Expression of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas.

BACKGROUND: Cholangiocellular carcinomas and gallbladder carcinomas are highly aggressive tumours with a poor prognosis and are generally regarded as chemoresistant tumours. Overexpression of ATP-binding cassette transporters of the multidrug resistance protein (MDR) and multidrug resistance-related protein (MRP) family in cancer cells is a major cause for the multidrug resistance phenotype in vitro and in vivo. To further define the role of MRP family members in biliary tract cancer, we studied the expression and localization of MRP2 and MRP3 in cholangiocellular carcinomas and gallbladder carcinomas. MATERIALS AND METHODS: The expression and cellular localization of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas and gallbladder carcinomas were analysed by immunohistochemistry using isoform-specific antibodies. Expression of MRP isoforms was studied in vitro in Mz-ChA-1 cells derived from gallbladder adenocarcinoma by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and immunofluorescence microscopy. RESULTS: Mz-ChA-1 cells constitutively expressed MDR P-glycoproteins, MRP1, MRP2 and MRP3 by RT-PCR, immunoblotting and immunofluorescence microscopy. MRP2 and MRP3 are expressed in the respective apical and basolateral membrane domains. MRP3 was the predominant MRP isoform in gallbladder carcinomas (93%) and cholangiocellular carcinomas (57%), whereas MRP2 expression was detected in only 29% of gallbladder carcinomas and was undetectable in cholangiocellular carcinomas. CONCLUSIONS: Our findings suggest that the intrinsic multidrug resistance of cholangiocellular and gallbladder carcinomas seems to be independent of MRP2 expression while the expression of MRP3 may contribute to the MDR phenotype.

ATP and the processivity of Xenopus laevis DNA polymerase alpha.

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase alpha from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase alpha to DNA and conclude that ATP increases the processivity of the enzyme.

Tissue distribution and cDNA cloning of a human fatty acid transport protein (hsFATP4).

Fatty acid uptake is partly controlled by the FATP gene family, of which at least five members are known in mice. Using the mmFATP1 cDNA as hybridization probe, a 1.6 kb partial cDNA clone was isolated from a human heart cDNA library. With 5' and 3' RACE procedures, the complete cDNA was isolated. Sequence comparisons with its mouse homologues identified this clone as hsFATP4.

Organization of the immunity region immI of bacteriophage P1 and synthesis of the P1 antirepressor.

The immI region of bacteriophage P1 includes the ant/reb gene, which encodes the antirepressor protein, and the c4 gene, which encodes a repressor molecule that negatively regulates antirepressor synthesis. The antirepressor interferes with the activity of the P1 repressor of lytic function, the product of the c1 gene. We have determined the DNA sequences of the immI region of P1 wild-type and the mutants virs, ant16, ant17, and reb22. Using suitable P1 immI DNA subfragments cloned into a vector of the T7 bacteriophage RNA polymerase expression system the antirepressor protein(s) was overproduced. On the basis of positions of immI mutations and the sizes of ant gene products, the following organizational feature of the P1 immI region is suggested: (1) the genes c4 and ant are cotranscribed in that order from the same promoter in the clockwise direction of the P1 genetic map; (2) an open reading frame for an unknown gene is located in between c4 and ant; (3) the site at which the c4 repressor acts is located within the c4 structural gene; (4) two antirepressor proteins of molecular weights 42,000 and 32,000 are encoded by a single open reading frame, with the smaller protein initiating at an in-frame start codon; (5) transcription of immI is regulated via a c1-controlled operator, Op51, indicating a communication between the immunity systems immC and immI.

The antirepressor of phage P1. Isolation and interaction with the C1 repressor of P1 and P7.

Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in-frame start codon. Another open reading frame, icd, 5' upstream of and overlapping ant1 is required for ant1 expression. Using appropriate ant gene-carrying plasmids we have overproduced and purified Ant1/2 in the form of a protein complex and Ant2 as a single protein. Sequence analysis confirmed the N-terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N-terminal methionine is missing in the Ant2 protein. Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co-precipitate with the Ant1/2 complex but not with Ant2 protein alone. The results suggest that the antirepressor may exert its C1-inactivating function by a direct protein-protein interaction.

Cloning, expression, and characterization of the icd gene in the immI operon of bacteriophage P1.

The immI operon of P1 contains the genes c4, icd (formerly called orfx), and ant which are constitutively transcribed in that order from a single promoter, P51b. C4 is an antisense RNA which is processed from the precursor transcript. C4 RNA acts as a translational repressor of icd, thereby also inhibiting antirepressor (ant) synthesis. We have cloned the icd and the overlapping icd and ant genes. We show, by means of plasmid deletion analysis, that icd is translationally coupled to ant. An internal in-frame deletion of icd making up 65% of the codons still allows antirepressor synthesis at a reduced rate, indicating that a functionally active icd gene product is dispensable for ant expression. We identify the product of the icd gene as a 7.3-kDa protein which interferes with cell division. The results suggest that constitutive expression of icd, in the absence of a functionally active antirepressor, prevents P1 lysogen formation because of its detrimental effect on the host cell.

