Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present study the effect of all-trans-retinoic acid (RA) on nuclear RNA polymerase activity in N-methyl-N-nitrosourea (MNU)-induced mammary tumors was investigated. Three experimental protocols were used. (1) The tumor mince was incubated with 1 microM RA for 30 min at 30 degrees C; the RNA polymerase activity was measured in the purified nuclei and compared with control nuclei. (2) In order to evaluate the influence of retinoic binding protein on enzyme activity, mammary tumor nuclei were incubated with RA bound cytosolic retinoic acid binding protein complex (RA-CRABP) at 25 degrees C for 30 min. This step allows the complex to translocate into the nuclei. The enzyme activity in these nuclei was compared with the nuclei pre-incubated with buffer or cytosol. (3) Finally, the influence of the addition of RA-CRABP complex directly into the RNA polymerase reaction mixture was determined and compared with appropriate controls. Results indicated that the RNA polymerase activity in the nuclei of RA treated tissue as well as in the nuclei subsequent to the translocation step was significantly reduced. However, the addition of RA-CRABP into the reaction mixture did not alter the enzyme activity. These results suggest that alteration of RNA polymerase activity may be an essential step in the retinoid action in mammary tissues.
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PMID:Effect of all-trans-retinoic acid on nuclear RNA polymerase activity in chemically-induced rat mammary tumors. 318 28

The insulin-like growth factor (IGF) regulatory system has a major impact on bone physiology. Among the modulators of IGFs, a family of structurally related proteins, the IGF-binding proteins (IGFBPs), have been shown to either potentiate or inhibit IGF actions on bone growth. However, the regulation of IGFBP expression in bone cells is not completely understood. In the present study, the expression of IGFBP-5 was analyzed in primary osteoblastic cells (Ob cells) isolated from 22-day-old fetal rat calvariae. Treatment of Ob cells with either IGF-I or all-trans-retinoic acid (RA) caused a time- and dose-dependent increase in IGFBP-5 messenger RNA (mRNA) levels, as determined by Northern blot analysis. Stimulation of IGFBP-5 mRNA was obtained at 100 nM IGF-I between 6 and 16 h (2- to 2.5-fold) and 100 nM RA between 16 and 24 h (3- to 4-fold). Concomitant treatment of Ob cells with IGF-I and RA revealed an additive effect and a 5- to 7-fold increase in IGFBP-5 mRNA levels after 16-24 h. The effect of IGF-I and RA and their combination on IGFBP-5 transcripts was similar in confluent and subconfluent cultures of Ob cells. IGF-I and RA did not change IGFBP-5 mRNA stability in Ob cells after transcription arrest with the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole. IGF-I and RA at 100 nM elevated the levels of IGFBP-5 heterogenous nuclear RNA, measured by reverse transcription-polymerase chain reaction. The effect was similar to that observed on mRNA levels. IGFBP-5 from rat Ob cells appeared as a single band of 31 kilodaltons in both the conditioned medium and the extracellular matrix as determined by Western immunoblots. IGF-I and RA, both at 100 nM, increased IGFBP-5 by 2- to 3-fold after 24 h. In conclusion, IGF-I and RA modify the synthesis and secretion of IGFBP-5 in rat Ob cells through pathways that may involve increased transcription and elongation and/or altered processing of heterogenous nuclear RNA. Our data suggest that IGFBP-5 may play a role in the osteoblastic-differentiated function regulated by IGF-I and RA.
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PMID:Insulin-like growth factor (IGF) I and retinoic acid induce the synthesis of IGF-binding protein 5 in rat osteoblastic cells. 753 61

Retinoids and transforming growth factor-beta 1 (TGF-beta 1) reduce the transcriptional activation of matrix metalloproteinases (MMPs) and increase the expression of the specific tissue inhibitor of MMPs (TIMP-1) in fibroblasts. In contrast, all-trans-retinoic acid (retinoic acid) increases MMP expression in osteoblasts. Therefore, the mechanistic aspects of TIMP-1 regulation by retinoic acid in primary cultures of rat calvarial bone cell populations were studied and compared with those of TGF-beta 1 to determine if modulation of TIMP-1 would augment MMP expression. Retinoic acid was found to reduce TIMP-1 mRNA levels after 24 and 72 hr of culture by up to 60% in a dose-dependent manner. Maximal inhibition occurred at 10(-6) M retinoic acid with half maximal repression at approximately 5 x 10(-8) M. To determine the half life of TIMP-1 mRNA, the specific RNA polymerase II inhibitor DRB was added to cultures and the chase RNA analyzed by slot blots. TIMP-1 mRNA had a half life of approximately 14 hr and this was unaltered by retinoic acid treatment, suggesting that retinoic acid exerts its effects on TIMP-1 transcriptionally. When retinoic acid was added to cycloheximide-treated cultures TIMP-1 mRNA levels were reduced at 5 hr compared with controls. This showed that ongoing protein synthesis was not required to mediate the retinoic acid repression of TIMP-1 mRNA levels and supports the evidence that retinoic acid acts at the transcriptional level to reduce TIMP-1 expression. In contrast, TGF-beta 1 increased TIMP-1 mRNA levels by 3.5-fold at 24 hr to > 10-fold at 72 hr without alterations in mRNA stability indicating that transforming growth factor (TGF)-beta 1 also acts at the transcriptional level to upregulate TIMP-1 expression in bone cells. Thus, these studies have revealed that TIMP-1 regulation by retinoic acid is different in osteoblasts from other cells and that retinoic acid has the property of generating resorptive and formative cell phenotypes in a tissue-specific manner. In bone, reduced TIMP-1 expression would favor bone matrix degradation and bone resorption that is a characteristic action of retinoids.
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PMID:Repression of tissue inhibitor of matrix metalloproteinase expression by all-trans-retinoic acid in rat bone cell populations: comparison with transforming growth factor-beta 1. 779 Mar 89

