A novel form of CYP3A from rat brain: cDNA cloning, protein characterization and gene regulation of cytochrome P450 3A9

One full length cDNA clone, designated 3aH15, was isolated from a rat brain cDNA library using a fragment of CYP3A2 cDNA as a probe. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to mouse Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported rat CYP3A sequences. Clone 3aH15 was thus named CYP3A9 by Cytochrome P450 Nomenclature Committee. CYP3A9 seems to the major CYP3A isozyme expressed in rat brain. Sexual dimorphism of the expression of CYP3A9 was shown for the first time in rat brain as well as in rat liver. CYP3A9 appears to be female specific in rat liver based on the standards proposed by Kato and Yamazoe who defined sex specific expression of P450s as being a 10-fold or higher expression level in one sex compared with the other. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Male rats treated with estrogen had a similar expression level of CYP3A9 mRNA both in the liver and brain. Ovariectomy of adult female rats drastically reduced the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. On the other hand, only a two-fold induction of CYP3A9 expression by dexamethasone was observed in male liver and no significant induction of CYP3A9 mRNA was observed in female liver or in the brains. These results suggest that estrogen may play an important role in the female specific expression of the CYP3A9 gene and that CYP3A9 gene expression is regulated differently from other CYP3A isozymes. ^ P450 3A9 recombinant protein was expressed in E. coli using the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity (10.1 nmol P450/mg protein) from solubilized fractions using two chromatographic steps. The purified P450 3A9 protein was active towards the metabolism of many clinically important drugs such as imipramine, erythromycin, benzphetamine, ethylmorphine, chlorzoxazone, cyclosporine, rapamycin, etc. in a reconstituted system containing lipid and rat NADPH-P450 reductase. Although P450 3A9 was active towards the catabolism of testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 17β-estradiol, P450 3A9 preferentially catalyzes the metabolism of progesterone to form four different hydroxylated products. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture and GSH. The possible mechanisms of the stimulatory effects of GSH on P450 3A9 activities are discussed. Sexually dimorphic expression of P450 3A9 in the brain and its involvement in many neuroactive drugs as well as neurosteroids suggest the possible role of P450 3A9 in some mental disorders and brain functions. ^

The evolution of paired domains

Pax genes are important developmental control genes. They are involved in nervous system development, organogenesis and oncogenesis. A DNA specific binding domain called the paired domain, which is well conserved during evolution, defines Pax genes. Furthermore, Pax genes are also conserved in terms of their functions. For example, the Pax-6 gene has been showed to be one of the master control genes for eye development both in Drosophila and vertebrates. All of these properties of Pax genes make them an excellent model for studying the evolution of gene function. ^ Molecular evolutionary studies of paired domain are carried out in this study. Five Pax genes from cnidarians, which are the most primitive organisms possessing a nervous system, were isolated and characterized for their DNA binding properties. By combining data obtained from Genbank and this study, the phylogenetic relationship between Pax genes was studied. It was found that Pax genes could be divided into five groups: Pax-1/9, Pax-3 /7, Pax-A, Pax-2/5/ 8/B, and Pax- 4/6. Furthermore, Pax-2/5/8/ B, Pax-A and Pax-4/6 could be clustered into a supergroup I, while Pax-1/9 and Pax-3/7 could be clustered into supergroup II. The phylogeny was also supported by studies on DNA binding properties of paired domains from different groups. A statistical method was applied to infer the critical amino acid residue substitutions between two supergroups and within the supergroup I. It was found that two amino acid residues were mainly responsible for the difference of DNA binding between two supergroups, while only one amino acid was critical for the evolution of novel DNA binding properties of Pax-4/6 group from ancestor. Evolutionary implications of these data are also discussed. ^

REGULATION OF THE EXPRESSION OF NAR OPERON ENCODING NITRATE REDUCTASE IN ESCHERICHIA COLI

The nar operon, which encodes the nitrate reductase in Escherichia coli, can be induced under anaerobic conditions without nitrate to a low level and with nitrate to a maximum level. The anaerobic formation of nitrate reductase is dependent upon the fnr gene product while the narL gene product is required for further induction by nitrate. The sequence was determined across the entire promoter and regulatory region of the nar operon. The translational start site of the first structural gene of the nar operon, narG gene, was established by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. The transcriptional start site and the level of the transcript was determined by S1 mapping procedure. One major transcript was identified which was initiated 50 base pair (bp) upstream from the translational start site of the first structural gene. The synthesis of the transcript was repressed aerobically, fully induced by nitrate anaerobically, and greatly reduced in a ${\rm Fnr\sp-}$ mutant. Deletions were created in the 5$\sp\prime$ nar regulatory sequence with either an intact nar operon or a nar::lacZ fusion. The expression of the plasmids with deletions were determined in a strain with wild type fnr and narL loci, a Fnr- mutant strain and a NarL- mutant strain. These experiments demonstrated that the $5\sp\prime$ limit of the nar operon lies at about $-210$ bp from the transcription start site. The region required for anaerobic induction by the fnr gene product is located around $-60$ bp. Two putative narL recognition sites were identified, one of which is around $-200$ and another immediately adjacent to the fnr recognition region. The deletion of the sequences around $-200$ rendered the remaining narL complex repressive and thus decreased the expression of nar operon, suggesting that the two potential narL sites interact with each other over a significant length of DNA. ^

