Differential sensitivities of the MRP gene family and gamma-glutamylcysteine synthetase to prooxidants in human colorectal carcinoma cell lines with different p53 status.

1Recent molecular cloning studies have identified six members in the multidrug-resistance protein (MRP) gene family. However, the regulation of expression of these genes is largely unknown. We previously reported that expression of MRP1, encoding multidrug-resistance associated protein, and gamma-GCSh, which encodes the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS), could be up-regulated by prooxidants [Yamane et al., J Biol Chem 1998;273:31075-85]. In the present study, we investigated whether different members of the MRP family exhibit different responses to induction by prooxidants, and whether p53 status influences the levels of induction. A panel of colorectal cancer cell lines with different p53 status, i.e. HCT116 containing wild-type p53, and HT29, SW480, and Caco2 containing mutant p53, was treated with tert-butylhydroquinone (t-BHQ) and pyrrolidinedithiocarbamate (PDTC). MRP1 and gamma-GCSh mRNA levels were determined by the RNase protection assay, using gene-specific probes. We report here that induction of MRP1 and gamma-GCSh expression by these prooxidants varied among the different cell lines, and p53 mutations were not always associated with elevated levels of induction. These results suggest that the effects of p53 on the induced expression of MRP1 and gamma-GCSh depend on the environment of the cell and/or nature of p53 mutations. In an isogenic HCT116 cell line containing p53(-/-) alleles, we demonstrated that, as for MRP1, expression of MRP2 and MRP3 was induced by the prooxidants, whereas expression of MRP4 and MRP5 was not. MRP6 mRNA was not detectable. Induction of MRP2 expression by prooxidants seemed to be independent of p53 status. Our results demonstrated the differential regulation of the MRP gene family by p53 mutation under oxidative stress.

2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-kappa B pathway.

Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression. However, the underlying mechanisms are largely unknown. In this study, we demonstrated that a NF-kappa B site on the mdr1b promoter was required for this induction. Overexpression of antisense p65 and I kappa B alpha partially abolished the induction. We then delineated the pathway through which 2-AAF activates NF-kappa B. 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of I kappa B beta (but not I kappa B alpha), and increase in NF-kappa B DNA binding activity. Consistent with the idea that ROS may participate in mdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF. Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-kappa B DNA binding. Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of I kappa B beta, and subsequent activation of NF-kappa B. This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.

Induction of MRP1 and gamma-glutamylcysteine synthetase gene expression by interleukin 1beta is mediated by nitric oxide-related signalings in human colorectal cancer cells.

Treatment of human colorectal cancer cells HT29 with interleukin 1beta (IL-1beta) induces expression of the multidrug resistance protein (MRP1) gene encoding the ATP-dependent glutathione S-conjugate export (GS-X) pump and the gamma-glutamylcysteine synthetase (gamma-GCSh) gene encoding heavy (catalytic) subunit of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the biosynthesis of glutathione (GSH). The induction can be suppressed by N(G)-methyl-L-arginine, a specific inhibitor of nitric oxide synthase (NOS). These results suggest that IL-1beta-mediated MRP1 and gamma-GCSh induction involve nitric oxide (NO) -related signaling. Further supports to the involvement of NO in the induction of MRP1 and gamma-GCSh expression are made by the following observations. (i) Expression of MRP1 and gamma-GCSh genes were induced by treating the cells with NO donors, i.e., S-nitro-N-acetyl-D,L-penicillamide (SNAP) and S-nitroso-L-glutathione, in a concentration-dependent manner. (ii) Ectopic expression of inducible NOS (iNOS) activity by transfecting expressible recombinant iNOS cDNA encoding functional iNOS but not the nonfunctional version resulted in elevated expression of MRP1 and gamma-GCSh. We also demonstrated that HT-29 cells treated with either 1L-1beta or SNAP induced ceramide production, and addition of C2 or C6 ceramides into cultured HT-29 cells resulted in induction of gamma-GCSh but not MRP1 expression. Collectively, our results demonstrate that induction of MRP1 and gamma-GCSh by IL-1beta is regulated, at least in part, by an NO-related signaling, and induction of gamma-GCSh is by NO-related ceramide signaling.