The translocation t(15;17)(q22;q21) is seen exclusively in patients with acute promyelocytic leukemia (APL) and in the promyelocytic blast crisis of chronic myeloid leukemia (CML). This translocation juxta-poses the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor-alpha (RARA) gene on chromosome 17, resulting in the formation of a chimeric mRNA transcript. We describe a patient with the microgranular variant form of APL, with no detectable cytogenetic abnormality of either chromosomes 15 or 17, who nevertheless had juxtaposition of PML and RARA genes and expressed a chimeric transcript. Conventional cytogenetics showed the karyotype 46,XY,d-er(3)t(3;8)(p25;q12). Fluorescent in situ hybridization (FISH) with paints for chromosomes 8, 15, and 17 confirmed the presence of structurally intact chromosomes 15 and 17 and trisomy for chromosome 8q. Nevertheless, FISH using cosmid probes for PML and RARA showed their juxtaposition on one chromosome 15 homolog. Both genes were also present on their normal homologs; in addition, part of the RARA gene was still present on the remaining chromosome 17. DNA analysis by Southern blotting, performed with a variety of probes including PML, RARA and retinoic acid receptor-beta (RARB), showed a rearrangement in PML. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed the existence of hybrid transcripts of 276, 455 bp and 623 bp, from PML-RARA on the der(15) chromosome, consistent with alternate exon splicing of the long form of the transcript occurring in 50% to 60% of patients with APL. Our results show that APL patients with cytogenetically normal chromosomes 15 and 17 may, nevertheless, have involvement of both PML and RARA genes defining a subgroup of APL, t(15;17)-negative/PML-RARA-positive which is analogous to Philadelphia chromosome-negative/BCR-ABL-positive CML. In this case, the presence of chimeric transcripts suggests that treatment with all-trans RA may be warranted in APL, even in the absence of detectable cytogenetic change, showing the usefulness of RT-PCR or FISH to aid diagnosis.
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PMID:Interstitial insertion of retinoic acid receptor-alpha gene in acute promyelocytic leukemia with normal chromosomes 15 and 17. 818 Mar 90

Unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of genes bearing thyroid hormone response elements in their promoters. Binding of hormonal ligand to the receptor releases the transcriptional silencing and leads to gene activation. Previous studies showed that the silencing activity of TR is located within the C-terminal ligand-binding domain (LBD) of the receptor. To dissect the role of the LBD in receptor-mediated silencing, we used a cell-free transcription system containing HeLa nuclear extracts in which exogenously added unliganded TRbeta repressed the basal level of RNA polymerase II-driven transcription from a thyroid hormone response element-linked template. We designed competition experiments with a peptide fragment containing the entire LBD (positions 145 to 456) of TRbeta. This peptide, which lacks the DNA-binding domain, did not affect basal RNA synthesis from the thyroid hormone response element-linked promoter when added to a cell-free transcription reaction mixture. However, the addition of the LBD peptide to a reaction mixture containing TRbeta led to a complete reversal of receptor-mediated transcriptional silencing in the absence of thyroid hormone. An LBD peptide harboring point mutations, which severely impair receptor dimerization, also inhibited efficiently the silencing activity of TR, indicating that the relief of repression by the LBD was not due to the sequestration of TR or its heterodimeric partner retinoid X receptor into inactive homo- or heterodimers. We postulate that the LBD peptide competed with TR for a regulatory molecule, termed a corepressor, that exists in the HeLa nuclear extracts and is essential for efficient receptor-mediated gene repression. We have identified the region from positions 145 to 260 (the D domain) of the LBD as a potential binding site of the putative corepressor. We observed further that a peptide containing the LBD of retinoic acid receptor (RAR) competed for TR-mediated silencing, suggesting that the RAR LBD may bind to the same corepressor activity as the TR LBD. Interestingly, the RAR LBD complexed with its cognate ligand, all-trans retinoic acid, failed to compete for transcriptional silencing by TRbeta, indicating that the association of the LBD with the corepressor is ligand dependent. Finally, we provide strong biochemical evidence supporting the existence of the corepressor activity in the HeLa nuclear extracts. Our studies demonstrated that the silencing activity of TR was greatly reduced in the nuclear extracts preincubated with immobilized, hormone-free glutathione S-transferase-LBD fusion proteins, indicating that the corepressor activity was depleted from these extracts through protein-protein interactions with the LBD. Similar treatment with immobilized, hormone-bound glutathione S-transferase-LBD, on the other hand, failed to deplete the corepressor activity from the nuclear extracts, indicating that ligand binding to the LBD disrupts its interaction with the corepressor. From these results, we propose that a corepressor binds to the LBD of unliganded TR and critically influences the interaction of the receptor with the basal transcription machinery to promote silencing. Ligand binding to TR results in the release of the corepressor from the LBD and triggers the reversal of silencing by allowing the events leading to gene activation to proceed.
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PMID:Transcriptional silencing by unliganded thyroid hormone receptor beta requires a soluble corepressor that interacts with the ligand-binding domain of the receptor. 862 57