A dnaB-like protein of Pseudomonas aeruginosa.

A dnaB-like protein from P. aeruginosa was purified to near homogeneity using as an assay the immunoprecipitation by E. coli dnaB antiserum in a solid-phase. In the chromatographic characteristics including the affinity to immobilized ATP the dnaB-like protein of P. aeruginosa is similar to the dnaB protein of E. coli with the exception that it does not bind to heparin-Sepharose. The dnaB-like protein has a native molecular weight of about 320,000 as determined by glycerol gradient sedimentation. It consists of several identical subunits of molecular weight of 56,000 as measured in a denaturing SDS gel. Associated with the enzyme is a DNA-dependent ATPase- and helicase activity. The dnaB-like protein is similar to the E. coli dnaB protein with regard to the binding of ATP gamma S and the formation of a ternary complex consisting of the enzyme, ATP gamma S, and phi X174 DNA. However, the enzyme of P. aeruginosa is inactive in a phi X174 DNA-dependent in vitro dnaB complementation assay using an E. coli dnaBts extract.

Circular single stranded phage M13-DNA as a template for DNA synthesis in protein extracts from Xenopus laevis eggs: evidence for a eukaryotic DNA priming activity.

Unfractionated protein extracts from activated Xenopus laevis eggs contain all functions required for the chain elongation reactions in replicative DNA synthesis (A.Richter, B. Otto and R.Knippers, 1981, Nucl.Ac.Res. 9, 3793-3807). In order to further explore the DNA synthesizing capacity of this in vitro system and to obtain information on the DNA priming activity in these extracts single stranded phage M13-DNA was used as template for in vitro DNA synthesis. The main results of this investigation are: (i) single stranded circular template DNA is converted to a double stranded DNA form in an alpha-amanitin-insensitive reaction which is absolutely dependent on ribonucleoside triphosphates; (ii) the in vitro synthesized complementary strands are DNA fragments of 1000-2000 nucleotides lengths; (iii) the DNA primase activity copurifies through several column steps and sucrose gradient centrifugation with a DNA polymerase alpha. These activities may therefore be closely associated in a quarternary enzyme complex.

Determination of catalase activity at physiological hydrogen peroxide concentrations.

A method for the determination of catalase activity (EC 1.11.1.6.) in homogenates and cell suspensions is described by following the decomposition of H2O2 at physiological H2O2 levels. This first chemiluminescence assay for catalase activity is based on the reaction of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and NaOCl. The chemiluminescence of this reaction specifically depends on the H2O2 concentration and shows fast kinetics of less than 2 s. Using a flow technique, the exponential decay of H2O2 in the presence of catalase is followed down to 10(-8) M H2O2 at pH 7.4 over three orders of magnitude. At these very low H2O2 concentrations neither oxygen is liberated in gaseous form nor enzyme inactivation or loss of cell viability is observed. Addition of the catalase inhibitor NaN3 completely inhibits H2O2 decomposition. Since the method is not influenced by sulfhydryl and amino group containing compounds, it is especially suited for crude tissue homogenates and suspensions of intact cells. Interestingly, application to cell suspensions shows that intact human erythrocytes and rat hepatocytes exhibit only 5.8 and 1.9% of catalase activity when compared to homogenized cells. These data suggest that the diffusion of H2O2 through membranes is lower than that assumed so far.

Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation.

A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.

Pathogenesis of genetic haemochromatosis.

Genetic haemochromatosis is an autosomal recessive inherited iron overload disease. The genetic defect and the underlying metabolic error are not known. Several observations indicate that the 2-4-fold increase of iron absorption is due to a regulatory defect of a membrane iron transport system in duodenal mucosal cells. The key pathophysiologic factor may be the increase of gut-derived non-transferrin bound iron liganded to low-molecular mass organic molecules. A putative membrane carrier protein for non-transferrin bound iron was identified and preliminary data suggest its enrichment in plasma membranes of human mucosal cells as well as in liver and other organs which are affected in genetic haemochromatosis. Cellular accumulation of ionic iron leads to peroxidative decomposition of organelle membrane phospholipids with the consequence of cell degeneration and cell death. Impairment of organ function and structural alterations such as cirrhosis of the liver are clinical manifestations.

Direct evidence for catalase as the predominant H2O2 -removing enzyme in human erythrocytes.