Thyroid hormone influences conditional transcript elongation of the apolipoprotein A-I gene in rat liver.

Chronic administration of thyroid hormone (T3) increases apoA-I gene expression in rat liver by enhancing mRNA maturation, but reduces apoA-I mRNA synthesis to 50% of control. To gain insight into the inverse relation of mRNA maturation and mRNA synthesis, we measured transcription in livers of control and T3-treated rats (50 micrograms/100 g body weight for 7 days) by nuclear run-on assays using overlapping antisense RNA probes encompassing the apoA-I gene. In control rats, after normalization for hybridization efficiency and probe length, the hybridization signals with intron 3 probes were reduced to 45% of those obtained with exon 1 to exon 3 probes (P < 0.01) indicating transcriptional arrest or pausing close to the exon 3-intron 3 border or 450 to 650 nucleotides downstream of the transcription start site. In T3-treated rats, the elongation block was nearly twice as effective, while the rate of transcription initiation was similar to control. In contrast, the distribution of nascent transcripts across the apoA-IV gene was symmetric, and T3-treatment suppressed apoA-IV mRNA synthesis by processes operating in the 5' region such as transcription initiation. Thus, conditional transcript elongation contributes to the regulation of apoA-I gene expression in rat liver.

In vivo gene therapy for hyperlipidemia: phenotypic correction in Watanabe rabbits by hepatic delivery of the rabbit LDL receptor gene.

Elevations of plasma total or LDL cholesterol are major risk factors for cardiovascular disease. Efforts directed at preventing and treating cardiovascular disease have often focused on reducing the levels of these substances in the blood. The Watanabe Heritable Hyperlipidemic Rabbit, which has exceedingly high plasma cholesterol levels resulting from an LDL receptor deficiency, provides an excellent animal model for testing new treatments. A recombinant adenoviral vector containing the rabbit LDL receptor cDNA was administered to Watanabe rabbits. Plasma total cholesterol levels in the treated animals were reduced from 825.5 +/- 69.8 (mean +/- SD) to 247.3 +/- 61.5 mg/dl 6 d after infusion. These animals also demonstrated a 300-400% increase in plasma levels of HDL cholesterol and apo AI 10 d after treatment. As a result, the LDL:HDL ratio exhibited a dramatic decrease. Because only the rabbit LDL receptor gene was used for treatment, the results strongly suggest that the elevations of plasma HDL cholesterol and apo AI were secondary to a reduction in plasma total cholesterol in the treated animals. These results suggest an inverse relationship between plasma LDL and HDL cholesterol levels and imply that reduction of LDL cholesterol levels may have a beneficial effect on plasma HDL cholesterol.

Thyroid hormone influences the maturation of apolipoprotein A-I messenger RNA in rat liver.

Chronic administration of thyroid hormone (T3) increases apolipoprotein (apo) A-I gene expression in rat liver. That transcriptional activity of the apoA-I gene is reduced to 50% of control, whereas abundance levels of nuclear and total cellular apoA-I mRNA are increased 3-fold, implies more effective apoA-I mRNA maturation. To study hormonal effects on apoA-I RNA processing, we quantified mRNA precursors in control and T3-treated rats (50 micrograms/100 g body weight for 7 days). Northern blotting, amplification of reverse-transcribed RNA, and ribonuclease protection assays showed that the splicing pathway is branched, in that either intron 1 or intron 2 is removed first from the primary transcript, whereas intron 3 is removed last. In T3-treated rats, abundance levels of the primary transcript, the intron 1-containing precursor devoid of intron 2, the intron 2-containing precursor devoid of intron 1, the intron 3-containing precursor lacking both introns 1 and 2, and nuclear mRNA were 65, 183, 78, 195, and 268% of controls. Compared with control rats, the half-life of the intron 1-containing precursor, measured after injection of actinomycin D, was increased 2-fold in T3-treated rats. In contrast, half-lives of the primary transcript and the intron 2-containing precursor were similar in control and T3-treated rats. Ribonuclease protection assays revealed an RNA species extending from the transcription start site close to the 3' end of intron 1. The abundance of this RNA fragment, probably representing a degradation product, was 2.5-fold higher in control than in T3-treated animals (p < 0.001). Sequences of apoA-I mRNA precursors were identical in control and T3-treated rats which excluded hormonal effects on splice-site selection or post-transcriptional editing of apoA-I transcripts. Compartmental modeling of apoA-I mRNA processing suggested that chronic thyroid hormone administration enhances apoA-I mRNA maturation more than 7-fold by protecting the intron 1-containing precursor devoid of intron 2 from degradation and by facilitating the splicing of intron 1 from this precursor.

