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PubMed1981

Production of alpha-fetoprotein, normal serum proteins, and human chorionic gonadotropin in stomach cancer: histologic and immunohistochemical analyses of 35 cases.

By immunoperoxidase histochemical staining of formalin-fixed paraffin-embedded sections, the production of alpha-fetoprotein(AFP), albumin(ALB), transferrin(TF), alpha-1-antitrypsin(AAT), and human chorionic gonadotropin(HCG) was examined in 35 operatively resected stomach cancers with elevated serum AFP levels (higher than 20 ng/ml as determined by radioimmunoassay). Cells positive for AFP were found in 19 cases (54%). In 29 cases (83%), some tumor cells contained normal serum proteins (ALB, TF, or AAT). All 19 tumors with AFP-positive cells also stained positively for two or three kinds of normal serum proteins. In some cases, AFP and normal serum proteins were localized in the same cells. There were two cases in which metastatic tumors produced AFP, whereas the primary sites did not. In nine cases (26%), HCG was present in tumor cells and HCG- and AFP-positive cells were coexistent in six tumors. Histologic examination of AFP-producing stomach tumors revealed medullary or papillotubular arrangements with marked nuclear atypia and eosinophilic granular or clear cytoplasms containing no glycogen or mucin. Some tumors with medullary patterns resembled liver cell carcinomas. Concordant phenotypic expression of AFP and normal serum protein production appears to be a general feature of AFP-producing tumors such as liver cell carcinoma, yolk sac tumor, and stomach cancer.

Blood Proteins↗
PubMed1987

Hepatocellular carcinoma with sarcomatous change. Clinicopathologic and immunohistochemical studies of 14 autopsy cases.

Among 355 autopsy cases of hepatocellular carcinoma (HCC), 14 cases exhibited sarcomatous appearance (incidence, 3.9%). A clinicopathologic study was performed in these 14 cases, and the immunohistochemical localization of keratin (KRT), vimentin (VMT), albumin (ALB), fibrinogen (FBG) and alpha-fetoprotein (AFP) was also examined using the avidin-biotin complex method. Clinically, the HCCs with sarcomatous appearance were characterized by negative or low serum AFP levels and high incidence of extrahepatic metastasis. Grossly they were of infiltrative, mixed expansive and infiltrative, and pedunculated types. Histologically, the tumor consisted mainly of spindle-shaped cells and partly of multinucleated cells, and showed a sinusoidal growth pattern at the tumor-nontumor boundary. Immunohistochemically, tumor cells in the regions showing sarcomatous appearance were frequently found to be positive to KRT and VMT, whereas the percentage of positivity to ALB, FBG, and AFP were not significantly different from those in ordinary HCC. These results strongly suggest that the lesion showing sarcomatous appearance represents the sarcomatous change of HCC rather than being regarded as the complication of HCC and sarcoma.

Adult↗
PubMed2000

Treatment of sulfur mustard (HD)-induced lung injury.

An in vivo sulfur mustard (HD) vapor exposure model followed by bronchoalveolar lavage was developed previously in this laboratory to study biochemical indicators of HD-induced lung injury. This model was used to test two treatment compounds--niacinamide (NIA) and N-acetyl cysteine (NAC)--for their ability to ameliorate HD-induced biochemical changes. Anesthetized rats were intratracheally intubated and exposed to 0.35 mg of HD in 0.1 ml of ethanol or ethanol alone for 50 min. At the beginning of the exposure (t = 0), the rats were treated with either NIA (750 mg kg(-1)) or NAC (816 mg kg(-1)), i.p. At 24 h post-exposure, rats were euthanized and the lungs were lavaged with saline (three 5-ml washes). One milliliter of the recovered lavage fluid was analyzed for cellular components. The remaining fluid was centrifuged (10 min at 300 g) and the supernatant was assayed on a Cobas FARA clinical analyzer for lactate dehydrogenase (LDH), gamma-glutamyltransferase (GGT), albumin (ALB), total protein (TP) and glutathione peroxidase (GP). The HD alone and HD+NIA treatment caused significant increases in all of the biochemical parameters compared with control levels. The NAC treatment yielded LDH, ALB and TP values that, although elevated, were not significantly different from the control. The GP levels were significantly higher than the control but significantly lower than the HD alone levels, indicating some protection compared with the HD alone group. The GGT levels were unaffected by NAC compared with HD alone. Cytological analysis of lavage fluid showed that the percentages of neutrophils were 5.3 +/- 1.0 (mean +/- SEM) for control, 46.6 +/- 4.5 for HD, 31.4 +/- 4.7 for HD + NIA and 21.6 +/- 4.7 for HD + NAC, respectively. The neutrophil counts were significantly higher for the three HD-exposed groups vs controls; however, the NAC-treated group had neutrophil counts lower than HD alone, indicating decreased inflammatory response. These results show that NAC may be useful as a potential treatment compound for HD-induced lung injury.