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K-0.5 = 416 mum) for Ala-Asp compared with Ala-Ala (K-0.5 = 54 mum) and Ala-Lys (K-0.5 = 112 mum). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.

Parvovirus B19 uptake is a highly selective process controlled by VP1u, a novel determinant of viral tropism

The VP1 unique region (VP1u) of human parvovirus B19 (B19V) is the immunodominant part of the viral capsid. Originally inaccessible, the VP1u becomes exposed upon primary attachment to the globoside receptor. To study the function of the exposed VP1u in B19V uptake, we expressed this region as a recombinant protein. Here, we report that purified recombinant VP1u binds and is internalized in UT7/Epo cells. By means of truncations and specific antibodies, we identified the most N-terminal amino acid residues of VP1u as the essential region for binding and internalization. Furthermore, the recombinant VP1u was able to block B19V uptake, suggesting that the protein and the virus undertake the same internalization pathway. Assays with different erythroid and nonerythroid cell lines showed that the N-terminal VP1u binding was restricted to a few cell lines of the erythroid lineage, which were also the only cells that allowed B19V internalization and infection. These results together indicate that the N-terminal region of VP1u is responsible for the internalization of the virus and that the interacting receptor is restricted to B19V-susceptible cells. The highly selective uptake mechanism represents a novel determinant of the tropism and pathogenesis of B19V.

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

Relative positioning of classical benzodiazepines to the γ2-subunit of GABAA receptors.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60, and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors.

Accelerated discovery of novel benzodiazepine ligands by experiment-guided virtual screening.

High throughput discovery of ligand scaffolds for target proteins can accelerate development of leads and drug candidates enormously. Here we describe an innovative workflow for the discovery of high affinity ligands for the benzodiazepine-binding site on the so far not crystallized mammalian GABAA receptors. The procedure includes chemical biology techniques that may be generally applied to other proteins. Prerequisites are a ligand that can be chemically modified with cysteine-reactive groups, knowledge of amino acid residues contributing to the drug-binding pocket, and crystal structures either of proteins homologous to the target protein or, better, of the target itself. Part of the protocol is virtual screening that without additional rounds of optimization in many cases results only in low affinity ligands, even when a target protein has been crystallized. Here we show how the integration of functional data into structure-based screening dramatically improves the performance of the virtual screening. Thus, lead compounds with 14 different scaffolds were identified on the basis of an updated structural model of the diazepam-bound state of the GABAA receptor. Some of these compounds show considerable preference for the α3β2γ2 GABAA receptor subtype.

Bridged bicyclic peptides as potential drug scaffolds: synthesis, structure, protein binding and stability

Double cyclization of short linear peptides obtained by solid phase peptide synthesis was used to prepare bridged bicyclic peptides (BBPs) corresponding to the topology of bridged bicyclic alkanes such as norbornane. Diastereomeric norbornapeptides were investigated by 1H-NMR, X-ray crystallography and CD spectroscopy and found to represent rigid globular scaffolds stabilized by intramolecular backbone hydrogen bonds with scaffold geometries determined by the chirality of amino acid residues and sharing structural features of β-turns and α-helices. Proteome profiling by capture compound mass spectrometry (CCMS) led to the discovery of the norbornapeptide 27c binding selectively to calmodulin as an example of a BBP protein binder. This and other BBPs showed high stability towards proteolytic degradation in serum.

The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production

The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/β) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/β induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/β production.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

Protective effect of a germline, IL-17-neutralizing antibody in murine models of autoimmune inflammatory disease.

