cells using RT-PCR. Expression of these forms varied among tissues and cells. Levels of PDGF mRNAs were very low in chicken thrombocytes, which are analogous to mammalian platelets. However, the level of PDGF-A chain mRNA expression in chicken thrombocytes peaked 4 h after exposure to type 1 collagen or thrombin, and then decreased gradually with continued incubation. These results suggest that chicken PDGF in thrombocytes plays an important role in the vascular system and in healing damaged tissue.

Characterization of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen.

The chicken riboflavin carrier protein (RCP) is an estrogen induced egg yolk and white protein. Eggs from hens which have a splice mutation in RCP gene fail to hatch, indicating an absolute requirement of RCP for the transport of riboflavin to the oocyte. In order to understand the mechanism of regulation of this gene by estrogen, the chicken RCP gene including 1 kb of the 5' flanking region has been isolated. Characterization of the gene structure shows that it contains six exons and five introns, including an intron in the 5' untranslated region. Sequence analysis of the 5' flanking region does not show the presence of any classical, palindromic estrogen response element (ERE). However, there are six half site ERE consensus elements. Four deletion constructs of the 5' flanking region with varying number of ERE half sites were made in pGL3 basic vector upstream of the luciferase-coding region. Transient transfection of these RCP promoter deletion constructs into a chicken hepatoma cell line (LMH2A) showed 6-12-fold transcriptional induction by a stable estrogen analogue, moxesterol. This suggests that the RCP gene is induced by estrogen even in the absence of a classical ERE and the half sites of ERE in this promoter may be important for estrogen induction

CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR).

Nuclear receptors constitute a large family of ligand-modulated transcription factors that mediate cellular responses to small lipophilic molecules, including steroids, retinoids, fatty acids, and exogenous ligands. Orphan nuclear receptors with no known endogenous ligands have been discovered to regulate drug-mediated induction of cytochromes P450 (CYP), the major drug-metabolizing enzymes. Here, we report the cloning of an orphan nuclear receptor from chicken, termed chicken xenobiotic receptor (CXR), that is closely related to two mammalian xenobiotic-activated receptors, the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Expression of CXR is restricted to tissues where drug induction of CYPs predominantly occurs, namely liver, kidney, small intestine, and colon. Furthermore, CXR binds to a previously identified phenobarbital-responsive enhancer unit (PBRU) in the 5'-flanking region of the chicken CYP2H1 gene. A variety of drugs, steroids, and chemicals activate CXR in CV-1 monkey cell transactivation assays. The same agents induce PBRU-dependent reporter gene expression and CYP2H1 transcription in a chicken hepatoma cell line. These results provide convincing evidence for a major role of CXR in the regulation of CYP2H1 and add a member to the family of xenobiotic-activated orphan nuclear receptors.

A Conserved Nuclear Receptor Consensus Sequence (DR-4) Mediates Transcriptional Activation of the Chicken CYP2H1 Gene by Phenobarbital in a Hepatoma Cell Line

Phenobarbital-responsive DNA elements were identified in the 5'-flanking region of the chicken CYP2H1 gene by in reporter gene assays in a chicken hepatoma cell line (leghorn male hepatoma (LMH)). A 264-base pair (bp) enhancer sequence (phenobarbital-responsive unit (PBRU)) responded to phenobarbital and a variety of phenobarbital-type inducers. Analysis of putative transcription factor binding sites within the 264-bp element revealed a nuclear receptor half-site repeat (DR-4) neighboring a putative nuclear factor-1 site. This motif resembles phenobarbital response elements in the flanking regions of three phenobarbital-inducible genes, rat CYP2B2, mouse Cyp2b10, and human CYP2B6. Activation of the 264-bp element was eliminated after site-directed mutagenesis of the DR-4 hexamer half-sites. Evidence for evolutionary conservation of this recognition site was indicated by activation in LMH cells of a mouse Cyp2b10 phenobarbital-responsive enhancer by the same spectrum of inducers that activate the CYP2H1 264-bp PBRU. Inhibition of this activation by okadaic acid may explain the reported inhibitory effects on induction of CYP2B1/2 and Cyp2b10 by this phosphatase inhibitor. We show that this inhibition occurs directly on the 264-bp PBRU, whereas the proximal promoter of CYP2H1 is induced by okadaic acid in reporter gene assays. These experiments exploit the unique phenobarbital inducibility of the hepatoma-derived cell line LMH.

Cloning and Functional Expression of a First Inducible Avian Cytochrome P450 of the CYP3A Subfamily (CYP3A37)

CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of ≅55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6β-hydroxylation. At a substrate concentration of 100 μM, progesterone, testosterone, and androstenedione were found to be 6β-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16α-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species.

Effects of Phenylarsine Oxide on Expression of Heme Oxygenase-1 Reporter Constructs in Transiently Transfected Cultures of Chick Embryo Liver Cells

Heme oxygenase catalyzes the first and rate-controlling step of heme catabolism. Induction of heme oxygenase-1 can be caused by numerous factors, including heme, other metalloporphyrins, transition metal ions, heat shock, ultraviolet light, phorbol esters, sodium arsenite, and phenylarsine oxide (PAO). Induction of this enzyme may protect cells from oxidative damage. Using heme oxygenase-1 promoter/reporter gene constructs, we have previously reported that the sodium arsenite-mediated induction of heme oxygenase-1 in chick embryo liver cells and chicken hepatoma (LMH) cells involves an AP-1 element. We have now investigated whether the PAO-mediated induction of heme oxygenase-1 also involves an AP-1 element. Primary cultures of chick embryo liver cells were transiently transfected with heme oxygenase-1 promoter/reporter gene constructs, treated with PAO, and reporter gene activities were measured. We found that the PAO-mediated increase in reporter gene activity was dose- and time-dependent. This activity was decreased by prior treatment with N-acetylcysteine. Studies with mutated constructs showed that both an AP-1 element and a metal responsive element are involved in the PAO-mediated induction of the heme oxygenase-1 reporter construct. Electrophoretic mobility shift assays showed that nuclear proteins from PAO-treated cells had increased binding to an AP-1 probe, and that this increase was abrogated by N-acetylcysteine. These findings support the hypothesis that the PAO-mediated induction of heme oxygenase-1 is caused by activation of AP-1 and MRE/cMyc elements and may involve nuclear proteins whose states of phosphorylation determine binding to regulatory elements, and thus the level of expression of heme oxygenase-1.

