Α-fetoprotein mRNA Research Articles

The SGLT2 Inhibitor Canagliflozin Prevents Carcinogenesis in a Mouse Model of Diabetes and Non-Alcoholic Steatohepatitis-Related Hepatocarcinogenesis: Association with SGLT2 Expression in Hepatocellular Carcinoma.

The aim of the present study is to investigate the effects of canagliflozin, a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, on non-alcoholic steatohepatitis (NASH) and NASH-related hepatocellular carcinoma (HCC) in a mouse model of diabetes and NASH-HCC. First, mice aged five weeks were divided into two groups (vehicle group and canagliflozin group) and were treated for three weeks. Then, mice aged five weeks were divided into three groups of nine animals each: the vehicle group, early canagliflozin group (treated from five to nine weeks), and continuous canagliflozin group (treated from five to 16 weeks). Canagliflozin was administered at a dose of 30 mg/kg in these experiments. In addition, the in vitro effects of canagliflozin were investigated using HepG2 cells, a human HCC cell line. At the age of eight or 16 weeks, the histological non-alcoholic fatty liver disease activity score was lower in the canagliflozin-treated mice than in vehicle-treated mice. There were significantly fewer hepatic tumors in the continuous canagliflozin group than in the vehicle group. Immunohistochemistry showed significantly fewer glutamine synthetase-positive nodules in the continuous canagliflozin group than in the vehicle group. Expression of α-fetoprotein mRNA, a marker of HCC, was downregulated in the continuous canagliflozin group when compared with the vehicle group. At 16 weeks, there was diffuse SGLT1 expression in the hepatic lobules and strong expression by hepatocytes in the vehicle group, while SGLT2 expression was stronger in liver tumors than in the lobules. In the in vitro study, canagliflozin (10 μM) suppressed the proliferation of HepG2 cells. Flow cytometry showed that canagliflozin reduced the percentage of HepG2 cells in the G2/M phase due to arrest in the G1 phase along with decreased expression of cyclin D and Cdk4 proteins, while it increased the percentage of cells in the G0/1 phase. Canagliflozin also induced apoptosis of HepG2 cells via activation of caspase 3. In this mouse model of diabetes and NASH/HCC, canagliflozin showed anti-steatotic and anti-inflammatory effects that attenuated the development of NASH and prevented the progression of NASH to HCC, partly due to the induction of cell cycle arrest and/or apoptosis as well as the reduction of tumor growth through the direct inhibition of SGLT2 in tumor cells.

Maternal nicotinamide supplementation causes global DNA hypomethylation, uracil hypo-incorporation and gene expression changes in fetal rats

Recent evidence shows that excess nicotinamide can cause epigenetic changes in developing rats. The aim of the present study was to investigate the effects of maternal nicotinamide supplementation on the fetus. Female rats were randomised into four groups fed a standard chow diet (control group) or diets supplemented with 1 g/kg of nicotinamide (low-dose group), 4 g/kg of nicotinamide (high-dose group) or 4 g/kg of nicotinamide plus 2 g/kg of betaine (betaine group) for 14-16 d before mating and throughout the study. Fetal tissue samples were collected on the 20th day of pregnancy. Compared with the control group, the high-dose group had a higher fetal death rate, and the average fetal body weight was higher in the low-dose group but lower in the high-dose group. Nicotinamide supplementation led to a decrease in placental and fetal hepatic genomic DNA methylation and genomic uracil contents (a factor modifying DNA for diversity) in the placenta and fetal liver and brain, which could be completely or partially prevented by betaine. Moreover, nicotinamide supplementation induced tissue-specific alterations in the mRNA expression of the genes encoding nicotinamide N-methyltransferase, DNA methyltransferase 1, catalase and tumour protein p53 in the placenta and fetal liver. High-dose nicotinamide supplementation increased fetal hepatic α-fetoprotein mRNA level, which was prevented by betaine supplementation. It is concluded that maternal nicotinamide supplementation can induce changes in fetal epigenetic modification and DNA base composition. The present study raises the concern that maternal nicotinamide supplementation may play a role in the development of epigenetic-related diseases in the offspring.