Monoclonal antibodies (mAbs) inhibiting cytokines have recently emerged as new drug modalities for the treatment of chronic inflammatory diseases. Interleukin-17 (IL-17) is a T-cell-derived central mediator of autoimmunity. Immunization with Qβ-IL-17, a virus-like particle based vaccine, has been shown to produce autoantibodies in mice and was effective in ameliorating disease symptoms in animal models of autoimmunity. To characterize autoantibodies induced by vaccination at the molecular level, we generated mouse mAbs specific for IL-17 and compared them to germline Ig sequences. The variable regions of a selected hypermutated high-affinity anti-IL-17 antibody differed in only three amino acid residues compared to the likely germline progenitor. An antibody, which was backmutated to germline, maintained a surprisingly high affinity (0.5 nM). The ability of the parental hypermutated antibody and the derived germline antibody to block inflammation was subsequently tested in murine models of multiple sclerosis (experimental autoimmune encephalomyelitis), arthritis (collagen-induced arthritis), and psoriasis (imiquimod-induced skin inflammation). Both antibodies were able to delay disease onset and significantly reduced disease severity. Thus, the mouse genome unexpectedly encodes for antibodies with the ability to functionally neutralize IL-17 in vivo.

Tolerance of allografts across MHC barriers by allochimeric class I MHC proteins

Class I MHC proteins have been shown to induce accelerated rejection or prolong survival of allografts in various experimental models. These immunological effects have been attributed to the highly polymorphic alpha helical regions of the extracellular portions of the class I MHC molecule. The present experiments were designed to elucidate the immunomodulatory effects of these polymorphic regions and delineate the mechanisms involved. Soluble allochimeric class I MHC proteins were produced by substituting the PVG class I MHC RT1.Ac amino acid residues within the a 1 helical region with those of the donor BN ( a 1hn-RT1.Ac), the a 2 helical region of BN ( a 2hn-RT1.Ac), and both the a 1 and a 2 helical regions (RT1.An). The class I MHC proteins were produced in an E. coli protein expression system. The a 2hn-RT1.Ac and RT1.An proteins, when administered subcutaneously into PVG hosts 7 days prior to transplantation, resulted in accelerated rejection of BN cardiac allografts. The a 1hn-RT1.Ac construct did not demonstrate such immunogenic effects. Intra-portal administration of a 1hn-RT1.Ac or RT1.An, in combination with perioperative CsA, induced tolerance to BN cardiac allografts. The a 1hn-RT1.Ac protein was able to induce tolerance in a larger majority of the PVG recipients and at a lower dose of protein when compared to the RT1.An protein. RT1.An administered orally to PVG recipients also induced long term survival of cardiac allografts. In vitro analysis revealed that lymphocytes from tolerant hosts were hyporesponsive to donor splenocytes, but responsive to 3rd party splenocytes. Evaluation of T cell cytokine expression patterns revealed that rejector PVG hosts displayed a Type I T-cell response when re-challenged with donor splenocytes, in contrast to tolerant animals that displayed a Type II T-cell response. FACS analysis of the T cells revealed that the ratio of CD4 to CD8 cells was 3:1 and was consistent in the groups tested suggesting a complex interaction between the subsets of T cells, yielding the observed results. Histologic analysis of the cardiac allografts revealed that tolerant PVG hosts maintained BN cardiac allografts without any evidence of acute or chronic rejection after 300 days post transplant. This body of work has demonstrated that the use of soluble donor/recipient allochimeric class I MHC proteins with a short peri-operative course of CsA resulted in transplant tolerance. This treatment regimen proffers a clinically relevant approach to the induction of tolerance across MHC barriers. ^

Studies of the structure and function of a fibrinogen binding MSCRAMM(RTM) from Staphylococcus epidermidis

