Total Liver RNA Research Articles

Characterization of tamoxifen and 4-hydroxytamoxifen glucuronidation by human UGT1A4 variants

IntroductionTamoxifen (TAM) is an antiestrogen widely used in the treatment and prevention of breast cancer in women. One of the major mechanisms of metabolism of TAM and one of its major active metabolites, 4-hydroxytamoxifen (4-OH-TAM), is via glucuronidation. In the present study, the glucuronidating activities of three common variant isoforms encoded by the human UDP-glucuronosyltransferase (UGT) 1A4 gene were examined against TAM, trans-4-OH-TAM and cis-4-OH-TAM.MethodsHPLC was used to detect glucuronide conjugates in microsomes from UGT1A4-overexpressing HK293 cells. The UGT1A4 wild-type cDNA was synthesized by RT-PCR using normal human liver total RNA. The UGT1A424Thr/48Leu and UGT1A424Pro/48Val variants were generated by site-directed mutagenesis of the pcDNA3.1/V5-His-TOPO plasmid expressing wild-type UGT1A424Pro/48Leu. Levels of UGT1A4 expression in UGT-overexpressing cell lines were measured by western blot analysis.ResultsMicrosomes from wild-type UGT1A424Pro/48Leu-overexpressing HK293 cells exhibited significant levels of activity against TAM, trans-4-OH-TAM and cis-4-OH-TAM, forming exclusively the tamoxifen quaternary ammonium glucuronide (TAM-N+-glucuronide) and the 4-hydroxytamoxifen quaternary ammonium glucuronides (trans-4-OH-TAM-N+-glucuronide and cis-4-OH-TAM-N+-glucuronide) with apparent Km values of 2.0 μM, 2.2 μM, and 2.1 μM, respectively. Higher glucuronidation activities were found by kinetic analysis for microsomes from the variant UGT1A424Pro/48Val-overexpressing cell line as compared with microsomes from wild-type UGT1A424Pro/48Leu-overexpressing cells against TAM and against both the trans and cis isomers of 4-OH-TAM. A significantly (P < 0.02) lower Km value (~1.6-fold to 1.8-fold) was observed for both 4-OH-TAM isomers, while a near-significant (P = 0.053) decrease in Km was observed for TAM for the UGT1A424Pro/48Val variant as compared with wild-type UGT1A4. The Vmax/Km ratio for the UGT1A424Pro/48Val variant was significantly (P ≤ 0.005) higher than that observed for the wild-type UGT1A4 isoform for both the trans and cis isomers of 4-OH-TAM after normalization for UGT1A4 expression by western blotting. No significant effect on enzyme kinetics was observed for the UGT1A424Thr/48Leu variant against either isomer of 4-OH-TAM or with TAM.ConclusionThese data suggest that the UGT1A4 codon 48 Leu>Val polymorphism significantly alters glucuronidation rates against TAM and its active hydroxylated metabolites, and that this polymorphism may play an important role in individual pharmacological response to TAM therapy.

Human Homologs of Ubc6p Ubiquitin-conjugating Enzyme and Phosphorylation of HsUbc6e in Response to Endoplasmic Reticulum Stress

Ubiquitin-conjugating enzyme Ubc6p is a tail-anchored protein that is localized to the cytoplasmic face of the endoplasmic reticulum (ER) membrane and has been implicated in the degradation of many misfolded membrane proteins in yeast. We have undertaken characterization studies of two human homologs, hsUbc6 and hsUbc6e. Both possess tail-anchored protein motifs, display high conservation in their catalytic domains, and are functional ubiquitin-conjugating enzymes as determined by in vitro thiol-ester assay. Both also display induction by the unfolded protein response, a feature of many ER-associated degradation (ERAD) components. Post-translational modification involving phosphorylation of hsUbc6e was observed to be ER-stress-related and dependent on signaling of the PRK-like ER kinase (PERK). The phosphorylation site was mapped to Ser-184, which resides within the uncharacterized region linking the highly conserved catalytic core and the C-terminal transmembrane domain. Phosphorylation of hsUbc6e also did not alter stability, subcellular localization, or interaction with a partner ubiquitin-protein isopeptide ligase. Assays of hsUbc6e(S184D) and hsUbc6e(S184E), which mimic the phosphorylated state, suggest that phosphorylation may reduce capacity for forming ubiquitin-enzyme thiol-esters. The occurrence of two distinct Ubc6p homologs in vertebrates, including one with phosphorylation modification in response to ER stress, emphasizes diversity in function between these Ub-conjugating enzymes during ERAD processes.