Effects of pre-operative transcatheter arterial chemoembolization for resectable hepatocellular carcinoma: Implication of circulating cancer cells by detection of α-fetoprotein mRNA

Transcatheter arterial chemoembolization (TACE) is useful for the treatment of multiple hepatocellular carcinomas (HCCs). Pre-operative TACE is used to reduce recurrence caused by peri- and post-operative spread of cancer cells; however, the efficacy is controversial. In this study, we evaluated the efficacy of pre-operative TACE for HCC and the implication of circulating cancer cells, retrospectively. We analyzed 495 patients with HCC who had undergone hepatectomy between 1980 and 2006, including 252 patients (50.9%) who received pre-operative TACE. The median follow-up period was 49.9 months. We compared the survival of TACE and non-TACE groups and also performed subgroup analysis. α-fetoprotein (AFP) mRNA was quantified to represent circulating cancer cells. Pre-operative TACE prolonged disease-free survival after hepatectomy in patients with HCCs greater than 5 cm (5-year disease-free survival of the pre-operative TACE and no-TACE groups was 37.3 vs. 14.8%, p<0.05). Patients with tumors showing 70% or greater necrosis had a significantly more favorable survival, and those with complete necrosis were all AFP mRNA-negative. The survival of the AFP mRNA-positive patients was worse than that of the AFP mRNA-negative patients. Pre-operative TACE may be beneficial for patients with tumors larger than 5 cm, and AFP mRNA quantification may be useful for the prediction of survival after surgery in TACE-treated patients.

Comparison Between Human-Telomerase Reverse Transcriptase mRNA and α-Fetoprotein mRNA as a Predictive Value for Recurrence of Hepatocellular Carcinoma in Living Donor Liver Transplantation

Objective In this study we compared the potential roles of preoperative human-telomerase reverse transcriptase (h-TERT) mRNA versus α-fetoprotein (AFP) mRNA expression in the peripheral blood as a tool to predict prognosis and tumor recurrence after living donor liver transplantation (LDLT) in hepatocellular carcinoma (HCC) patients. Patients and Methods We examined 14 patients with unresectable HCC who underwent LDLT. Six patients displayed stage IVA HCC that deviated from the Milan criteria, while the rest of the patients fell within the limitations of the Milan criteria. We analyzed the relationship between preoperative h-TERT mRNA or AFP mRNA expression in the peripheral blood and survival without recurrence. Results There was no significant difference between the survival curves without recurrence of those patients who did versus did not meet the Milan criteria. There was also no significant difference between the survival curves without recurrence among patients with positive versus negative AFP mRNA expression. However, there was a significant difference ( P = .005) between the survival curves without recurrence of those patients with positive preoperative h-TERT mRNA expression versus those who either had an initially negative preoperative h-TERT mRNA or who converted from positive to negative after neoadjuvant immunochemotherapy. Conclusions h-TERT mRNA seemed to prove more valuable than AFP mRNA not only to assess preoperative treatment modalities and postoperative patient surveillance, but also to evaluate prospective LDLT patients with HCC. Moreover, use of h-TERT mRNA could potentially expand the indications for transplantation to patients outside the Milan criteria.

α-Fetoprotein mRNA in the circulation as a predictor of postsurgical recurrence of hepatocellular carcinoma: A prospective study