Acetylcysteine↗
PubMed1985

The relationship between levels of cerebrospinal fluid myelin basic protein and IgG measurements in patients with multiple sclerosis.

Cerebrospinal fluid (CSF) myelin basic protein (MBP) levels, CSF/serum albumin ratio (CSF/S alb), and 4 CSF IgG measurements--absolute CSF IgG level (CSF IgG), CSF IgG/albumin ratio, the Tibbling-Link IgG index, and the daily rate of intrathecal IgG synthesis--were measured in patients with multiple sclerosis and control subjects. In four clinical subgroups of patients, including 22 with polysymptomatic exacerbations, 22 with monosymptomatic exacerbations, 41 with chronic progressive disease, and 21 in remission, there was no correlation between CSF MBP and either CSF/S alb or the CSF IgG measurements. This finding was also observed in longitudinal studies of patients. CSF MBP levels, as determined in a cross-sectional study of 325 patients with multiple sclerosis, are an excellent indicator of disease activity.

Cross-Sectional Studies↗
PubMed2005

Analysis of gene expression patterns in the developing chick liver.

The chick embryo has been used widely for studying liver development. However, in the past 30 years, the usage has decreased markedly due to lack of appropriate marker genes for differentiation in the developing chick liver. To use the chick embryo for analyzing the molecular mechanism of liver development, we surveyed marker genes in the developing chick liver by examining the expression pattern of genes that are well-characterized in the developing mammalian liver. By whole-mount in situ hybridization, Fibrinogen-gamma (FIB) expression was first detected at stage 12, specifically in the anterior intestinal portal, and its liver-specific expression persisted in the later stages. Albumin (ALB) expression was first detected at stage 30, when the liver starts maturing. Cytokeratin 19 (CK19) was first detected at stage 37 in the ductal plate of the liver, and its expression continued in the intrahepatic bile ducts derived from the ductal plate. Hex, a transcription factor, is an additional marker of bile duct differentiation. Hence, FIB, ALB, and CK19 expression can be used to trace hepatic induction, maturation, and bile duct differentiation, respectively.

Animals↗
PubMedDate not supplied

Electrophoretic behavior and partial characterization of disease-associated serum forms of gamma-glutamyltransferase.

We have recently devised an improved procedure for the rapid electrophoretic separation of multiple forms of serum gamma-glutamyltransferase (GGT). This procedure is based on the separation on cellulose acetate strips, usually employed for lipoprotein electrophoresis, followed by visualization with a fluorescent reagent. The method is highly sensitive and the fractions are more clearly resolved than with other procedures. Reference intervals have been evaluated in the sera from 142 healthy subjects and the patterns (two GGT forms comigrating with alpha 1 and alpha 2-globulin) are reproducible. In 150 sera from patients with various hepatobiliary diseases (including neoplasias), acute pancreatitis and non liver-involving neoplasias, we observed some disease-specific GGT forms: an albumin comigrating enzyme (Alb-GGT) specific of liver neoplasia; a gamma-globulin comigrating GGT (gamma-GGT) and a nonmigrating isoform (dep-GGT) both specifically associated to extrahepatic jaundice. Multiple lipoprotein fraction precipitation showed that beta-, gamma- and dep-GGT are complexes between GGT and low density lipoprotein and very low density lipoproteins (LDL + VLDL), and that some of the alpha 1-GGT from cirrhotic patients is a complex between GGT and high density lipoprotein (HDL). GGT fractions from normal subjects and Alb-GGT from patients with liver neoplasia do not appear to be complexed with lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers, Tumor↗
PubMed2004

Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver.