Associations of allelic differences at the A-I/C-III/A-IV gene cluster with carotid artery intima-media thickness and plasma lipid transport in hypercholesterolemic-hypertriglyceridemic humans.

Individuals with elevated levels of plasma cholesterol and triglyceride may be at higher risk for coronary artery disease than those with isolated elevations of either cholesterol or triglyceride. Sequence variation in the A-I/C-III/A-IV gene cluster has been implicated in the etiology of some disorders associated with premature atherosclerosis and/or hypertriglyceridemias with or without elevations of cholesterol. This led to the hypothesis that allelic variation at this gene locus alters plasma lipid transport and affects susceptibility for atherosclerosis. The study population, from the Atherosclerosis Risk in Communities (ARIC) Study, consisted of 50 normolipidemic individuals, 48 subjects with elevated plasma cholesterol, 47 subjects with elevated plasma triglyceride, and 123 subjects with both elevated plasma cholesterol and triglyceride who were used to evaluate associations between an Xmn I polymorphic site 2.5 kilobase pairs (kbp) upstream of the structural gene for apolipoprotein (apo) A-I, intimal-medial thickening of the extracranial carotid arteries, and several plasma lipid factors. The relative allele frequencies of the 8.3-kbp allele and the 6.6-kbp allele were .86 and .14, respectively, in the entire study population and did not differ among the lipid phenotypes. In the group with elevated plasma cholesterol and triglyceride, subjects possessing the 6.6-kbp allele exhibited a greater carotid artery intimal-medial thickness (P = .034) and higher plasma levels of apoA-I, high-density lipoprotein (HDL) cholesterol, and HDL3 cholesterol (P < .02) than subjects homozygous for the 8.3-kbp allele. In contrast, subjects with the 6.6-kbp allele displayed lower mean ratios of apolipoproteins C-II to C-III, C-II to A-IV and E to A-IV in plasma (P < .05) and a lower mean ratio of apolipoprotein C-II to C-III in the triglyceride-rich lipoproteins (P = .026). Sequence variation in or near the genes encoding apolipoproteins A-I, C-III, and A-IV may therefore identify a group of hypercholesterolemic-hypertriglyceridemic persons who are at higher risk for atherosclerosis than others with the same lipoprotein phenotype.

Altered regulation of apolipoprotein A-IV gene expression in the liver of the genetically obese Zucker rat.

Apolipoprotein (apo) A-IV, a structural component of chylomicrons and high-density lipoproteins, may play a role in the catabolism of triglyceride-rich lipoproteins and in reverse cholesterol transport. To study the regulation of apoA-IV gene expression by genetic and nutritional factors, we determined the effect of a fish oil-rich and a sucrose-rich diet on apoA-IV gene transcription and nuclear and total cellular apoA-IV mRNA abundance in livers of genetically obese, hyperlipoproteinemic (fa/fa) Zucker rats and their lean (Fa/-) littermates. In obese rats fed chow, hepatic apoA-IV gene expression was more than twofold higher than in lean rats because of a post-transcriptional mechanism. apoA-I gene expression and apoC-III mRNA levels, studied as controls, were similar in both groups. The fish oil-rich diet reduced total cellular apoA-IV mRNA abundance transcriptionally to 34 +/- 4% of basal values in lean rats, but did not alter apoA-IV gene expression in obese rats. In contrast, this diet reduced apoA-I gene expression in both lean and obese animals. The sucrose-rich diet increased apoA-IV gene expression twofold in both lean and obese rats. Thus, genetic obesity alters the response of hepatic apoA-IV gene expression to a lipid-lowering diet rich in fish oil by a mechanism affecting transcriptional regulation.