Cloning and characterization of chicken growth hormone binding protein (cGHBP)

Growth hormone (GH) is indispensable for the growth of animals and its biological activity is mediated by binding to the growth hormone receptor (GHR) [Harvey S, Scanes CG, Daughaday WH. Growth hormone. Boca Raton: CRC Press; 1995]. GHR is a transmembrane protein responsible for signal transduction upon GH binding. GH also binds to the growth hormone binding protein (GHBP) which is the soluble form of GHR extracellular domain existing in circulation. Actions of GHBP include prolongation of GH bioavailability and prevention of GH signaling system from over-stimulation. To date, little is known about the mechanisms generating the chicken GHBP (cGHBP). Elucidating the genomic structure of cGHR will provide insights into such underlying mechanisms. Using polymerase chain reaction and library screening methods, we have characterized the genomic organization of chicken GHR (cGHR). The full-length coding region of the cGHR transcript is composed of eight exons (exons 2–10), lacking a human homolog exon 3 and spans at least 71 kb on the genome. A novel transcript of size 1.2 kb was isolated from chicken liver total RNA using 5′ and 3′ rapid cDNA ends amplification (RACE). It was generated by utilizing a previously unknown polyadenylation signal located at the intron 6. Semi-quantitative reverse transcription polymerase chain reaction showed that this transcript is widely expressed in a variety of tissues. This transcript has an open reading frame comprising 203 amino acids. In vitro binding assay using ELISA demonstrated that Escherichia coli expressed recombinant protein encoded by this transcript was able to bind with chicken GH. Hence, this transcript is a potential candidate for cGHBP.

Studies on LXR- and FXR-mediated effects on cholesterol homeostasis in normal and cholic acid-depleted mice

As previously reported by us, mice with targeted disruption of the CYP8B1 gene (CYP8B1-/-) fail to produce cholic acid (CA), upregulate their bile acid synthesis, reduce the absorption of dietary cholesterol and, after cholesterol feeding, accumulate less liver cholesterol than wild-type (CYP8B1+/+) mice. In the present study, cholesterol-enriched diet (0.5%) or administration of a synthetic liver X receptor (LXR) agonist strongly upregulated CYP7A1 expression in CYP8B1-/- mice, compared to CYP8B1+/+ mice. Cholesterol-fed CYP8B1-/- mice also showed a significant rise in HDL cholesterol and increased levels of liver ABCA1 mRNA. A combined CA (0.25%)/cholesterol (0.5%) diet enhanced absorption of intestinal cholesterol in both groups of mice, increased their liver cholesterol content, and reduced their expression of CYP7A1 mRNA. The ABCG5/G8 liver mRNA was increased in both groups of mice, but cholesterol crystals were only observed in bile from the CYP8B1+/+ mice. The results demonstrate the cholesterol-sparing effects of CA: enhanced absorption and reduced conversion into bile acids. Farnesoid X receptor (FXR)-mediated suppression of CYP7A1 in mice seems to be a predominant mechanism for regulation of bile acid synthesis under normal conditions and, as confirmed, able to override LXR-mediated mechanisms. Interaction between FXR- and LXR-mediated stimuli might also regulate expression of liver ABCG5/G8.

Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4alpha (HNF4alphaDeltaL) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4alpha-floxed (HNF4alphaF/F) mice. The expression of genes involved in the hydroxylation and side chain beta-oxidation of cholesterol, including oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4alphaDeltaL mice. Cholesterol 7alpha-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4alphaDeltaL mice, whereas expression in the light cycle was not different between HNF4alphaDeltaL and HNF4alphaF/F mice. Because CYP8B1 expression was reduced in HNF4alphaDeltaL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4alphaDeltaL mice. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo.

