Tsung-Sheng Su

Evidence of autocrine regulation in human hepatoma cell lines

Human hepatoma cell lines were studied for the expression of platelet-derived growth factor (PDGF), insulin-like growth factor-I (IGF-I) and their receptors at the mRNA level. Transcripts of PDGF were consistently detected in these cell lines. In addition, some cell lines also expressed PDGF receptor RNA. Moreover, RNA of IGF-I and its receptor were detected in every cell line examined. These results suggest that autocrine regulation may be an important mechanism for the maintenance of the transformed state of human hepatoma cells.

Selective suppression of insulin-induced proliferation of cultured human hepatoma cells by somatostatin.

The effects of somatostatin (SRIF), insulin, and triiodothyronine (T3) on the growth of human hepatoma cells were investigated on the well-differentiated human hepatoma cell line Hep3B. Results showed that both insulin and T3 can stimulate cell growth of serum starved Hep3B cells at physiological concentrations. SRIF alone showed little growth-promoting activity. When added concurrently with insulin, however, SRIF suppressed the insulin-induced cell proliferation in a dose-dependent manner. On the other hand, SRIF had no inhibitory effect on T3-induced cell proliferation. SRIF is labile in the medium, with a half-life of about 2 h during culture incubation. SRIF did not disturb the insulin binding to its surface receptors nor inhibit the insulin-dependent receptor kinase activity of Hep3B cells in vitro. These results suggest that postreceptor regulation may be involved. The selective suppression by SRIF of insulin-induced cell growth provides an unique approach to the study of insulin actions on proliferation of human hepatoma cells.

Expression of c-myc gene in human hepatoma

The level of c-myc transcript was examined in liver samples from seven hepatoma patients. Transcripts were detected in all the normal liver parts examined; in contrast, in two hepatoma parts, there was a dramatic reduction in c-myc transcripts. The restriction enzyme pattern of c-myc gene appeared the same among samples. The data suggest that c-myc gene expression might not be required for the maintenance of the tumor state in human liver carcinogenesis.

Expression of class I and class II major histocompatibility antigens on human hepatocellular carcinoma.

Previous reports indicate that human hepatocytes do not express class I and class II MHC antigens. Our analyses on 10 human hepatocellular carcinoma (HCC) cell lines by immunofluorescence tests and RIA, demonstrate that all the human HCC cell lines tested express class I MHC antigens and among them, three poorly differentiated human HCC cell lines also express class II MHC antigens. Results of immunoprecipitation and/or Western blotting experiments indicate similarity in the chemical nature of both the class I and class II MHC antigens expressed by the human HCC cell lines and by a human B lymphoblastoid cell line Raji. Furthermore, a new variant form of class I antigen was detected in some of these HCC cell lines. Immunohistochemical studies of HCC tissues using the peroxidase-antiperoxidase staining method indicated that class I and class II antigens were detectable in 7 out of 11 and 3 out of 11 HCC tissues from patients, respectively. The availability of MHC class I antigen-positive cultured HCC cell lines, including the poorly differentiated lines that also express MHC class II antigen, has provided us with interesting models to study the relationship between expression of MHC antigen and transformation and differentiation of human hepatocytes. These studies will also allow us some insight into the role of MHC class I and class II antigen in the immunosensitivity and immunogenicity of HCC cells to the host-immune response.

Evidence that mutational activation of theras genes may not be involved in aflatoxin B1-induced human hepatocarcinogenesis, based on sequence analysis of theras andp53 genes

Exposure to aflatoxin B1 (AFB1) is one of the risk factors for developing hepatoma. In rats, activation of the ras gene is a prevalent event in AFB1‐induced hepatocarcinogenesis. It is not clear whether a similar event occurs in humans. By analysis of codon 249 of the p53 gene, six of 36 human hepatoma samples were found to show a G→T transversion, suggesting that AFB1 may be a risk factor for hepatocarcinogenesis. However, analysis at codons 12, 13, and 61 in the ras family genes revealed a A→T transversion at codon 61 of the N‐ras gene in a single tumor. Apparently, ras activation is rare in human hepatoma, and the mutation detected might not be induced by AFB1. This suggests that activation of the ras gene may not be a major event in AFB1‐related human hepatocarcinogenesis. Mol. Carcinog. 26:69–73, 1999.

Study of restriction fragment length polymorphisms at the human phenylalamine hydroxylase locus and evaluation of its potential application in prenatal diagnosis of phenylketonuria in Chinese

SummaryUsing a human phenylalanine hydroxylase cDNA probe, the restriction fragment length polymorphisms at the human phenylalanine hydroxylase locus have been determined with the restriction enzymes BglII, PvuII, EcoRI+BamHI, MspI, XmnI, HindIII and EcoRV. The frequency of the observed heterozygosity of the restriction site polymorphisms at this locus in a Chinese population is approximately 54%, which is significantly lower than that in Caucasians. No DNA rearrangement or deletion of the phenylalanine hydroxylase locus was detected among mutant phenylalanine hydroxylase genes in seven Chinese classical phenylketonuria (PKU) families. Haplotype analysis of these seven families revealed that the mutant alleles belonged to five different haplotypes, i.e. haplotype 4, 11 and three unreported haplotypes. The majority of normal and mutant phenylalanine hydroxylase genes are confined to hyplotype 4. These results indicate that approximately 42% of Chinese PKU families are informative for prenatal diagnosis of PKU when eight restriction sites linked to the phenylalanine hydroxylase locus are examined.

Phenylketonuria mutation in Chinese haplotype 44 identical with haplotype 2 mutation in northern-European Caucasians

SummaryDNA amplification with the polymerase chain reaction was employed to identify the phenylketonuria (PKU) mutation in Chinese PKU families. The amplified DNA was hybridized with oligonucleotides corresponding to the two most common mutant alleles, i.e., mutations associated with PKU haplotype 2 and 3 among Caucasians of northern-European ancestry. The results of analysis demonstrate that the mutation in Chinese haplotype 44 is a single-base substitution corresponding to the mutation associated with haplotype 2 in Caucasians, whereas the mutations of the phenylalanine hydroxylase gene in haplotypes 4, 7, 11 and 28 among Chinese do not correlate with either of the two mutations identified in northern-European Caucasians.

Identification of a missense phenylketonuria mutation at codon 408 in Chinese

SummaryA single base transition of G to A at codon 408 of the phenylalanine hydroxylase gene is identified. This missense mutation results in the substitution of Arg408 for Gln408 (R408Q) and accounts for about 5% of phenylketonuria (PKU) chromosomes among Chinese. This mutation is in linkage disequilibrium with restriction fragment length polymorphism haplotype 4. In addition, another mutation (R408W), at the same codon and prevalent on haplotype 2 PKU chromosomes in Caucasians, is identified in a PKU allele of haplotype 41. Previously, this mutation has been observed on a haplotype 44 background in Chinese PKU patients.

A transgenic approach to study argininosuccinate synthetase gene expression

BackgroundArgininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed.ResultsTwo lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene.ConclusionsWe demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene.

Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse model

BackgroundArgininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter.ResultsWe established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples.ConclusionsThe BAC transgenic mouse model described is a valuable tool for studying ASS gene expression. Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing arginine-depleting agents and for studies of the role of ASS in tumorigenesis.