Coagulase-negative staphylococci (CNS) are recognized as important pathogens and are particularly associated with foreign body infections. S. epidermidis accounts for approximately 75% of the infections caused by CNS. Three genes, sdrF, sdrG, and sdrH, were identified by screening a S. epidermidis genomic library with a probe encompassing the serine-aspartate dipeptide repeat-encoding region (region R) of clfA from S. aureus. SdrG has significant amino acid identity to ClfA, ClfB and other surface proteins of S. aureus. SdrG is also similar to a protein (Fbe) recently described by Nilsson, et al. (Infection and Immunity, 1998, 66:2666–73) from S. epidermidis. The N-terminal domain (A region) of SdrG was expressed as a his-tag fusion protein in E. coli. In an ELISA, this protein, rSdrG(50-597) was shown to bind specifically to fibrinogen (Fg). Western ligand blot analysis showed that SdrG binds the Bβ chain of Fg. To further characterize the rSdrG(50-597)-Fg interaction, truncates of the Fg Bβ chain were made and expressed as recombinant proteins in E. coli. SdrG was shown to bind the full-length Bβ chain (1462), as well as the N-terminal three-quarters (1-341), the N-terminal one-half (1-220) and the N-terminal one-quarter (1-95) Bβ chain constructs. rSdrG(50-597) failed to bind to the recombinant truncates that lacked the N-terminal 25 amino acid residues of this polypeptide suggesting that SdrG recognizes a site within this region of the Bβ chain. Inhibition ELISAs have shown that peptide mimetics, including β1–25, and β6–20, encompassing this 25 residue region can inhibit binding of rSdrG(50-597) to Fg coated wells. Using fluorescence polarization we were able to determine an equilibrium constant (KD) for the interaction of rSdrG(50-597) with the Fg Bβ chain peptide β1–25. The labeled peptide was shown to bind to rSdrG(50-597) with a KD of 0.14 ± 0.01μM. Because rSdrG(50-597) recognizes a site in the Fg Bβ chain close to the thrombin cleavage site, we investigated the possibility of the rSdrG(50-597) site either overlapping or lying close to this cleavage site. An ELISA showed that rSdrG(50-597) binding to thrombin-treated Fg was significantly reduced. In a clot inhibition assay rSdrG(50-597) was able to inhibit fibrin clot formation in a concentration dependent manner. Furthermore, rSdrG(50-597) was able to inhibit clot formation by preventing the release of fibrinopeptide B as determined by HPLC. To further define the interaction between rSdrG(50-597) and peptide β6–20, we utilized an alanine amino acid replacement strategy. The residues in β6–20 that appear to be important in rSdrG(50-597) binding to Fg, were confirmed by the rSdrG(273-597)-β6–20 co-crystal structure that was recently solved by our collaborators at University of Alabama-Birmingham. Additionally, rSdrG(50-597) was not able to bind to Fg from different animal species, rather it bound specifically to human Fg in an ELISA. This suggests that the sequence variation between Fg Bβ chains of different species, specifically with in the N-terminal 25 residues, affects the ability of rSdrG(50-597) binding to Fg, and this may explain why S. epidermidis is primarily a human pathogen. ^

Studies of the expression of DNA repair related genes

This dissertation examines the biological functions and the regulation of expression of DNA ligase I by studying its expression under different conditions.^ The gene expression of DNA ligase I was induced two- to four-fold in S-phase lymphoblastoid cells but was decreased to 15% of control after administration of a DNA damaging agent, 4-nitroquinoline-1-oxide. When cells were induced into differentiation, the expression level of DNA ligase I was decreased to less than 15% of that of the control cells. When the gene of DNA ligase I was examined for tissue specific expression in adult rats, high levels of DNA ligase I mRNA were observed in testis (8-fold), intermediate levels in ovary and brain (4-fold), and low levels were found in intestine, spleen, and liver (1- to 2-fold).^ In confluent cells of normal skin fibroblasts, UV irradiation induced the gene expression of DNA ligase I at 24 and 48 h. The induction of DNA ligase I gene expression requires active p53 protein. Introducing a vector containing the wild type p53 protein in the cells caused an induction of the DNA ligase I protein 24 h after the treatment.^ Our results indicate that, in addition to the regulation by phosphorylation/dephosphorylation, cellular DNA ligase I activity can be regulated at the gene transcription level, and the p53 tumor suppresser is one of the transcription factors for the DNA ligase I gene. Also, our results suggest that DNA ligase I is involved in DNA repair as well as in DNA replication.^ Also, as an early attempt to clone the human homolog of the yeast CDC9 gene which has been shown to be involved in DNA replication, DNA repair, and DNA recombination, we have identified a human gene with mRNA of 1.7 kb. This dissertation studies the gene regulation and the possible biological functions of this new human gene by examining its expression at different stages of the cell cycle, during cell differentiation, and in cellular response to DNA damage.^ The new gene that we recently identified from human cells is highly expressed in brain and reproductive organs (BRE). This BRE gene encodes an mRNA of 1.7-1.9 kb, with an open reading frame of 1,149 bp, and gives rise to a deduced polypeptide of 383 amino acid residues. No extensive homology was found between BRE and sequences from the EMBL-Gene Banks. BRE showed tissue-specific expression in adult rats. The steady state mRNA levels were high in testis (5-6 fold), ovary and brain (3-4 fold) compared to the spleen level, but low in intestine and liver (1-2 fold). The expression of this gene is responsive to DNA damage and/or retinoic acid (RA) treatment. Treatment of fibroblast cells with UV irradiation and 4-nitroquinoline-1-oxide caused more than 90% and 50% decreases in BRE mRNA, respectively. Similar decreases in BRE expression were observed after treatment of the brain glioma cell line U-251 and the promyelocytic cell line HL-60 with retinoic acid. (Abstract shortened by UMI). ^