805. Fatality in Mice Due to Oversaturation of Cellular Micro/Short Hairpin RNA Pathways

Induction of RNA interference (RNAi) by expression of short hairpin RNA (shRNA) holds significant promise for functional genomics and human gene therapy. Therefore, numerous non-viral and viral shRNA expression vectors are currently being developed and evaluated in pre-clinical models of human disease. Here we report efficient expression of shRNAs in entire murine livers from intravenously delivered Adeno-associated virus (AAV) serotype 8, carrying a novel stabilized double-stranded (sds) vector genome with the shRNA cassette flanked by an intact AAV-2 and a truncated AAV-4 terminal repeat (ITR). As opposed to conventional dsAAV with two AAV-2 ITRs, the use of heterologous repeats prevented recombination of the genome ends and thus markedly enhanced vector stability and yield. We engineered a panel of 49 distinct sdsAAV-8 vectors to express shRNAs unique in stem length (19 to 29 nucleotides), sequence, loop structure, and target (human alpha-1-antitrypsin or luciferase gene, or human Hepatitis B or C virus genome). Strikingly, their in vivo evaluation in various wildtype mice, or in animals transgenic for the targets, showed that 36 shRNAs resulted in dose-dependent and often massive liver injury. Typical observations were rises in liver enzymes, emergence of icteric sera, drops in liver-derived albumin, and as a result of the latter, frequent occurrence of ascites. Moreover, histological analyses revealed the presence of megakaryocytes, indicative of liver damage and hepatocyte repopulation. In fact, shRNA-induced liver injury was often associated with a transient RNAi effect, peaking within weeks after vector delivery and completely lost thereafter. Immunohistochemical and Southern blot analyses confirmed that shRNA expression caused individual cell death and liver regeneration with hepatocytes lacking the AAV/shRNA, explaining the transient phenotype. Most strikingly, 23 of the tested shRNA vectors ultimately caused death of the treated animals within a month after the injection. Northern blot analysis of total liver RNA showed that morbidity correlated with high shRNA expression levels, which were in turn frequently associated with down-regulation of liver-derived micro-RNAs. The latter remained stable in alternative models of liver injury, implying a global competition mechanism specifically from shRNA overexpression. In fact, subsequent in vitro and in vivo studies hinted at the karyopherin Exportin-5 as a limiting factor in the common pathway. At this point, we hypothesize that this carrier is used by shRNAs and miRNAs for nuclear export, and that its saturation with over-abundant shRNAs is a main cause for the observed liver toxicity and animal morbidity. Our findings thus strongly imply that control over intracellular shRNA expression levels will be mandatory for the clinical RNAi use, and generally underscore the inherent risks of this technology. These can likely be minimized by optimizing shRNA dose and sequence, as shown by our findings of persistent and therapeutic RNAi against human Hepatitis B virus in vivo.

Dynamic alteration of telomerase expression and its diagnostic significance in liver or peripheral blood for hepatocellular carcinoma

To investigate the dynamic alteration of telomerase expression during development of hepatocellular carcinoma (HCC) and its diagnostic implications in liver tissues or peripheral blood mononuclear cells for HCC. Dynamic expressions of liver telomerase during malignant transformation of hepatocytes were observed in Sprague-Dawly (SD) rats fed with 0.05% of 2-fluoenyacetamide (2-FAA). Total RNA and telomerase were extracted from rat or human liver tissues. The telomerase activities in livers and in circulating blood were detected by a telomeric repeat amplification protocol-enzyme-linked immunosorbent assay (TRAP-ELISA), and its diagnostic value was investigated in patients with benign or malignant liver diseases. The hepatoma model displayed the dynamic expression of hepatic telomerase during HCC development. The telomerase activities were consistent with liver total RNA levels (r = 0.83, P<0.01) at the stages of degeneration, precancerosis, and cancerization of hepatocytes. In HCC patients, the telomerase levels in HCC tissues were significantly higher than in their adjacent non-cancerous tissues, but liver total RNA levels were lower in the former than in the latter. Although the circulating telomerase of HCC patients was abnormally expressed among patients with chronic liver diseases, the telomerase activity was a non-specific marker for HCC diagnosis, because the incidence was 15.7% in normal control, 25% in chronic hepatitis, 45.9% in liver cirrhosis, and 85.2% in HCC, respectively when absorbance value of telomerase activity was more than 0.2. If the value was over 0.6, the incidence was 60% in HCC group and 0% in any of the others (P<0.01) except in two cases with liver cirrhosis. However, the combination of circulating telomerase with serum alpha-fetoprotein level could increase the positive rate and the accuracy (92.6%, 125 of 135) of HCC diagnosis. The overexpression of telomerase is associated with HCC development, and its abnormality in liver tissues or in peripheral blood could be a useful marker for diagnosis and prognosis of HCC.