Previously, we reported a system to enrich mouse fetal hepatic progenitor cells (HPCs) by forming cell aggregates. In this study, we sorted two cell populations, CD49f(+)Thy1(-)CD45(-) cells (CD49f-positive cells) and CD49f(+/-)Thy1(+)CD45(-) cells (Thy1-positive cells), from the cell aggregates using a flow cytometer. CD49f-positive cells stained positive for endodermal specific markers such as alpha-fetoprotein (AFP), albumin (ALB), and cytokeratin 19 (CK19), and are thus thought to be HPCs. However, Thy1-positive cells were a morphologically heterogeneous population; reverse-transcription polymerase chain reaction (RT-PCR) and immunocytochemical analyses revealed the expression of mesenchymal cell markers such as alpha-smooth muscle actin, desmin, and vimentin, but not of AFP, ALB, or CK19. Therefore, Thy1-positive cells were thought to be of a mesenchymal lineage. When these two cell populations were co-cultured, the CD49f-positive colonies matured morphologically and stored a significant amount of glycogen. Furthermore, real-time RT-PCR demonstrated an increased expression of tyrosine amino transferase and tryptophan oxygenase mRNA, and transmission electron microscopy confirmed that co-cultured cells produced mature hepatocytes. However, when CD49f-positive cells were cultured alone or when the two populations were cultured separately, the CD49f-positive cells did not mature. These results indicate that CD49f-positive cells are primitive hepatic endodermal cells with the capacity to differentiate into hepatocytes, and that Thy1-positive cells promote the maturation of CD49f-positive cells by direct cell-to-cell contact. In conclusion, we were able to isolate CD49f-positive primitive hepatic endodermal cells and Thy1-positive mesenchymal cells and to demonstrate the requirement of cell-to-cell contact between these cell types for the maturation of the hepatic precursors.

Animals↗
PubMed2005

Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity.

Epithelial cells in embryonic day (ED) 12.5 murine fetal liver were separated from hematopoietic cell populations using fluorescence-activated cell sorting (FACS) and were characterized by immunocytochemistry using a broad set of antibodies specific for epithelial cells (alpha-fetoprotein [AFP], albumin [ALB], pancytokeratin [PanCK], Liv2, E-cadherin, Dlk), hematopoietic/endothelial cells (Ter119, CD45, CD31), and stem/progenitor cells (c-Kit, CD34, Sca-1). AFP(+)/ALB(+) cells represented approximately 2.5% of total cells and were positive for the epithelial-specific surface markers Liv2, E-cadherin, and Dlk, but were clearly separated and distinct from hematopoietic cells (Ter119(+)/CD45(+)). Fetal liver epithelial cells (AFP(+)/E-cadherin(+)) were Sca-1(+) but showed no expression of hematopoietic stem cell markers c-Kit and CD34. These cells were enriched by FACS sorting for E-cadherin to a purity of 95% as defined by co-expression of AFP and PanCK. Purified fetal liver epithelial cells formed clusters in cell culture and differentiated along the hepatocytic lineage in the presence of dexamethasone, expressing glucose-6-phosphatase (G6P) and tyrosine amino transferase. Wild-type ED12.5 murine fetal liver cells were transplanted into adult dipeptidyl peptidase IV knockout mice and differentiated into mature hepatocytes expressing ALB, G6P, and glycogen, indicating normal biochemical function. Transplanted cells became fully incorporated into the hepatic parenchymal cords and showed up to 80% liver repopulation at 2 to 6 months after cell transplantation. In conclusion, we isolated and highly purified a population of epithelial cells from the ED12.5 mouse fetal liver that are clearly separate from hematopoietic cells and differentiate into mature, functional hepatocytes in vivo with the capacity for efficient liver repopulation. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Animals↗
PubMed2005

Oral adeno-associated virus-sTRAIL gene therapy suppresses human hepatocellular carcinoma growth in mice.