α-fetoprotein (AFP) messenger RNA (mRNA) has been proposed as a marker of hepatocellular carcinoma (HCC) cells disseminated into the circulation, but its clinical significance remains controversial. We prospectively assessed the prognostic value of AFP mRNA in patients undergoing curative hepatic resection for HCC. Peripheral blood samples were taken from 87 patients before and after surgery to determine the presence of AFP mRNA by use of a reverse-transcription polymerase chain reaction. A primary endpoint was recurrence-free interval. AFP mRNA was detectable preoperatively in 31 patients (36%) and postoperatively in 30 patients (34%). With a median follow-up period of 28 months (range, 3-41 months), HCC recurred in 46 patients (53%). Among 4 groups separated according to preoperative and postoperative AFP mRNA status, patients with consistent positivity of AFP mRNA showed the highest recurrence rate (85%) and trend to distant or multiple recurrence. The recurrence-free interval was significantly shorter in patients with postoperative positivity of AFP mRNA than in those without (53% [95% CI, 36-71] vs. 88% [95% CI, 79-96] at 1 year, 37% [95% CI, 17-57] vs. 60% [95% CI, 46-75] at 2 years; P = .014), whereas the preoperative positivity of AFP mRNA provided no significance (P = .100). Cox's proportional-hazards model identified the postoperative positivity of AFP mRNA as an independent prognostic factor for HCC recurrence (relative risk, 2.33; 95% CI, 1.26-4.34; P = .007). In conclusion, postsurgical recurrence of HCC can be predicted by detecting AFP mRNA–expressing cells in peripheral blood. (HEPATOLOGY 2002;35:853-860.)

Increased GABAergic activity inhibits α-fetoprotein mRNA expression and the proliferative activity of the HepG 2 human hepatocellular carcinoma cell line

Background/Aims: Gamma aminobutyric acid (GABA) is a potent inhibitory neurotransmitter with growth regulatory properties. Recent data indicate that increased GABAergic activity inhibits hepatocyte proliferation in regenerating livers. In the present study, we aimed to investigate whether GABA inhibits the growth of malignant hepatocytes. Methods: Increasing concentrations of muscimol (0.05–50 μM), a specific GABA A receptor agonist, were added to HepG2 human hepatocellular carcinoma cells and alpha-fetoprotein (AFP) and albumin mRNA expression were determined for varying periods of time (maximum 24 h) thereafter. Cell proliferation was also documented after 48 h of exposure to muscimol. Results: Muscimol significantly ( p<0.0001) decreased AFP mRNA expression (maximum decrease: 65% below baseline values) without affecting albumin mRNA expression. However, the effect on AFP mRNA was transient (maximum duration: 3–6 h) and not associated with changes in cell proliferation. Because preliminary data indicate that GABA A receptor activity is markedly downregulated in malignant hepatocytes, transfection studies were performed wherein HepG2 cells were cotransfected with GABA A receptor β 2 and γ 2 subunit genes in a pCDM8 expression vector or vector alone followed by re-exposure to either muscimol (5 μM) or saline. In this series of experiments, in addition to AFP mRNA inhibition being as extensive and more prolonged (maximum duration: 6–12 h) in muscimol-treated, GABA A receptor-transfected cells, proliferative activity was also significantly inhibited when compared to saline-treated GABA A receptor-transfected controls ( p<0.01) and muscimol-treated cells transfected with vector alone ( p<0.005). Conclusion: The results of this study indicate that increased GABAergic activity inhibits AFP mRNA expression and cell proliferation in this malignant hepatocyte cell line.

Presence of α-fetoprotein mRNA in blood correlates with outcome in patients with hepatocellular carcinoma

Background/Aims: Since hematogenous spread of tumor cells may adversely affect the prognosis of patients with hepatocellular carcinoma, we prospectively analyzed whether the presence of alpha-fetoprotein (AFP) messenger RNA (mRNA) in blood, used as a marker of circulating hepatocellular carcinoma cells, correlates with outcome. Methods: Eighty-eight patients were enrolled between December 1993 and August 1995, and 81 were followed until the end of 1997. All patients were treated with percutaneous ethanol injection therapy and/or transarterial embolization during follow-up. The status of AFP mRNA in blood was serially determined. Cumulative metastasis-free survival and overall survival were analyzed in relation to AFP mRNA and other clinical and laboratory variables. Results: Among 81 patients followed, 54 were positive for AFP mRNA at entry and 27 were negative. Extrahepatic metastasis developed more frequently among the AFP mRNA-positive patients (13 of 54) than among the AFP mRNA-negative patients (2 of 27) ( p=0.0296). After treatment, AFP mRNA became negative in 24 of 54 patients (44%). Cumulative metastasis-free survival and overall survival were significantly better in the 24 patients whose AFP mRNA became negative after treatment than in the 30 patients with persistently positive AFP mRNA ( p=0.0001 and p Conclusions: The presence or absence of AFP mRNA in blood is a predictor of outcome in patients with hepatocellular carcinoma.

CDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of α-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

CDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of alpha-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

Analysis of α-fetoprotein mRNA in peripheral blood: detection and semi-quantitation by reverse transcription polymerase chain reaction

The presence of α-fetoprotein (AFP) mRNA in mononuclear cells of the peripheral blood was examined in patients with hepatocellular carcinoma (HCC), liver cirrhosis, chronic hepatitis and normal volunteers using nested reverse transcription polymerase chain reaction (RT-PCR). Then, the AFP mRNA levels were quantitatively analyzed in AFP mRNA-positive patients with HCC. AFP mRNA was detected in 23 of 38 (60.5%) cases with HCC, 2 of 12 (16.7%) cases with cirrhosis, and 1 of 9 (11.1%) cases with chronic hepatitis, respectively. AFP mRNA was not demonstrated in six healthy volunteers. The frequency of positive cases in TNM stage III–IV (16/21, 76.2%) was significantly higher than that in stage I–II (7/17, 41.2%)( P<0.05). AFP mRNA was detected more frequently in patients with positive levels of serum protein induced by vitamin K antagonist II (PIVKA-II) (≥40 mAU/ml, 18/25, 72.0%) than in those with negative level (40 mAU/ml, 5/13, 38.5%) ( P<0.05). However, there was no significant correlation between detectable rate of AFP mRNA and serum AFP level. The frequency of AFP mRNA positive result was not closely associated with L. culinaris agglutinin A, a reactive α-fetoprotein (AFP L3). On the other hand, the expression levels of AFP mRNA were measured in 23 AFP mRNA-positive patients with HCC. Quantitative analysis of AFP mRNA was performed by comparing the amplification kinetic curve of AFP mRNA with that of β-actin mRNA as an internal control. The AFP mRNA levels were expressed as Δ cycle values by this method. Δ cycle values were significantly increased in patients with HCC in stages III–IV (4.7±2.4) compared with patients with HCC in stages I–II (−1.3±7.3) ( P<0.01). These results suggested that the detection and quantitation of AFP mRNA in peripheral blood may indicate the hematogenous spreading of tumor cells in patients with HCC.

Quantitative analysis of alpha-fetoprotein mRNA in circulating peripheral blood of patients with hepatocellular and alpha-fetoprotein-producing gastric carcinomas

In conjunction with strategies introduced in recent years to identify cancer micrometastasis through amplification of cancer-associated mRNA, we developed a highly sensitive system to detect α -fetoprotein mRNA in circulating peripheral blood of hepatocellular carcinoma patients. The aim of the present study was to make our original system quantitative. Peripheral venous blood from patients with hepatocellular carcinoma and α -fetoprotein-producing gastric carcinoma was subjected to reverse transcription followed by our original three-step polymerase chain reaction co-amplifying both the original sequence and our synthetic competitor. We succeeded in modifying our system for quantitative analysis, and investigated the perioperative change, the postoperative change and the change after chemotherapy in order to illustrate the possible application of this method. The quantitative analysis of α-fetoprotein mRNA present in the peripheral blood represents a useful tool for analyzing the relationship of surgery to recurrence, the effect of chemotherapy, and to predict impending recurrence in patients with hepatocellular and α-fetoprotein-producing gastric carcinomas.

Activity and Inducibility of Drug-Metabolizing Enzymes in Immortalized Hepatocyte-like Cells (mhPKT) Derived from al-PK/Tag1 Transgenic Mouse