Amplification of Nanogram Amounts of Total RNA by the SMART-Based PCR Method for High-Density Oligonucleotide Microarrays

The SMART (switching mechanism at the 5′ end of RNA templates of reverse transcriptase) method (1)(2), in combination with PCR, has been used to amplify minute samples obtained from sources such as laser capture microdissections and biopsies for gene expression analysis using oligonucleotide microarray gene chip technology (3)(4)(5)(6)(7). Amplifications >105-fold can be achieved in a short time. However, reports in the literature have rarely discussed the feasibility and reproducibility of PCR-based methods when the amount of starting total RNA in a sample is only a very few nanograms or less, which is often the case for clinical samples. We developed a modified SMART-based PCR protocol with which reliable and robust gene expression data can be obtained from 1 ng of starting total RNA. We also investigated sources of array data variations. The modified protocol (see Supplement 1 in the Data Supplement that accompanies the online version of this Technical Brief at http:www.clinchem.org/content/vol51/issue12) has three major changes compared with the published standard procedure (5): the volume of the reverse transcription reaction is reduced to 5 μL; 50 ng of poly(dG/dC) carrier (Sigma) is added to the reverse transcription reaction; and PowerScript reverse transcriptase (BD Biosciences Clontech) is used instead of SuperScript II reverse transcriptase (Invitrogen). When we used the modified protocol to reverse-transcribe and amplify 10, 1, 0.1, and 0.01 ng of human heart or liver total RNA (BD Biosciences Clontech), we obtained ample cDNA from one round of 23, 26, 29, and 32 PCR cycles, respectively (Fig. 1A⇓ ). The addition of poly(dG/dC) facilitates the reverse transcription of longer transcripts as indicated by larger ranges of cDNA sizes and clearer band patterns, which are important for getting consistent good array data, but it does not adversely interfere …

Effects of 3-Methylcholanthrene on Gene Expression Profiling in the Rat Using cDNA Microarray Analyses

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are bioactivated by the cytochrome P450 (P450) 1A family of enzymes to metabolites that are capable of covalently binding to DNA, a critical step in the initiation of carcinogenesis. We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P4501A expression for up to 45 days. Here, we tested the hypothesis that MC elicits persistent induction of other genes that are regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 micromol/kg) ip once daily for 4 days, and gene expression patterns were investigated using total liver RNA isolated from animals at 1, 15, and 28 days after MC withdrawal. Gene expression was studied by cDNA microarray analyses using 4608 unique clones from liver-derived expressed sequence tag (EST) libraries fortified with clones of known liver genes representing approximately 4000 genes. Several phase I (P4501A1, -1A2) and phase II [e.g., glutathione-S-transferase (GST)-M1, UDP-glucuronosyl transferases (UGT)] genes were persistently induced (3-10-fold) by MC for 15-28 days. The persistent induction of P4501A1 gene expression was confirmed by real time reverse transcriptase polymerase chain reaction (RT-PCR) experiments. MC also elicited a 5-fold persistent augmentation of acute phase genes such as orosomucoid 1 and alpha-1-acid glycoprotein (AGP), and this was accompanied by sustained liver damage and inflammation in the MC-exposed rats. In conclusion, our results strongly suggest that sustained induction of P4501A1 by MC is accompanied by persistent expression of other genes belonging to the Ah gene battery, as well as certain other genes involved in toxic responses. Elucidating the mechanisms of persistent induction of P4501A1 and other genes by MC might lead to a better understanding of the mechanisms of toxicity mediated by PAHs.

Gene Expression Profiles in Rat Liver Treated With Perfluorooctanoic Acid (PFOA)

Perfluorooctanoic acid (PFOA; Pentadecafluorooctanoic acid) is widely used in various industrial applications. It is persistent in the environment and does not appear to undergo further degradation or transformation. PFOA is found in tissues including blood of wildlife and humans; however, the environmental fate and biological effects of PFOA remain unclear. Microarray techniques of gene expression have become a powerful approach for exploring the biological effects of chemicals. Here, the Affymetrix, Inc. rat genome 230 2.0 GeneChip was used to identify alterations in gene regulation in Sprague-Dawley rats treated with five different concentrations of PFOA. Male rats were exposed by daily gavage to 1, 3, 5, 10, or 15 mg PFOA/kg, body weight (bw)/day for 21 days and at the end of the exposure, liver was isolated and total liver RNA were used for the gene chip analysis. Over 500 genes, whose expression was significantly (p < 0.0025) altered by PFOA at two-fold changes compared to control, were examined. The effects were dose-dependent with exposure to 10 mg PFOA/kg, bw/day, causing alteration in expression of the greatest number of genes (over 800). Approximately 106 genes and 38 genes were consistently up- or down-regulated, respectively, in all treatment groups. The largest categories of induced genes were those involved in transport and metabolism of lipids, particularly fatty acids. Other induced genes were involved in cell communication, adhesion, growth, apoptosis, hormone regulatory pathways, proteolysis and peptidolysis and signal transduction. The genes expression of which was suppressed were related to transport of lipids, inflammation and immunity, and especially cell adhesion. Several other genes involved in apoptosis; regulation of hormones; metabolism; and G-protein coupled receptor protein signaling pathways were significantly suppressed.

