The limits of the DNase I-sensitive domain of the human apolipoprotein B gene coincide with the locations of chromosomal anchorage loops and define the 5' and 3' boundaries of the gene

In eukaryotic cells, chromatin is organized as domains or loops that are generated by periodic attachment of the chromatin fiber to protein components of a nuclear matrix, or scaffold. These chromosomal loops may have a function in gene regulation. The length of the chromatin domain encompassing the human apolipoprotein B gene was studied by determining the locations of nuclear matrix attachment sites as well as the boundaries of the DNase I-sensitive domain in cells that express the gene (such as HepG2 and CaCo-2 cells) and in those that do not (HeLa cells). Three nuclear matrix attachment regions (MARs) of the human apolipoprotein B gene have been localized: a 3' -proximal MAR, between nucleotides +43,186 and +43,850; a 5' -proximal MAR, between nucleotides -2,765 and -1,801; and a 5' -distal MAR, between nucleotides -5,262 and -4,048. Both the 3' -proximal and the 5' -distal MARS were present in cells that express the gene (HepG2 and CaCo-2 cells) as well as in cells that do not (HeLa cells), whereas the 5' -proximal MAR was detected only in HepG2 cells. These MARs were located at the bases of chromosomal loops in histone-extracted nuclei in all three cell lines. Various classes of A/T-rich sequences resembling the recognition site for topoisomerase II were present within the MAR-containing fragments. The boundaries of the DNase I-sensitive domain coincide with the positions of the 3' -proximal and 5' -distal matrix attachment sites. These results suggest the existence of a 47.5-kilobase domain that represents a topologically sequestered functional unit containing the coding region and all known cis-acting regulatory elements of the human apolipoprotein B gene.     

Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene

Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.     

Insulin decreases the secretion of apoB-100 from hepatic HepG2 cells but does not decrease the secretion of apoB-48 from intestinal CaCo-2 cells

We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 µM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.     

HBV X基因的表达及在真核细胞中对内质网压力的作用

运用PCR技术获得HBx基因,分别克隆到原核表达载体pET-his和真核表达载体pcDNA3.1(-)上。重组质粒pET-his-HBx转化大肠杆菌BL21(DE3)后,IPTG诱导表达,利用Ni柱纯化后的蛋白免疫家兔,获得特异性的抗-HBx兔抗血清。重组质粒pcDNA3.1(-)-HBx分别转染HepG2和Hep3B细胞系后,经RT-PCR和Westernblot检测,证明HBx可以在这两种细胞系中表达。通过报告基因的表达研究了HBx对XBP1和GRP78启动子的激活活性,结果表明瞬时转染HBx的细胞系中,XBP1和GRP78启动子介导的荧光素酶活性比相应的对照细胞增加了3～7倍。通过RT-PCR分析证明,转染了HBx的细胞中XBP1mRNA发生了剪切。因此,可以初步推断HBx在HepG2和Hep3B细胞中的表达可以引起内质网压力反应,为进一步阐明HBx表达对内质网的影响和肝脏病原发生机制奠定了基础。     

Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG2

A total of six established human hepatoma-derived cell lines, including Hep3B, NPLC/PRF/5 (NPLC), Tong/HCC, Hep 10, huH1, and huH2, were screened for their ability to accumulate significant quantities of lipoproteins in serum-free medium. Only two cell lines, Hep3B and NPLC, secreted quantitatively significant amounts of lipoproteins. In a 24-h period the accumulated mass of apolipoproteins (apo) A-I, A-II, B, and E and albumin for Hep3B cells was 1.96, 1.01, 1.96, 1.90, and 53.2 micrograms/mg cell protein per 24 h, respectively. NPLC cells secreted no detectable albumin but the 24-h accumulated mass for apolipoproteins A-I, A-II, B, and E was 0.45, 0.05, 0.32, and 0.68 micrograms/mg cell protein per 24 h, respectively. Twenty four-hour serum-free medium of Hep3B cells contained lipoproteins corresponding to the three major density classes of plasma; percent protein distribution among the lipoprotein classes was 4%, 41%, and 56% for very low density lipoprotein ("VLDL"), low density lipoprotein ("LDL"), and high density lipoprotein ("HDL"), respectively. NPLC was unusual since most of the lipoprotein mass was in the d 1.063-1.235 g/ml range. Hep3B "LDL", compared with plasma LDL, contained elevated triglyceride, phospholipid, and free cholesterol. Nondenaturing gradient gel electrophoresis revealed that Hep3B "LDL" possessed a major component at 25.5 nm and a minor one at 18.3 nm. Immunoblots showed that the former contained only apoB while the latter possessed only apoE. Like plasma VLDL, Hep3B "VLDL" particles (30.5 nm diameter) isolated from serum-free medium contained apoB, apoC, and apoE. "HDL" harvested from Hep3B and NPLC medium were enriched in phospholipid and free cholesterol and poor cholesteryl ester which is similar to the composition of HepG2 "HDL." "HDL" from Hep3B and NPLC culture medium on gradient gel electrophoresis had peaks at 7.5, 10, and 11.9 nm which were comparable to major components found in HepG2 cell medium. Hep3B cells, in addition, possessed a particle that banded at 8.2 nm which appeared to be an apoA-II without apoA-I particle by Western blot analysis. The cell line also produced a subpopulation of larger-sized "HDL" not found in HepG2 medium. NPLC "HDL" had a distinct peak at 8.3 nm which by Western blot was an apoE-only particle. Electron microscopy revealed that "HDL" harvested from Hep3B and NPLC medium consisted of discoidal and small, spherical particles like those of HepG2. The "HDL" apolipoprotein content of each cell line was distinct from that of HepG2. ApoA-II at 35% of apolipoprotein distinguishes Hep3B "HDL" from HepG2, which contains only 10%.(ABSTRACT TRUNCATED AT 400 WORDS)     

A solution hybridization/RNase protection assay with riboprobes to determine absolute levels of apoB, A-I, and E mRNA in human hepatoma cell lines

A solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.     

Increased expression of N-acetylglucosaminyltransferase-V in human hepatoma cells by retinoic acid and 1alpha,25-dihydroxyvitamin D3

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.