The effects of retinoic acids (RAs) on development of seminal vesicles (SVs) of neonatal mice were investigated in vitro. SVs from 0-day-old male mice were cultured for 2-6 days in serum-free, chemically defined medium containing transferrin and BSA supplemented with 5alpha-dihydrotestosterone (DHT; 10(-8) M) and insulin (10 microg/ml), alone and in combination. Before culture, SVs from 0-day-old mice consisted of an unbranched epithelium surrounded by mesenchyme. SVs cultured in medium with DHT plus insulin or DHT alone formed numerous epithelial branches after day 2 of culture, whereas epithelial branching did not occur in SVs cultured with insulin alone. All-trans-RA or 13-cis-RA (10(-9)-10(-6) M) added to medium containing DHT plus insulin or DHT alone inhibited epithelial branching in a dose-dependent manner. This inhibitory effect was reversible after removal of the retinoids from the medium on day 4 of culture. These RAs also decreased [3H]thymidine labeling indexes of both epithelium and mesenchyme of SVs cultured in medium with DHT plus insulin or DHT alone and inhibited the increase in their protein contents. 9-Cis-RA was less inhibitory than all-trans-RA or 13-cis-RA on epithelial branching, [3H]thymidine labeling indexes of epithelium and mesenchyme, and protein content of SVs cultured in medium with DHT and insulin. In the absence of DHT (insulin alone), all-trans-RA did not affect either the [3H]thymidine labeling indexes of epithelium and mesenchyme or the protein content of cultured SVs. Reverse transcriptase-PCR demonstrated strong expression of transcripts for mouse RA receptors (RARalpha, RARgamma, and RXRalpha), with lower levels of expression of RARbeta, RXRbeta, and RXRgamma in neonatal SVs. The present results indicate that RAs reversibly inhibit androgen-dependent development of neonatal mouse SVs, most likely through RARs.
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PMID:Inhibitory effects of retinoic acids on androgen-dependent development of neonatal mouse seminal vesicles in vitro. 877 Sep 10

In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic acid (ATRA), HL60 cells differentiate into granulocyte-like cells. Membrane-associated phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or phorbol myristate acetate (PMA) was upregulated by these treatments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that both hPLD1a and hPLD1b mRNAs were expressed in HL60 cells and that their expression levels increased during differentiation. hPLD2 mRNA levels rose dramatically during differentiation. These results suggest that the PLD genes undergo changes in transcriptional regulation during granulocytic differentiation of HL60 cells.
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PMID:Increased mRNA expression of phospholipase D (PLD) isozymes during granulocytic differentiation of HL60 cells. 951 45