Physiological Regulation of Phospholipid Methylation Alters Plasma Homocysteine in Mice

Biological methylation reactions and homocysteine (Hcy) metabolism are intimately linked. In previous work, we have shown that phosphatidylethanolamine N-methyltransferase, an enzyme that methylates phosphatidylethanolamine to form phosphatidylcholine, plays a significant role in the regulation of plasma Hcy levels through an effect on methylation demand (Noga, A. A., Stead, L. M., Zhao, Y., Brosnan, M. E., Brosnan, J. T., and Vance, D. E. (2003) J. Biol. Chem. 278, 5952-5955). We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine. Our data show that plasma Hcy is elevated by 20-40% in mice lacking hepatic CTalpha. CTalpha-deficient hepatocytes secrete 40% more Hcy into the medium than do control hepatocytes. Liver activity of betaine:homocysteine methyltransferase and methionine adenosyltransferase are elevated in the knockout mice as a mechanism for maintaining normal hepatic S-adenosylmethionine and S-adenosylhomocysteine levels. These data suggest that phospholipid methylation in the liver is a major consumer of AdoMet and a significant source of plasma Hcy.

Protease-resistant Prion Protein Amplification Reconstituted with Partially Purified Substrates and Synthetic Polyanions

Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27-30 and PrPC molecules at a molar ratio of 1:250 yielded approximately 2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to approximately 10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27-30, PrPC, and a stimulatory polyanionic compound.

High Mobility Group Protein-B1 Interacts with Sterol Regulatory Element-binding Proteins to Enhance Their DNA Binding

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that are predominately involved in the regulation of lipogenic and cholesterogenic enzyme gene expression. To identify unknown proteins that interact with SREBP, we screened nuclear extract proteins with 35S-labeled SREBP-1 bait in Far Western blotting analysis. Using this approach, high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel SREBP interacting protein. In vitro glutathione S-transferase pull-down and in vivo coimmunoprecipitation studies confirmed an interaction between HMGB1 and both SREBP-1 and -2. The protein-protein interaction was mediated through the helix-loop-helix domain of SREBP-1, residues 309-344, and the A box of HMGB1. Furthermore, an electrophoretic mobility shift assay demonstrated that HMGB1 enhances SREBPs binding to their cognate DNA sequences. Moreover, luciferase reporter analyses, including RNA interference technique showed that HMGB1 potentiates the transcriptional activities of SREBP in cultured cells. These findings raise the intriguing possibility that HMGB1 is potentially involved in the regulation of lipogenic and cholesterogenic gene transcription.

Hypolipidemic action of the SERM acolbifene is associated with decreased liver MTP and increased SR-BI and LDL receptors

This study aimed to identify the mechanisms of the hypolipidemic action of the selective estrogen receptor modulator (SERM) acolbifene (ACOL). Four weeks of treatment with ACOL reduced fasting and postprandial plasma triglycerides (TGs), an effect associated with lower VLDL-TG secretion rate (-25%), and decreased mRNA of microsomal triglyceride transfer protein (MTP; -29%). ACOL increased liver TG concentration (+100%) and amplified the feeding-induced increase in the master lipogenic regulators sterol-regulatory element binding protein-1a (SREBP-1a) and SREBP-1c. ACOL decreased total, HDL, and non-HDL cholesterol (CHOL) by 50%. SREBP-2 mRNA and HMG-CoA reductase activity were minimally affected by ACOL. However, in the fasted state, liver concentration of scavenger receptor class B type I (SR-BI) protein, but not mRNA, was 3-fold higher in ACOL-treated than in control animals and correlated with plasma HDL-CHOL levels (r = 0.80, P < 0.002). Liver LDL receptor (LDLR) protein, but not mRNA, was increased 2-fold by ACOL, independently of the nutritional status. This study demonstrates that ACOL possesses the unique ability among SERMs to reduce VLDL-TG secretion, likely by reducing MTP expression, and strongly suggests that the robust hypocholesterolemic action of ACOL is related to increased removal of CHOL from the circulation as a consequence of enhanced liver SR-BI and LDLR abundance.