Role of thyroid hormone in the expression of apolipoprotein A-IV and C-III genes in rat liver.

The genes coding for apolipoproteins A-I, C-III, and A-IV are closely linked to one another in the rat genome. Thyroid hormone stimulates apoA-I expression in rat liver by an unusual mechanism that enhances the maturation of mRNA. This hormone also increases apoA-IV mRNA abundance by a mechanism not yet studied, and its role in the expression of apoC-III has not been defined but may be of relevance to the metabolism of triglyceride-rich lipoproteins. We therefore measured the transcriptional activity of the apoA-IV and apoC-III genes and the abundance of their nuclear RNA and total cellular mRNA in livers of control rats and rats made hyper- and hypothyroid. After a single receptor-saturating dose of triiodothyronine (3 mg/100 g body weight), apoA-IV gene transcription increased at 20 min and reached a maximum of 260% of control at 6 h. Increases of transcription were reflected in increases of nuclear and total apoA-IV mRNA levels. ApoC-III gene transcription was temporarily increased to 160% at 2 h without changes in the abundance of its nuclear or total mRNA over 24 h. Lower hormone doses (20-500 micrograms/100 g body weight) stimulated apoA-IV mRNA transcription as well, but tended to reduce transcription from the apoC-III gene. Upon chronic administration of thyroid hormone, apoA-IV transcription decreased to 55% and nuclear apoA-IV RNA levels to 87% of control. However, total cellular apoA-IV mRNA levels increased to 279% of control, implying stabilization of mRNA in the cytoplasm. ApoC-III transcription decreased to 28% of control, but abundance of nuclear and total cellular apoC-III mRNA was reduced to a lesser extent. In hypothyroid rats, apoA-IV gene expression was decreased fourfold at the transcriptional level. In contrast, apoC-III gene transcription increased to 178% of control, but the abundance of nuclear and total cellular apoC-III mRNA did not differ from control rats. Thus, thyroid hormone affects the abundance of apoA-IV mRNA by changing its synthesis and its rate of degradation and enhances the efficiency of apoC-III mRNA maturation, thereby blunting the net effect of altered mRNA synthesis on mRNA abundance.

Effect of sucrose diet on expression of apolipoprotein genes A-I, C-III and A-IV in rat liver.

A sucrose-rich diet stimulates hepatic lipogenesis and induces net production of very low density lipoproteins in the liver. To study changes of hepatic apolipoprotein gene expression in response to such a diet, we measured the mRNA abundance of apolipoproteins A-I, C-III and A-IV in livers of rats fed a sucrose-rich diet or a control diet for 3 weeks. In livers of sucrose-fed rats, the abundance of cellular and nuclear apo A-IV mRNA increased to 185% +/- 21% and 142% +/- 22% of control values (P less than 0.01), respectively. In sucrose-fed rats, the transcriptional activity of the apo A-IV gene, measured in a cell-free transcription system using isolated liver nuclei, increased to 144% +/- 23% of control (P less than 0.05). In contrast, this diet neither affected the abundance of cellular and nuclear apo A-I and apo C-III mRNA nor the transcriptional activity of these genes in liver. These results are consistent with specialization of the regulatory elements of the genes coding for apolipoproteins A-I, C-III and A-IV. Alternatively, enhanced transcription of the apo A-IV gene may preclude increased synthesis of apo A-I and/or apo C-III mRNA due to the close linkage of the three genes in the rat genome.