Decomposition of hydrogen peroxide (H2O2 ) at physiological levels was studied in human erythrocytes by means of a recently developed sensitive H2O2 assay. The exponential decay of H2O2 in the presence of purified erythrocyte catalase was followed down to 10(-9) mol/L H2O2 at pH 7.4. H2O2 decomposition by purified erythrocyte glutathione peroxidase (GPO) could be directly observed down to 10(-7) mol/L H2O2 . No enzyme inhibition was observed at these low H2O2 concentrations. Catalase and GPO activities can be determined separately in a titrated mixture of purified enzymes, which simulates the conditions of H2O2 removal by the erythrocyte. Experiments with fresh human hemolysate allowed us to determine H2O2 decomposition by catalase and GPO using these enzymes in their original quantitative ratio. The different kinetics of these enzymes are shown: H2O2 decomposition by catalase depends linearly on H2O2 concentration, whereas that by GPO becomes saturated at concentrations above 10(-6) mol/L H2O2. Even at very low H2O2 concentrations GPO reaches only approximately 8% of the rate at which catalase simultaneously degrades H2O2. These data indicate an almost exclusive role for catalase in the removal of H2O2 in normal human erythrocytes.

The c1 repressor of bacteriophage P1 operator-repressor interaction of wild-type and mutant repressor proteins.

The c1 repressor gene of bacteriophage P1 and the temperature-sensitive mutants P1c1.100 and P1c1.162 was cloned into an expression vector and the repressor proteins were overproduced. A rapid purification procedure was required for the isolation of the thermolabile repressor proteins. Identification of the highly purified protein of an apparent molecular weight of 33,000 as the product of the c1 gene was verified by (i) the coincidence of partial amino acid sequences determined experimentally to that deduced from the c1 DNA sequence, and (ii) the temperature-sensitive binding to the operator DNA of the thermolabile repressor proteins. Analysis of the products of c1-c1.100 recombinant DNAs relates the thermolability to an unknown alteration in the C-terminal half of the c1.100 repressor. Binding to the operator DNA of c1 repressor is sensitive to N-ethylmaleimide. Since the only three cysteine residues are located in the C-terminal half of the repressor it is suggested that this part of the molecule is important for the binding to the operator DNA. This assumption is supported by the findings that a 14-kDa C-terminal repressor fragment obtained by cyanogen bromide cleavage retains DNA binding properties.

A newly detected class of mammalian single strand-specific DNA-binding proteins. Effects on DNA polymerase alpha-catalyzed DNA synthesis.

Using a rapid purification scheme, we have isolated from calf thymus cells two single strand-specific DNA-binding proteins, with apparent molecular masses of 48 and 61 kDa. These proteins remained undetected in earlier studies on DNA-binding proteins from calf thymus because they are extremely sensitive to proteolytic breakdown occurring during purification. The proteins are immunologically and biochemically related. They also share a number of functional properties. Both proteins bind noncooperatively to single-stranded DNA and almost totally ignore double-stranded DNA. Both proteins stimulate DNA synthesis catalyzed by mammalian DNA polymerase alpha in the presence of an excess of "activated" DNA as primer-template. The stimulation factor was between 2 and 100 depending on the protein/DNA ratio. We have analyzed this effect in more detail with specifically primed single-stranded phage fd DNA as template and concluded that the proteins block nonproductive polymerase-binding sites on single-stranded DNA sequences, thereby increasing the probability for an association of polymerase with 3'-OH primer ends.

Characterization and partial purification of a ferrireductase from human duodenal microvillus membranes.

Reduction of ferric iron in the presence of HuTu 80 cells or duodenal microvillus membranes (MVMs) was investigated. With both systems, NADH-dependent reduction of Fe3+/NTA (nitrilotriacetic acid) was demonstrated, using the ferrous iron chelator ferrozine. Uptake of Fe3+ from Fe3+/NTA by HuTu 80 cells was strongly inhibited by addition of ferrozine, indicating that Fe2+ is the substrate for the iron uptake system. With isolated plasma membranes it is shown that the reductase activity is sensitive to trypsin and incubation at 65 degrees C. The reductase activity could be extracted from the plasma membrane and partially purified by ammonium sulphate precipitation and isoelectric focusing. From the purification and inhibition characteristics we conclude that reduction of ferric iron on the surface of duodenal plasma membranes is catalysed by a membrane protein.

The lytic replicon of bacteriophage P1 is controlled by an antisense RNA.