Synovial Sarcoma Translocation (SYT) Encodes a Nuclear Receptor Coactivator

We previously cloned and characterized a novel RNA-binding motif-containing coactivator, named coactivator activator (CoAA), as a thyroid hormone receptor-binding protein-interacting protein using a Sos-Ras yeast two-hybrid screening system. A database search revealed that CoAA is identical with synovial sarcoma translocation (SYT)-interacting protein. Thus, we hypothesized that SYT could also function as a coactivator. Subsequently, we isolated a cDNA encoding a larger isoform of SYT, SYT-long (SYT-L), from the brain and liver total RNA using RT-PCR. SYT-L possesses an additional 31 amino acids in its C terminus compared with SYT, suggesting that these two SYT isoforms may be expressed from two mRNAs produced by alternative splicing of a transcript from a single gene. By Northern blot analysis, we found that SYT-L mRNA is expressed in several human embryonic tissues, such as the brain, liver, and kidney. However, we could not detect SYT-L in adult tissues. Glutathione-S-transferase pull-down studies showed that SYT binds to the C-terminus of CoAA, but not to the coactivator modulator. Both isoforms of SYT function as transcriptional coactivators of nuclear hormone receptors in a ligand- and dose-dependent manner in CV-1, COS-1, and JEG-3 cells. However, the pattern of transactivation was different between SYT and SYT-L among these cells. SYT synergistically activates transcription with CoAA. In addition, SYT activates transcription through activator protein-1, suggesting that SYT may function as a general coactivator. These results indicate that SYT activates transcription, possibly through CoAA, to interact with the histone acetyltransferase complex.

Impaired maturation of myeloid progenitors in mice lacking novel Polycomb group protein MBT-1

Polycomb group (PcG) proteins participate in DNA-binding complexes with gene-repressing activity, many of which have been highlighted for their involvement in hematopoiesis. We have identified a putative PcG protein, termed MBT-1, that is associated with Rnf2, an in vivo interactor of PcG proteins. MBT-1 structurally resembles the H-L(3)MBT protein, whose deletion is predicted to be responsible for myeloid hematopoietic malignancies. The human MBT-1 gene is located on chromosome 6q23, a region frequently deleted in leukemia cells, and shows a transient expression spike in response to maturation-inducing stimuli in myeloid leukemia cells. MBT-1(-/-) myeloid progenitor cells exhibit a maturational deficiency but maintain normal proliferative activities. This results in the accumulation of immature myeloid progenitors and hence, a marked decrease of mature myeloid blood cells, causing the MBT-1(-/-) mice to die of anemia during a late embryonic stage. Together, we conclude that MBT-1 specifically regulates the maturational advancement of myeloid progenitor cells during transitions between two developmental stages. We also show that MBT-1 appears to influence myelopoiesis by transiently enhancing p57(KIP2) expression levels.

Hepcidin: Regulation in different animal models

Hepcidin in a hepatic iron regulatory protein recently discovered and is thought to be involved in iron regulation by affecting its absorption in the intestine. We used different rat models to observe the changes of hepcidin gene expression in the liver induced by damaging noxae: whole liver irradiation, CCl4 administration. The turpentine oil induced acute phase reaction model was used as reference. Changes of hepcidin gene expression were also studied in isolated rat hepatocytes treated with different cytokines. Total liver RNA was studied by real time PCR. Induction of hepcidin gene expression was found with a maximum of 9 folds at 6 hours of exposure to the X-rays. Maximal level of hepcidin specific mRNA (16 fold) was observed also after 6 hrs of CCl4 administration. In case of TO model, hepcidin expression was maximum (51 fold) after 6 hr of TO injection in muscles. A dose dependent increase of the hepcidin mRNA with maximum of 57 fold induction at 1000ng/ml dose was induced in rat hepatocytes treated with IL–6. No significant effect on hepcidin gene expression was observed after treatment of hepatocytes with IL–1ß, TNF alpha, IFN gamma or IFN alpha. Conclusion: From this data we can conclude that the local production of IL–6 could be responsible for the induction of hepcidin in different rat models of liver injury. Hepcidin may have some protective role within liver tissue.