Transcriptional and posttranscriptional regulation of apolipoprotein E, A-I, and A-II gene expression in normal rat liver and during several pathophysiologic states.

Assessment of the relative transcription rates and mRNA steady-state levels for apolipoprotein genes E, A-I, and A-II has been performed in normal rat liver, during liver regeneration and following induction of cirrhosis, as well as in rats with inherited analbuminemia associated with hyperlipidemia. Apo E exhibits primarily transcriptional control with an additional component of posttranscriptional control, whereas Apo A-I is controlled primarily at the posttranscriptional level, thus indicating that these genes are regulated independently. The level of control for Apo A-II has not been determined, because of difficulty experienced in measuring the transcription rate of this gene. During liver regeneration, cirrhosis, and analbuminemia, there is a marked increase in the ratio of Apo A-I to Apo E mRNA, resulting from an increase in the Apo A-I mRNA steady-state level and a decrease in Apo E mRNA. These changes are similar in the three pathophysiologic states and seem to occur through a combination of transcriptional and posttranscriptional mechanisms.

Role of thyroid hormones in apolipoprotein A-I gene expression in rat liver.

To study the regulation of hepatic apo A-I gene expression, we measured synthesis and abundance of cellular apo A-I mRNA and its nuclear precursors in livers of hypothyroid and hyperthyroid rats. In hypothyroid animals, both synthesis and abundance of apo A-I mRNA was reduced to half of control values. After injection of a receptor-saturating dose of triiodothyronine into euthyroid rats, apo A-I gene transcription increased at 20 min, reached a maximum of 179% of control (P less than 0.01) at 3.5 h, and remained elevated for up to 48 h. The abundance of nuclear and total cellular apo A-I mRNA increased at 1 and 2 h, respectively, and exceeded the levels expected from enhanced transcription more than two fold at 24 h after hormone injection. Upon chronic administration of thyroid hormones, levels of nuclear and cytoplasmic apo A-I mRNA remained elevated but transcription of the apo A-I gene fell to 42% of control (P less than 0.01). Thus, thyroid hormones rapidly stimulate apo A-I gene transcription. Posttranscriptional events leading to increased stability of nuclear apo A-I RNA precursors become the principal mechanism for enhanced gene expression in chronic hyperthyroidism and may cause feedback inhibition of apo A-I gene transcription. Our results furthermore imply that the majority of hepatic nuclear apo A-I RNA precursors are degraded in euthyroid animals.

Effect of sucrose diet on apolipoprotein biosynthesis in rat liver. Increase in apolipoprotein E gene transcription.

A sucrose-rich diet stimulates the biosynthesis of very low density lipoproteins in rat liver. This diet also increases the triglyceride content of hepatic very low density lipoproteins and changes their apolipoprotein composition. To study the changes of hepatic apolipoprotein biogenesis in response to such a diet, we measured secretory rates of apolipoproteins A-I, B, and E in cultured rat hepatocytes. In cultures from rats fed the sucrose-rich diet the production of apolipoprotein E was increased 2-fold as compared to controls, whereas the production of apolipoproteins A-I and B was unchanged. The enhanced production of apolipoprotein E could be accounted for by a 2-fold increase in hepatic apolipoprotein E mRNA, as measured by slot blot hybridization. To characterize the mechanisms leading to the increase of liver apolipoprotein E mRNA levels we measured the transcriptional activity of the apolipoprotein E gene in a cell-free transcription system using isolated liver cell nuclei. Transcriptional activity of the apolipoprotein E gene was 7% that of albumin gene transcription in control animals. In rats fed a sucrose-rich diet the transcription rate of the apolipoprotein E gene increased to 140 +/- 11% of controls. There was no change in albumin gene transcription. Thus, a sucrose-rich diet enhances apolipoprotein E biosynthesis in rat liver, at least in part, by stimulating transcription of the apolipoprotein E gene.

Apolipoprotein E gene mapping and expression: localization of the structural gene to human chromosome 19 and expression of ApoE mRNA in lipoprotein- and non-lipoprotein-producing tissues.