The extracellular domain of the tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) may function as a soluble cytokine to selectively kill various cancer cells without toxicity to most normal cells. We constructed a series of recombinant adeno-associated virus (AAV) vectors expressing the extracellular domain of human TRAIL fused with signal peptides of human insulin, interferon, human growth hormone, and serum albumin and designated them as AAV-ISN-T, AAV-IFN-T, AAV-HGH-T, and AAV-Alb-T, respectively. Transduction of human SMMC-7721 liver cancer cells with AAV-ISN-T led to higher levels of TRAIL(95-281) protein expression in the cell culture media and produced more apoptosis of the cells in vitro than those with AAV-IFN-T, AAV-HGH-T, and AAV-Alb-T. The therapeutic potential of AAV-ISN-T was then evaluated in a transplanted mouse model established by injection of human liver cancer SMMC-7721 cells subcutaneously. Subsequent oral or intraperitoneal administration of AAV-ISN-T resulted in a rapid, high level and long time expression of soluble TRAIL in the sera and livers of the animals, as well as effective suppression of tumor growth, with no toxicity to normal hepatocytes. These data strongly suggest that it is possible to increase soluble TRAIL expression to make full use of tumoricidal activity of TRAIL as a therapeutic strategy. In conclusion, we provide evidence that oral administration of AAV-TRAIL might be an important alternative route with practical significance for cancer gene therapy.

Animals↗
PubMed1997

Measurement of transferrin receptor kinetics in the baboon liver using dynamic positron emission tomography imaging and [18F]holo-transferrin.

We have evaluated the use of [18F]holo-transferrin ([18F]Tf) and positron emission tomography (PET) to measure in vivo Tf receptor expression and recycling using the baboon liver as a model. [18F]Tf was intravenously injected in three baboons and dynamic PET was performed over the region containing liver and spleen. In two of the three baboons, [18F]albumin ([18F]Alb), labeled with the same technique, was administered 3 hours later. Time activity curves (TACs) were obtained from liver and spleen for both tracers. TACs for [18F]Tf over the liver were fit to a pharmacokinetic model including vascular radioactivity and an extravascular tissue compartment corresponding to transferrin uptake and release. [18F]Alb data provided an independent estimate of plasma volume. Kinetic analysis showed the presence of a tissue compartment for [18F]Tf that rapidly reaches equilibrium (half time 7-10 minutes). In this organ, the measured rates for Tf turnover obtained with quantitative PET are similar to previously published data using cell culture systems. A model for [18F]Tf in the spleen was not statistically improved by adding a tissue compartment. These data and the pharmacokinetic modeling provide in vivo evidence of a high flux equilibrium binding compartment in the liver, consistent with Tf internalization and recycling.

Albumins↗
PubMed1997

Transgenic expression of hepatitis C virus structural proteins in the mouse.

Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the role of viral cytopathic effects remains unclear. To study the biosynthesis of HCV structural proteins and their pathogenic role, we constructed transgenic mice, expressing type 1b HCV structural proteins (core, E1, and E2) in liver tissues. Two liver-specific promoters were used. The mouse major urinary protein (MUP) promoter has been shown to be developmentally regulated with little or no expression in utero but high-level expression after birth. The albumin (Alb) promoter provides constitutive, high levels of transgenes in live. Expression of both HCV transgenes was detected in several lines by Northern blots, HCV-specific reverse transcriptase-polymerase chain reactions (RT-PCR), and Western immunoblotting. Alb HCV lines showed higher levels of HCV expression than the MUP HCV lines. Immunohistochemical analysis revealed a predominantly cytoplasmic presence of core protein with occasional nuclear staining, and both cytoplasmic and membrane expression of the E2 protein in the transgenic livers. In both transgenes, the highest levels of both antigens were seen in perivenular hepatocytes, suggesting potential processing specificity in those cells. At six months of age, the livers of all transgenic lineages remained histologically normal. We concluded that HCV structural proteins are not directly cytopathic in this animal model.