A Novel Immunocompetent Rat Model of HCV Infection and Hepatitis

Hepatitis C virus (HCV) infects millions of people worldwide. Therapy is limited, and treatment does not produce a sustained response in the majority of patients. Development of new agents has been hampered by the lack of a convenient animal model. The aim of this study was to determine whether an immunocompetent rat, tolerized and transplanted with a human hepatoma cell line (Huh 7 cells), could be used to sustain an HCV infection. Fetal rats were tolerized in utero with 10(5) Huh 7 cells. One day after birth, rats were transplanted with 5 x 10(6) Huh 7 cells and, a week later, inoculated with HCV, genotype 1. In tolerized, transplanted, and HCV-infected rats, Huh 7 cells were found in the liver, and HCV viral replication was detected by the presence of negative strand HCV RNA. HCV levels in serum were measured at 11,000 copies/mL at week 4, peaked at 22,500 copies/mL by week 12. In tolerized, transplanted, inoculated rats, but not controls, serum alanine aminotransferase (ALT) values increased to 60 IU/L by week 4 and reached a peak of approximately 120 IU/L by week 13. Histology showed foci of mononuclear infiltrates in portal and central regions. HCV-inoculated immunocompetent rats tolerized and transplanted with Huh 7 cells support HCV gene expression, viral replication, and develop biochemical and histologic evidence of hepatitis.

Effect of the tumor promoter phenobarbital on the pattern of global gene expression in liver of connexin32-wild-type and connexin32-deficient mice

The antiepileptic drug phenobarbital (PB) is used frequently as a model tumor promoter in rodent liver. It is believed to increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. The molecular mechanism underlying this process is only partly understood but seems to require the function of connexin32 (Cx32), one of the 2 gap junction proteins expressed in hepatocytes. PB mediates transcriptional activation of various genes in liver but which of these are relevant for tumor promotion is unknown. We have used oligonucleotide microarrays to identify genes differentially modulated in expression by PB in liver of Cx32-wild-type and Cx32-null mice. Mice of both strains were kept on PB containing (0.05%) or control diet for 2 weeks. Total liver RNA was isolated from 3 mice per experimental group and reverse transcribed; cDNAs were hybridized to oligonucleotide microarrays and a gene-by-gene linear model was used for statistical analysis of data. Five genes were identified as induced or repressed in untreated Cx32-null as compared to untreated Cx32-wild-type mice. PB affected the expression of 53 genes, of which 13 code for members of Phase-I/II of drug metabolism, and 12 genes were differentially affected in expression by PB in Cx32-null as compared to Cx32-wild-type mice. Among the differentially affected genes that could be verified by quantitative RT-PCR or Western analysis were the insulin like growth factor binding protein-1, retinol dehydrogenase-6 and the Y-chromosomally located gene Dby, among which may be a candidate of relevance for PB-mediated tumor promotion.

An RT-PCR-based Strategy to Estimate Full-Length CYP2D6 mRNA Copy Number

The goal of this study is to develop an analytical tool to assess cytochrome P450 2D6 (CYP2D6) levels in the form of full-length transcripts. CYP2D6 RNA in test samples was evaluated by co-amplification with an internal RNA control in a reverse-transcribed polymerase chain reaction (RT-PCR). The internal CYP2D6 RNA control was constructed by internally deleting 474 bp of CYP2D6 RNA, allowing simultaneous amplification of the test RNA together with the internal control RNA in a single RT-PCR reaction. With sequential dilution of test RNA, the CYP2D6 mRNA transcript levels in test samples were estimated. The full-length RT-PCR strategy allowed semiquantitative assessments of CYP2D6 RNA transcripts with a sensitivity limit of 500 copies for CYP2D6 RNA transcripts, 2500 copies/microg total human liver RNA, and 10% intraday coefficient of variation (CV). In a method validation study, the CYP2D6 activity appeared to relate more closely to full-length CYP2D6 mRNA concentration than a short-sequence of CYP2D6 RNA estimated with a real-time quantitative RT-PCR assay. We have developed an efficient semiquantitative assay and demonstrated its suitability for estimating full-length CYP2D6 mRNA transcripts in cells and tissues.