Apolipoprotein E (apoE) binds to specific cell-surface receptors and appears to be an important determinant in lipoprotein metabolism in man. Cloned human apoE cDNA (pAE155) was used as a probe in chromosome mapping studies to detect the structural gene sequences in human--Chinese hamster cell hybrids. Southern blot analysis of HincII-digested DNAs from 13 hybrids localized the gene to human chromosome 19. This observation indicates that apoE is syntenic to at least two other genes related to lipid metabolism, those for the low-density lipoprotein (LDL) receptor (the LDLR) and apoC-II. The cloned apoE cDNA was further used to detect the presence of apoE mRNA in RNA extracts of various human and baboon tissues. Northern gel analysis using the 32P-labeled pAE155 as a probe demonstrated the presence of hybridizable apoE mRNAs in human liver and in baboon liver, intestine, spleen, kidney, adrenal gland, and brain but not in baboon skeletal muscle. The apoE mRNAs appear to be intact and migrate on an agarose gel under denaturing conditions at approximately 18 S. To assay for the biological activity of the apoE mRNAs in these tissues, they were translated in a reticulocyte lysate system in vitro. Immunoprecipitation with an apoE-specific antiserum followed by sodium dodecyl sulfate gel electrophoresis and fluorography demonstrated that immunoreactive apoE with the expected apparent size was a product of translation of mRNAs from baboon liver, intestine, kidney, spleen, and brain but not that from baboon skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal Biosynthesis of apolipoproteins in the rat: apoE and apoA-I mRNA translation and regulation.

Rat intestinal poly(A) RNA was translated in wheat germ and reticulocyte lysate systems in vitro. ApoA-I and apoE were demonstrated to be specific products by immunoprecipitation and fractionation on sodium dodecyl sulfate acrylamide gels. They were identical in size to the respective products from rat liver. In pulse-labeling studies, apoE was shown to be synthesized by slices of rat intestine in situ. Furthermore, a high cholesterol diet stimulated the synthesis of apoE and apoA-I at the pretranslational level.

Characterization, cell-free synthesis, and processing of apolipoprotein A-I of rat high-density lipoproteins.

Rat apolipoprotein A-I (apoA-I) was isolated from delipidated high-density lipoproteins by sequential chromatography on Sephacryl S-200 and Sephadex G-150 columns in guanidine buffer. The purified protein had an apparent Mr of 27 000 and was homogeneous by NaDodSO4 and urea gel electrophoresis. Its amino acid composition was similar to that previously reported by Swaney et al. [Swaney, J. B., Wraithwaite, F., & Eder, H. G. (1977) Biochemistry 16, 271-278]. Microsequencing yielded an N-terminal sequence of Asp-Glu-Pro-Pro-Val-(Ser)-Glu-. Rabbit antisera were generated against the purified rat apoA-I and were shown to be monospecific against the protein by immunodiffusion and immunoelectrophoresis. Total poly(A) RNA was isolated from the rat liver by extraction in guanidine hydrochloride buffer and oligo(dT)-cellulose chromatography. In vitro translation of the RNA was performed in both wheat germ and nuclease-treated reticulocyte lysate systems, using [35S]Met as the radioactive amino acid precursor. Immunoreactive 35S- labeled apoA-I synthesized in vitro was precipitated by a rabbit antirat apoA-I serum. It was analyzed on an NaDodSO4- acrylamide slab gel and visualized by fluorography. The in vitro product was found to have an apparent Mr of 28 500, being larger than the authentic plasma protein by approximately 1500 daltons. When translation was performed in the presence of dog pancreatic microsomal membranes, the immunoprecipitable material was cotranslationally cleaved to a product identical in size (Mr 27 000) with plasma apoA-I. Thus, we have synthesized in vitro a putative precursor to rat apoA-I, designated preapoA-I. The preapoA-I has been processed in a cell-free system to its mature plasma counterpart by the addition of exogenous microsomal membranes.