The lytic replicon of phage P1 is used for DNA replication during the lytic cycle. It comprises about 2% of the P1 genome and contains the P1 C1 repressor-controlled operator-promoter element Op53.P53 and the kilA and the repL genes, in that order. Transcription of the lytic replicon of P53 and synthesis of the product of repL, but not kilA, are required for replicon function. We have identified an additional promoter, termed P53as (antisense), at the 5'-end of the kilA gene from which a 180 base transcript is constitutively synthesized and in the opposite direction to the P53 transcript. By using a promoter probe plasmid we show that transcription from P53 is strongly repressed by the C1 repressor, whereas that of P53as remains unaffected. Accordingly, the C1 repressor inhibits binding of Escherichia coli RNA polymerase to P53, but not to P53as, as shown by electron microscopy. Under non-repressed conditions transcription from P53 appears to be inhibited by P53as activity and vice versa. An inhibitory effect of P53as on the P1 lytic replicon was revealed by the construction and characterization of a P53as promoter-down mutant. Under non-repressed conditions transcription of repL and, as a consequence, replication of the plasmid is strongly enhanced when P53as is inactive. The results suggest a regulatory role for P53as on the P1 lytic replicon.

Effect of tauroursodeoxycholic acid on bile-acid-induced apoptosis and cytolysis in rat hepatocytes.

BACKGROUND/AIMS: In cholestatic liver disease, bile acids may initiate or aggravate hepatocellular damage. Cellular necrosis and cell death may be due to detergent effects of bile acids, but apoptosis may also play a role. In cholestasis, the conditions determining either apoptotic or cytolytic cell death are still unclear. Primary rat hepatocytes in culture represent a suitable model to study bile-acid-induced liver damage. METHODS: Glycochenodeoxycholic acid, a hydrophobic bile acid, was used to induce cell damage. Tauroursodeoxycholic acid, a hydrophilic bile acid, served as substrate to study possible protective effects of such compounds. To study the time and concentration dependency of bile-acid-induced cytolysis and apoptosis, morphologic alterations, hepatocellular enzyme release and nucleosomal DNA fragmentation were evaluated. RESULTS: Bile-acid-induced cytolysis, as indicated by hepatocellular enzyme release and by morphologic signs of membrane destruction, increased with concentration and time. Addition of tauroursodeoxycholic acid to the incubation medium reduced cytolysis significantly, indicating a direct hepatoprotective effect of this bile acid against the detergent action of hydrophobic bile acids. In contrast to cytolysis, apoptosis with DNA fragmentation was induced by low concentrations of glycochenodeoxycholic acid a few hours after incubation. Coincubation with tauroursodeoxycholic acid in equimolar concentrations significantly reduced apoptosis, indicating another direct hepatoprotective effect of tauroursodeoxycholic acid. CONCLUSIONS: It seems likely that in severe cholestasis, bile-acid-induced injury of hepatocytes is due mainly to cytolysis, whereas in moderately severe cholestasis apoptosis represents the predominant mechanism of bile acid toxicity. Tauroursodeoxycholic acid may reduce both bile-acid-induced apoptosis and cytolysis.

Downregulation of ileal bile acid absorption in bile-duct-ligated rats.

BACKGROUND/AIMS: Accumulation of toxic bile acids in cholestasis contributes to liver injury and depends on their synthesis, secretion and intestinal absorption. In the present study, we investigated the effect of cholestasis on the active ileal absorption of bile acids in vivo and the adaptation of transporters involved in ileal bile acid absorption. METHODS: Male Wistar rats underwent ligation of the common bile duct or biliary diversion. Sham-operated rats served as controls. Active ileal bile acid absorption of taurocholate was measured by an intestinal perfusion technique. Transporter mRNA levels of the Na+/bile acid cotransporting protein (IBAT), ileal lipid binding protein (ILBP) and organic anion transporter subtype 3 (Oatp3) and protein expression of IBAT and ILBP were determined in the distal ileum. RESULTS: After bile duct ligation the intestinal absorption rates of taurocholate were lower (p<0.05) and after biliary diversion absorption rates were higher compared to sham-operated animals (p<0.05). The absorption rates were inversely correlated to serum bile acid concentrations. Levels of IBAT-, ILBP- and Oatp3- mRNA were not different between the groups. However, in cholestatic rats, the expression of the 99-kDa dimer of IBAT was decreased compared to controls (p<0.05), whereas the 46-kDa monomeric protein of IBAT and the expression of ILBP was unchanged. After biliary diversion a similar pattern of protein expression was observed, despite an increased absorption rate. CONCLUSIONS: Cholestasis leads to a decreased active ileal absorption of taurocholate. The changes in protein expression may not account for the different absorption rates. The intestinal absorption of bile acids seems to be regulated by their systemic concentration.

HFE downregulates iron uptake from transferrin and induces iron-regulatory protein activity in stably transfected cells.

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular "labile iron pool." The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.