Heme biosynthesis in a chicken hepatoma cell line (LMH): comparison with primary chick embryo liver cells (CELC)

5-Aminolevulinic acid synthase (ALA synthase), the rate-controlling enzyme of hepatic heme biosynthesis, is feed-back repressed by heme. In the liver, chemicals such as barbiturates markedly induce ALA synthase, especially in the presence of partial defects of heme biosynthesis. The inducibility and regulation of ALA synthase have been investigated using a variety of models, including intact animals and liver cell culture systems. A widely used model that closely approximates what occurs in vivo and in humans is that of primary cultures of chick embryo liver cells (CELCs). However, CELCs have some limitations: the cells obtained are somewhat heterogeneous; isolation and culture must be repeated every week resulting in weekly variations; and cells are short-lived limiting the feasibility of time-course and transfection studies. The aim of this study was to determine if LMH cells, a chick hepatoma cell line, are a good model comparable to that of CELCs. In both cells similar patterns of response of, ALA synthase activities and mRNA levels, and of porphyrin accumulation were obtained following treatments known to affect heme biosynthesis. Similarly, heme repressed ALA synthase mRNA levels in both cell types and ALA synthase activities in LMH cells. We conclude that LMH cells are a useful model for the study of hepatic heme biosynthesis and regulation of ALA synthase.

A New Avian Hepadnavirus Infecting Snow Geese (Anser caerulescens) Produces a Significant Fraction of Virions Containing Single-Stranded DNA

We describe the identification and functional analysis of an evolutionary distinct new avian hepadnavirus. Infection of snow geese (Anser caerulescens) with a duck hepatitis B virus (DHBV)-related virus, designated SGHBV, was demonstrated by detection of envelope proteins in sera with anti-DHBV preS and S antibodies. Comparative sequence analysis of the PCR-amplified SGHBV genomes revealed unique SGHBV sequence features compared with other avian hepadnaviruses. Unlike DHBV, SGHBV shows an open reading frame in an analogous position to orthohepadnavirus X genes. Four of five cloned genomes were competent in replication, gene expression, and virus particle secretion in chicken hepatoma cells. Primary duck hepatocytes were permissive for infection with SGHBV, suggesting a similar or identical host range. SGHBV was found to secrete a significant fraction of virion-like particles containing single-stranded viral DNA. This was observed both in cell culture medium of SGHBV DNA-transfected LMH cells and in viremic sera of several birds, suggesting that it is a stable trait of SGHBV. Taken together, SGHBV has several unique features that expand the knowledge of the functional and evolutionary diversity of hepadnaviruses and offers new experimental opportunities for studies on the life cycle of hepadnaviruses.

Double-stranded linear duck hepatitis B virus (DHBV) stably integrates at a higher frequency than wild-type DHBV in LMH chicken hepatoma cells.

Integration of hepadnavirus DNAs into host chromosomes can have oncogenic consequences. Analysis of host-viral DNA junctions of DHBV identified the terminally duplicated r region of the viral genome as a hotspot for integration. Since the r region is present on the 5' and 3' ends of double-stranded linear (DSL) hepadnavirus DNAs, these molecules have been implicated as integration precursors. We have produced a LMH chicken hepatoma cell line (LMH 66-1 DSL) which replicates exclusively DSL duck hepatitis B virus (DHBV) DNA. To test whether linear DHBV DNAs integrate more frequently than the wild type open circular DHBV DNAs, we have characterized the integration frequency in LMH 66-1 DSL cells by using a subcloning approach. This approach revealed that 83% of the LMH 66-1 DSL subclones contained new integrations, compared to only 16% of subclones from LMH-D2 cells replicating wild-type open circular DHBV DNA. Also, a higher percentage of the LMH 66-1 DSL subclones contained two or more new integrations. Mathematical analysis suggests that the DSL DHBV DNAs integrated stably once every three generations during subcloning whereas wild-type DHBV integrated only once every four to five generations. Cloning and sequencing of new integrations confirmed the r region as a preferred integration site for linear DHBV DNA molecules. One DHBV integrant was associated with a small deletion of chromosomal DNA, and another DHBV integrant occurred in a telomeric repeat sequence.

PH-sensitive liposomes for receptor-mediated delivery to chicken hepatoma (LMH) cells

pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesterol hemisuccinate (3:2 mol/mol) bearing the N-acetylglucosamine derivative of bovine serum albumin (N-Ac-BSA) were applied for receptor-mediated delivery in chicken hepatoma (LMH) cells expressing the N-Ac-BSA-binding asialoglycoprotein receptor. Fluorescently labeled dextran was entrapped in liposomes by a modified freeze-thawing method (encapsulation efficiency of 23%). A novel method of coupling proteins onto the surface of preformed liposomes yielded a coupling efficiency of 60–70%. The association of pH-sensitive and lecithin liposomes with LMH cells was monitored by fluorescence-activated cell sorting and confocal microscopy. Prerequisites for receptor-mediated delivery to LMH cells were both the pH sensitivity of liposomes and the presence of N-Ac-BSA on the liposomal surface.