This report describes the establishment and characterization of the mhPKT cell line derived from the liver of a transgenic mouse harboring the simian virus (SV40) large T and small t antigens placed under the control of the 5′ regulatory sequence of the ratl-type pyruvate kinase (l-PK) gene. mhPKT cells had a prolonged life span, expressed the SV40-encoded nuclear large T antigen when grown in glucose-enriched medium, and induced tumors when injected subcutaneously into athymic (nu-nu) mice. Growth on petri dishes or filters yielded multiple layers of cuboid cells, with numerous spaces between adjacent cells that were closed by junctional complexes. These bile canaliculi-like structures exhibited numerous microvilli in which villin, an actin-binding brush-border protein, colocalized with actin. These bile canaliculi-like structures appeared to be functional as they accumulated fluorescein. mhPKT cells conserved the expression of the liver-specific transcription factors HNF1, HNF3, HNF4, and DBP together with substantial levels ofl-PK and albumin but not α-fetoprotein mRNA transcripts. mhPKT cells mainly metabolized testosterone into androstenedione and 6β-hydroxytestosterone, asin vivo.3-Methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) markedly increased ethoxyresorufin-O-deethylase activity and the related cytochrome P450 (CYP) 1A1/2 protein, whereas α-naphtoflavone antagonized the TCDD-elicited induction. Phenobarbital slightly increased the CYP2B-mediated activities of pentoxyresorufin-O-depentylase, 2β- and 16β-testosterone hydroxylase. mhPKT cells also had substantial sulfotransferase, UDP-glucuronyltransferase, and glutathioneS-transferase activities. This model may serve as a tool for long-termin vitrostudies of xenobiotic metabolism, potent CYP inducers, and hepatocyte damage due to drugs and other factors.

Modulation of the Expression of Liver-Specific Functions in Novel Human Hepatocyte Lines Cultured in a Collagen Gel Sandwich Configuration

Liver-specific functions were examined in two novel human hepatocyte lines established from normal liver tissue (HH29, HHY41) and one human hepatoma line (Hep G2), cultured either in a collagen gel sandwich configuration or on plain plastic dishes. At confluence, levels of albumin and α-fetoprotein mRNA were markedly increased in all lines cultured in the collagen gel sandwich over those on plastic, while levels of mRNA for mitochondrial elongation factor (EFTU), a ubiquitous enzyme, were equivalent. Secretion of albumin by cells cultured in the collagen gel sandwich was significantly increased in parallel with the abundance of albumin mRNA. Hepatocytes in the collagen gel sandwich configuration secreted α-fetoprotein of a higher molecular weight in addition to α-fetoprotein of standard molecular weight. The three-dimensional configuration of the collagen gel sandwich enhances the expression of liver-specific functions of human hepatocytes from continuous cell lines, probably by preserving proper cellular polarity in vitro.

Highly-sensitive identification of α-fetoprotein mRNA in circulating peripheral blood of hepatocellular carcinoma patients

In order to capture hepatocellular carcinoma (HCC) cells in circulating peripheral blood, we made analysis to see if a -fetoprotein (AFP) mRNA exists in the peripheral blood obtained from patients with HCC and also, as a control, from hepatitis-viral-marker-positive patients without HCC and a healthy volunteer. As the number of HCC cells in peripheral blood and the quantity of AFP mRNA are expected to be very small, the analysis was performed by the reverse transcription followed by an original three-step polymerase chain reaction. By this highlysensitive method, 5 of 7 HCC patients were positive for AFP mRNA. These 5 positive patients consisted of three with clinically apparent recurrence, one preoperative patient with tumor thrombus in the portal vein and one recurrence-free patient who developed clinically detectable recurrence three months after this analysis. Neither 4 patients with positive viral markers nor a healthy volunteer was positive. The results suggest that detection of AFP mRNA from HCC patients' peripheral blood by our highly-sensitive RT-PCR may be a practical and powerful tool to diagnose the preoperative spreading of HCC and to monitor its recurrence.

Detection of α-fetoprotein mRNA, an indicator of hematogenous spreading hepatocellular carcinoma, in the circulation: A possible predictor of metastatic hepatocellular carcinoma