Animals↗
PubMed1999

Chronic ethanol consumption impairs receptor-mediated endocytosis of formaldehyde-treated albumin by isolated rat liver endothelial cells.

Receptor-mediated endocytosis (RME) by a scavenger receptor on sinusoidal liver endothelial cells (LECs) for formaldehyde-treated bovine serum albumin (f-Alb) has previously been shown to be impaired following chronic ethanol consumption. These studies were initially performed by in situ perfusion, making it difficult to determine the point in the process at which RME is affected. Therefore, it was the purpose of this study to use isolated LECs to begin elucidating at what point in the process chronic ethanol consumption affects RME. Initial studies showed that degradation at the single-cell level were similarly decreased at levels that had been observed for in situ studies, suggesting that the ethanol effects can be repeated using isolated LECs, making them useful for in vitro studies. Binding studies with 125I-formaldehyde-treated bovine serum albumin (125I-f-Alb) demonstrated there was a slight, but significantly different, decrease in binding by LECs from ethanol-fed rats when compared with pair-fed or chow-fed rats. However, the affinity of these receptors was not different between these groups. In contrast, a defect in the initial stages of receptor-ligand internalization was indicated as less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated animals as compared with controls. Additionally, once the data were adjusted for the amount of ligand internalized, the degradation of the internalized ligand was only slightly impaired. These results indicate that chronic ethanol feeding impairs the process of RME by the liver; the major cause of this impairment appears to be caused by a decreased ability of these cells to internalize all of the surface-bound ligand, with a minimal defect in postinternalization events.

Alcohol Drinking↗
PubMed1987

Adsorption of fibronectin derived from serum and from human endothelial cells onto tissue culture polystyrene.

Human endothelial cells (HEC) suspended in a culture medium containing 20% human serum (CMS) adhere and spread on(to) moderately wettable polymers, such as tissue culture polystyrene (TCPS). We have previously shown that serum derived-fibronectin, which is a cell adhesion promoting protein, has a high affinity for TCPS, but that the amount of fibronectin which adsorbed from CMS was relatively small. In this study we investigated whether fibronectin derived from HEC contributes to the adhesion and spreading of the cells on(to) TCPS. Therefore, HEC were seeded in the presence of fibronectin-depleted CMS. The amount of fibronectin detected on TCPS increased with both cell seeding density and incubation time. Although initial HEC adhesion is delayed on TCPS which has been precoated with albumin (Alb), high density lipoprotein (HDL) or immunoglobulin G (IgG), maximal numbers of adhering and spreading HEC were found on these surfaces 6 h after seeding of HEC. Fibronectin was detected on these surfaces, but an exchange of preadsorbed Alb, HDL, or IgG for fibronectin could not be demonstrated. We conclude that HEC deposit fibronectin onto TCPS, irrespective of the presence of a preadsorbed layer of proteins which delay cell adhesion.

Adsorption↗
PubMed1993

Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and alpha 2HS-glycoprotein) proteins in rat bone.