The sites of expression in the small intestine and the function of CYP2J4, a recently identified rat cytochrome (P450) isoform found to be predominantly expressed in the small intestine, were characterized. Immunoblot analysis with a polyclonal antibody to heterologously expressed CYP2J4 revealed that expression of CYP2J4 was at the highest level in the distal duodenum and jejunum and decreased toward the ileum. Villous cells expressed higher levels of CYP2J4 than crypt cells. Isoform-specific RNA polymerase chain reaction indicated that a related P450 isoform, CYP2J3, was only a minor form in rat small intestine. Since the intestinal mucosa is exposed to high levels of dietary nutrients, we hypothesized that CYP2J4 may be active toward diet-derived factors. We determined that purified, heterologously expressed CYP2J4 is active toward all-trans- and 9-cis-retinal in reconstituted systems, producing the corresponding retinoic acids as the major products. Apparent K(m) values for the formation of retinoic acids were 54 and 49 microM, respectively, and apparent Vmax values were 20 and 21 nmol/min/nmol P450, respectively. These activities were readily inhibited by a polyclonal anti-CYP2J4 antibody. Rat enterocyte microsomes were also active with all-trans-retinal to produce all-trans-retinoic acid in the presence of NADPH, and the majority of retinoic acid synthesis activity was inhibited by the polyclonal anti-CYP2J4 antibody. These findings suggest that CYP2J4 plays a major role in intestinal microsomal metabolism of retinal to retinoic acid and may be involved in the maintenance of retinoid homeostasis in the small intestine in vivo.
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PMID:Characterization of the cytochrome P450 CYP2J4: expression in rat small intestine and role in retinoic acid biotransformation from retinal. 960 60

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an EGF family member expressed by numerous cell types that binds to EGF receptor 1 (HER-1) or 4 (HER-4) inducing mitogenic and/or chemotactic activities. Membrane-bound HB-EGF retains growth activity and adhesion capabilities and the unique property of being the receptor for diphtheria toxin (DT). The interest in studying HB-EGF in acute leukemia stems from these mitogenic, chemotactic, and receptor functions. We analyzed the expression of HB-EGF in L428, Raji, Jurkat, Karpas 299, L540, 2C8, HL-60, U937, THP-1, ML-3, and K562 cell lines and in primary blasts from 12 acute myeloid leukemia (AML) cases, by reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blot and by the evaluation of sensitivity to DT. The release of functional HB-EGF was assessed by evaluation of its proliferative effects on the HB-EGF-sensitive Balb/c 3T3 cell line. HB-EGF was expressed by all myeloid and T, but not B (L428, Raji), lymphoid cell lines tested, as well as by the majority (8 of 12) of ex vivo AML blasts. Cell lines (except for the K562 cell line) and AML blasts expressing HB-EGF mRNA underwent apoptotic death following exposure to DT, thus demonstrating the presence of the HB-EGF molecule on their membrane. Leukemic cells also released a fully functional HB-EGF molecule that was mitogenic for the Balb/c 3T3 cell line. Factors relevant to the biology of leukemic growth, such as tumor necrosis factor-alpha (TNF-alpha), 1alpha,25-(OH)2D3, and especially all-trans retinoic acid (ATRA), upregulated HB-EGF mRNA in HL-60 or ML-3 cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced HB-EGF mRNA and acquisition of sensitivity to DT in one previously HB-EGF-negative leukemia case. Moreover, the U937 and Karpas 299 cell lines expressed HER-4 mRNA. This work shows that HB-EGF is a growth factor produced by primary leukemic cells and regulated by ATRA, 1alpha, 25-(OH)2D3, and GM-CSF.
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PMID:Heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor expression by acute myeloid leukemia cells. 1002 1

Hormones such as 1 alpha, 25-dihydroxy vitamin D3 (D3), all-trans retinoic acid, and 9-cis retinoic acid stimulate differentiation of myeloid progenitor cells via their interaction with specific hormone receptors. However, the sensitivity of cells to these agents is not merely governed by the expression of their receptors and the availability of ligand to bind them. Recent studies from our group suggested that the actions of D3 and retinoids on myelopoiesis also are influenced by endogenous mechanisms involving other steroid hormones. In this study we examined the influence of local estrogen metabolism on the differentiation of HL60 cells and normal primitive myeloid progenitor cells. Quantitative thin-layer chromatography (TLC) analyses showed that HL60 and normal cells are able to generate estrone (E1) from estradiol (E2). Neither cell population generated significant amounts of E2 from E1. Reverse transcriptase polymerase chain reaction and Northern analyses confirmed that normal and leukemic myeloid progenitor cells expressed mRNA for the type I and IV isoforms of 17 beta-hydroxysteroid dehydrogenase. Conversion of E2 to E1 was upregulated within 24 hours when HL60 cells were treated with either all-trans retinoic acid or D3 at doses that induce their differentiation toward neutrophils or monocytes, respectively. Similarly, D3-induced monocyte differentiation of normal myeloid progenitor cells was associated with increased capacity to generate E1 from E2. When HL60 cells or normal myeloid progenitor cells were exposed to exogenous E1 they became more sensitive to the differentiation-inducing effects of D3. Data presented provide further evidence for the local modulation of myelopoiesis by intracrine mechanisms. In particular, our findings suggest that local metabolism of steroids by normal as well as leukemic myeloid cells influences their responsiveness to D3 and retinoids.
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PMID:Estrone potentiates myeloid cell differentiation: a role for 17 beta-hydroxysteroid dehydrogenase in modulating hemopoiesis. 1008 7


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