We attempted to detect circulating hepatocellular carcinoma by demonstrating hepatocyte-associated mRNA in the nuclear cell component of peripheral blood using nested reverse transcription-polymerase chain reaction because of the extremely small number of tumor cells in the circulation. Albumin mRNA was demonstrated not only in the liver tissue (hepatocytes) and HepG2 cells but also in nuclear cells of the blood from normal healthy volunteers (neutrophils and lymphocytes) by reverse transcription-polymerase chain reaction. In contrast, alpha-fetoprotein mRNA was demonstrated in the liver tissue, as well as in HepG2 cells, but not in peripheral blood of normal healthy volunteers, indicating the possibility of using alpha-fetoprotein mRNA for detection of benign and malignant hepatocytes among the population of neutrophils and lymphocytes. alpha-Fetoprotein mRNA in peripheral blood was detected in 17 of 33 cases of hepatocellular carcinoma (52%), 2 of 13 cases of cirrhosis (15%) and 2 of 17 cases of chronic hepatitis (12%). alpha-Fetoprotein mRNA was not demonstrated in 26 cases of normal healthy volunteers (0%). Among the patients with hepatocellular carcinoma, total volume of tumor tissue, maximum size of tumor and serum alpha-fetoprotein level were markedly increased in the patients with alpha-fetoprotein mRNA in blood. In addition, alpha-fetoprotein mRNA was detected in the blood of all 6 patients showing metastasis at extrahepatic organs (100%), in contrast to 11 of 27 cases without metastasis (41%). From these results, we conclude that the presence of alpha-fetoprotein mRNA in peripheral blood may be an indicator of circulating malignant or benign hepatocytes, which might predict hematogenous spreading metastasis of tumor cells in patients with hepatocellular carcinoma.

Molecular Basis of Reduced Albumin Gene Expression in Hepatoma Cell Lines with Different Growth Rates

To study the relationship between expression of a differentiated function and cellular proliferation we analyzed the molecular mechanisms controlling albumin gene expression in rat liver and in rat hepatoma cell lines of different growth rates: low (MH 1C 1), intermediate (FAO), and high (3924A), as evaluated by both cell doubling time and histone H3 gene expression. In comparison with the liver, albumin accumulation was reduced in FAO cells and absent in MH 1C 1 and 3924A cells. Thus, these hepatoma cell lines do not show the inverse relationship between cellular growth rate and expression of a differentiation marker that has been often described in several hepatic and nonhepatic cellular systems. This conclusion is further reinforced by the finding that all cell lines, irrespective of their growth rate, failed to express α-fetoprotein mRNA, a dedifferentiation marker, and that two other liver-abundant genes, transferrin and apolipoprotein A-1, are regulated in a way similar to that of albumin. The defect in albumin accumulation was due to decreased or lacking synthesis which, in turn, was accompanied by reduction or absence of albumin mRNA. Run-on transcription assays provided evidence for diminished or absent albumin gene transcription. Southern blot analysis revealed that the structure of the albumin gene is preserved in all the tumor cell lines; however, we found a different methylation state of the albumin gene that correlated well with albumin expression.

Regulation of albumin and α-fetoprotein gene expression by colloid osmotic pressure in human hepatoma cells

Background: Colloid osmotic pressure has been thought to regulate albumin synthesis; however, the exact mechanism remains obscure. In the present study, the effect of colloid osmotic pressure on the albumin and α-fetoprotein gene expression in HuH-7 human hepatoma cells was analyzed. Methods: HuH-7 cells were treated with albumin or dextran (mean mol wt, 70,000), and changes in the levels of albumin and α-fetoprotein messenger RNA (mRNA) were analyzed by Northern blotting. Furthermore, in transient chloramphenicol acetyltransferase (CAT) plasmid transfection experiments, effects of colloid osmotic pressure on CAT activities were studied. Results: By Northern blot analysis, the levels of both albumin and α-fetoprotein mRNA were dose-dependently suppressed by the elevation of colloid osmotic pressure and returned to pretreatment levels 48 hours after the culture medium containing dextran was replaced with a dextran-free fresh medium. In transient CAT plasmid transfection experiments, the increased level of colloid osmotic pressure resulted in the repression of both albumin and α-fetoprotein promoter activities. In contrast, α-fetoprotein enhancer activity, which possibly regulates not only α-fetoprotein but also albumin gene expression, was not affected by changes in colloid osmotic pressure. Conclusions: These results suggest that colloid osmotic pressure regulates both albumin and α-fetoprotein gene transcription through the modulation of their promoter activities.