The high-resolution, postembedding protein A-gold immunocytochemical technique was used to visualize the distribution of two noncollagenous bone proteins, osteopontin (OPN) and osteocalcin (OC), and two plasma proteins, alpha 2HS-glycoprotein (alpha 2HS-GP) and albumin (ALB), in sections of Lowicryl K4M-embedded rat tibial and alveolar bone. In the primary spongiosa of the metaphysis, a seam of organic material (lamina limitans) that labeled intensely with OPN and OC antibodies was observed at the bone/calcified cartilage interface just below the zone of vascular invasion of the growth plate. With deposition of bone matrix proper by osteoblasts in this region and its subsequent mineralization, extensive areas of bone were heavily labeled with anti-OPN, anti-OC, and anti-alpha 2HS-GP antibodies, where the majority of gold particles were associated with amorphous, electron-dense patches of organic material throughout the mineralized bone. In the unmineralized osteoid, substantially less labeling was observed, and where occasional mineralization loci were dispersed throughout the osteoid layer, these sometimes showed a concentration of gold particles. ALB labeling, on the other hand, was moderate and generally diffuse throughout the mineralized bone matrix and the osteoid. In alveolar bone, labeling patterns were generally similar to those found in tibial bone. Particularly striking in alveolar bone, however, was an intense anti-OPN labeling of (1) the lamina limitans at cell-lined bone surfaces, including that surrounding cell processes and osteocytes, (2) cement (reversal, resting) lines, and (3) the perilacumar matrix of some osteocytes. In summary, these data suggest that certain plasma proteins, such as alpha 2HS-GP, interact with bone matrix proteins, such as OPN and OC, at sites of tissue mineralization and that the presence of OPN in mineralized bone and at bone surfaces (lamina limitans) and cement lines has a multifunctional role, including regulation of mineralization and mediation of cell dynamics during endochondral and intramembranous bone modeling and remodeling.

Animals↗
PubMed1987

Regulation of amino acid transport in isolated rat hepatocytes during development.

The effect of amino acid depletion or supplementation and the effect of glucagon and insulin on the amino acid transport mediated by system A were investigated by determining the uptake of either 2-amino [1-14C]isobutyric acid (AIB) or N-methyl 2-amino [1-14C]isobutyric acid (MeAIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the Na+-dependent uptake was higher at the earliest developmental stages, and steadily decreased until the adult level. The hormones increased AlB and MeAIB uptake enhancing the Vmax, while the Km was unchanged. This effect was evident in cells from adult and 18-20-day-old fetuses, while no response was present before the 18th day of fetal life and in the perinatal period. Actinomycin D or cycloheximide abolished this hormone-dependent increase. A decrease in AlB and MeAIB transport after incubation in an amino acid-rich medium was demonstrated at all ages tested, but was particularly evident in the prenatal life. The increase in the activity of the system following amino acid starvation was shown to be mostly dependent from de novo protein synthesis in the fetal life; on the contrary in the adult the increase appeared to be more linked to the release from transinhibition of the transport.

Aging↗
PubMed2005

Induction of umbilical cord blood-derived beta2m-c-Met+ cells into hepatocyte-like cells by coculture with CFSC/HGF cells.

Several studies have indicated that adult stem cells derived from bone marrow (BM) and cord blood (CB) can differentiate into hepatocyte-like cells. This ability is important for the treatment of hepatic diseases with BM or CB as a potential approach. However, methods are still being developed for the efficient induction of stem cell differentiation and expansion to get enough cells to be useful. In the present study, we enriched a subset of umbilical cord blood beta(2)m(-)c-Met(+) cells (UCBCCs) and investigated the combination effect of liver nonparenchymal cells (cirrhotic fat-storing cells [CFSCs]) and hepatocyte growth factor (HGF) on the induction of UCBCCs into hepatocyte-like cells. UCBCCs were cocultured with CFSC/HGF feeder layers either directly or separately using insert wells. Flow cytometric analysis showed that most UCBCCs were CD34(+/-)CD90(+/-)CD49f(+)CD29(+)Alb(+)AFP(+). After cocultured with transgenic feeder layers for 7 days, UCBCCs displayed some morphologic characteristics of hepatocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence cell staining proved that the induced UCBCCs expressed several hepatocyte specific genes including AFP, Alb, CYP1B1 and cytokeratins CK18 and CK19. Furthermore, the induced cells displayed liver specific functions of indocyanine green (ICG) uptake, ammonium metabolism and albumin secretion. Hence, our data have demonstrated that UCBCCs might represent a novel subpopulation of CB-derived stem/progenitor cells capable of successful differentiation into hepatocyte-like cells when incubated with CFSC/HGF cells. In conclusion, not only HGF but also CFSCs and/or the secreted extracellular matrix (ECM) have been shown to be able to serve as essential microenvironment for hepatocyte differentiation.