The effects of retinoic acid on the expression of α-fetoprotein and albumin genes in rat hepatoma cell lines

Abstract Many transcriptional regulators can stimulate or repress gene expression depending on the cellular or genetic contexts. Thus dexamethasone increases the amount of α-fetoprotein mRNA in Morris rat hepatoma derived cell line McA-RH 8994 cells, but decreases it in McA-RH 7777 cells. In the present study, we demonstrated that retinoic acid, whose receptors belong to the steroid/thyroid hormone receptor gene family, also enhanced the expression of α-fetoprotein and albumin gene in McA-RH 8994 cells, but had no effect on α-fetoprotein gene expression in McA-RH 7777 cells. In contrast to the effect of dexamethasone on the α-fetoprotein gene expression, which requires ongoing protein synthesis, cycloheximide, a protein synthesis inhibitor, enhanced the effect of retinoic acid. Actinomycin D inhibited the retinoic acid mediated increase in α-fetoprotein and albumin mRNA expression. Since the McA-RH 8994 cells did not express retinoic acid receptor β mRNA, the observed regulatory effects of retinoic acid on α-fetoprotein and albumin gene expression were not mediated through retinoic acid receptor β. We also conclude that the regulation was at the level of transcription and that retinoic acid and dexamethasone probably regulate the expression of liver specific genes through different mechanisms.

Abundance of mRNAs encoding urea cycle enzymes in fetal and neonatal mouse liver

The relative abundances of mRNAs encoding the five urea cycle enzymes during development of mouse liver have been determined and compared with those of mRNAs encoding four other liver-specific proteins (phospho enolpyruvate carboxykinase, tyrosine aminotransferase, α-fetoprotein, and albumin). Urea cycle enzyme mRNAs in fetal liver are expressed at 2–14% of the abundance in adult liver as early as 6 days before birth. Expression of the urea cycle enzyme mRNAs is not coordinate during the fetal and neonatal period. However, profiles of three urea cycle enzyme mRNAs are quite similar to that of α-fetoprotein mRNA, suggesting the possibility of a common response to regulatory signals during fetal development. With the exception of ornithine transcarbamylase mRNA, the urea cycle enzyme mRNAs have been shown previously to be inducible by cAMP and glucocorticoids. However, only argininosuccinate lyase mRNA exhibits any significant change in abundance at birth, resembling postnatal expression of tyrosine aminotransferase mRNA. The results indicate that the urea cycle enzyme mRNAs are potentially useful markers for elucidating various features of hepatocyte differentiation in mammals.

Establishment of a rat placental cell line expressing characteristics of extraembryonic membranes

A cell line was derived from midgestation chorioallantoic placental explants of the outbred Holtzman rat. The cell line was found to express characteristics of extraembryonic membranes and to grow when introduced into allogeneic hosts. Growth in allogeneic hosts was detected following intraperitoneal injection of the cells but not following subcutaneous injection. The transplanted cells grew as cystic structures free in the peritoneum and as solid masses adhered to various abdominal organs. Cystic structures had a homogeneous morphology consisting of an epithelial-like cell layer surrounding a fluid-filled sac. Solid masses had a heterogeneous morphology, containing parts resembling normal components of the extraembryonic membranes (trophoblast, parietal, and visceral yolk sacs). Biochemical analysis of the placenta-derived cell line and transplanted structures derived from the cell line indicated that the cells had the potential to produce a variety of proteins characteristic of extraembryonic tissues. Cultured cells and both types of in vivo transplants produced the basement membrane protein, laminin. Peritoneal cystic structures also contained α-fetoprotein mRNA and very high levels of c-fos mRNA. Solid masses demonstrated elevated alkaline phosphatase activity, a marker of trophoblast cells. Cells grown in vitro expressed elevated c- myc mRNA levels, whereas, c- myc mRNA levels were reduced in the in vivo transplants. The behavior of the cell line in vitro and following in vivo transplantation suggests it contains elements capable of differentiation toward various components of the extraembryonic membranes. The results indicate that the rat placental cell line will be valuable for future studies on the differentiation of trophoblast cells and other components of the extraembryonic membranes.