Base Sequence↗
PubMed2003

Biallelic expression of the H19 gene during spontaneous hepatocarcinogenesis in the albumin SV40 T antigen transgenic rat.

Previous studies in this laboratory have demonstrated that the earliest cytogenetic alteration in the development of hepatic neoplasms in a transgenic strain of rats bearing the albumin Simian virus 40 T antigen (Alb SV40 T Ag) construct was a duplication of the chromosome 1q4.1-1q4.2 band. In this region, in the rat genome a cluster of linked imprinted genes occurs. One of these imprinted genes, H19, which is expressed in fetal liver but not in adult liver, was found to be expressed in virtually all neoplasms investigated. A single-nucleotide polymorphic marker in the H19 coding sequence was identified in two rat strains and utilized for the investigation of H19 imprinting. Our results reveal monoallelic expression of the maternal gene in fetal liver, but biallelic expression of the H19 gene in liver neoplasms, thus demonstrating the basis for the deregulation of the imprinted gene expression during hepatocarcinogenesis. These results suggest that the loss of genomic imprinting of the H19 gene found in the liver neoplasms of the Alb SV40 T Ag rat may result not from allelic loss, but from adverse changes in the epigenetic imprints present in the 5'-upstream region of the H19 promoter of the parental alleles.

Albumins↗
PubMed2004

Changes in the DNA methylation profile of the rat H19 gene upstream region during development and transgenic hepatocarcinogenesis and its role in the imprinted transcriptional regulation of the H19 gene.

Monoallelic expression of the imprinted H19 and insulin-like growth factor-2 (Igf2) genes depends on the hypomethylation of the maternal allele and hypermethylation of the paternal allele of the H19 upstream region. Previous studies from our laboratory on liver carcinogenesis in the F1 hybrid of Fischer 344 (F344) and Sprague-Dawley Alb SV40 T Ag transgenic rat (SD) strains revealed the biallelic expression of H19 in hepatomas. We undertook a comparative study of the DNA methylation status of the upstream region of H19 in fetal, adult, and neoplastic liver. Bisulfite DNA sequencing analysis of a 3.745-kb DNA segment extending from 2950 to 6695 bp of the H19 upstream region revealed marked variations in the methylation patterns in fetal, adult, and neoplastic liver. In the fetal liver, equal proportions of hyper- and hypomethylated strands revealed the differentially methylated status of the parental alleles, but in neoplastic liver a pronounced change in the pattern of methylation was observed with a distinct change to hypomethylation in the short segments between 2984 and 3301 bp, 6033-6123 bp, and 6518-6548 bp. These results indicated that methylation of all cytosines in this region may contribute to the imprinting status of the rat H19 gene. This phenomenon of differential methylation-related epigenetic alteration in the key cis-regulatory domains of the H19 promoter influences switching to biallelic expression in hepatocellular carcinogenesis. Similar to mouse and human, we showed that the zinc-finger CCTCC binding factor (CTCF) binds to the unmethylated CTCF binding site in the upstream region to influence monoallelic imprinted expression in fetal liver. CTCF does not appear to be rate limiting in fetal, normal, and neoplastic liver. 3' to the CTCF binding sites, another DNA region exhibits methylation of CpG's in both DNA strands in adult liver, retention of the imprint in fetal liver, and complete demethylation in neoplastic liver. In this region is also a putative binding site for a basic helix-loop-helix leucine-zipper transcription factor, TFEB. The differential CpG methylation seen in the adult that involves the TFEB binding site may explain the lack of expression of the H19 gene in adult normal liver. Furthermore, these findings demonstrate that the loss of imprinting of the H19 gene in hepatic neoplasms of the SD Alb SV40 T Ag transgenic rat is directly correlated with and probably the result of differential methylation of CpG dinucleotides in two distinct regions of the gene that are within 4 kb 5' of the transcription start site. Cytogenetic analysis of hepatocytes in the transgenic animal prior to the appearance of nodules or neoplasms indicates a role of such loss of imprinting in the very early period of neoplastic development, possibly the transition from the stage of promotion to that of progression.

5' Untranslated Regions↗