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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Detection of the PML/RARalpha fusion gene by RT-PCR in acute promyelocytic leukemia (APL) blasts is not only critical to commence promptly the specific therapy with all-trans retinoic acid (ATRA) or arsenic trioxide (As(2)O(3)), but also essential for the definition of PML breakpoint type and subsequent monitoring of minimal residual disease (MRD). The current PML/RARalpha amplification techniques with conventional nested PCR are laborious and time consuming, which fails to meet the requirements for rapid diagnosis of APL in clinical practice. Therefore, an easily handled RT-PCR methodology for the rapid and accurate amplification of PML/RARalpha fusion transcripts is needed. A modified one round RT-PCR protocol was described with a few variations which includes rapid extraction of high quality cellular total RNA, cDNA synthesis with random hexamer and M-MLV reverse transcriptase, optimal concentrations of MgCl(2) (1 mmol/L), PCR primers (0.4 micro mol/L) and Taq polymerase (0.01 U/ micro l), hot-start procedure, and concomitant amplification of PML/RARalpha fusion gene and RARalpha internal control under the identical thermocycle parameters. The results in 40 patients with newly diagnosed APL showed that the improved RT-PCR protocol allowed the rapid detection of PML/RARalpha fusion gene and the accurate discrimination of its transcript types, and simultaneous amplification of RARalpha internal control under the identical program in less than 6 hours. There were no false positive or negative results found with the assay. In conclusion, the assay reported here is proved to be a simple, easily handled, and highly specific procedure for the diagnosis of APL cases, particularly those requiring such urgent therapeutic intervention as ATRA or As(2)O(3) and meriting its further application in APL management.
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PMID:[Improved RT-PCR for detection of PML/RARalpha fusion gene in rapid diagnosis of acute promyelocytic leukemia]. 1470 40

The effect of all-trans retinoid acid (ATRA) on the expression of Notch1 gene by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in acute promyelocytic leukemia cells (APL), NB4, and HL-60 lacking t(15;17) was studied. The cells were treated with ATRA 0.5 microM for up to 96 hours. The increased transcript level of Notch1 was in concert with that of CD11b in NB4 cells, but not in HL-60 cells. The expression of Notch1 gene might be specific for APL cells. As Notch1 gene is involved in the differentiation and leukemogenesis in lymphoid neoplasm, observations suggest that Notch1 is involved in ATRA-modulated differentiation process in APL.
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PMID:All-trans retinoid acid increases Notch1 transcript expression in acute promyelocytic leukemia. 1505 51

Patients with acute promyelocytic leukemia (APML) with the t(11;17) translocation usually respond poorly to all-trans retinoic acid (ATRA) and chemotherapy. We describe a patient with promyelocytic leukemia zinc finger/retinoic acid receptor alpha (PLZF/RARalpha) APML who was treated with combination chemotherapy after poor response to arsenic trioxide. He achieved hematological remission in 4 weeks followed by achievement of molecular remission in 8 weeks. Four cycles of consolidation chemotherapy followed by four cycles of maintenance therapy were given over a period of 9 months. At a follow-up of 32 months after achieving hematological remission, he continues to remain in hematological and molecular remission with normal blood parameters and negative reverse transcriptase polymerase chain reaction (RT-PCR) results. Combination chemotherapy can achieve sustained remission in patients with PLZF/RARalpha APML.
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PMID:Hematological and molecular remission with combination chemotherapy in a patient with PLZF-RARalpha acute promyelocytic leukemia (APML). 1559 71

The role of all-trans retinoic acid (ATRA) in pediatric acute promyelocytic leukemia (APL) is the topic of several ongoing studies. The results of the Italian pediatric experience with the multicentric Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA)-Italian Pediatric Hematology and Oncology Group (AIEOP) "AIDA" (ATRA and idarubicin) trial are presented. Of the 983 patients with APL enrolled in this protocol between January 1993 and June 2000, 124 (13%) had younger than 18 years. Treatment consisted of ATRA and idarubicin induction followed by 3 polychemotherapy consolidation courses. Molecular response by reverse transcriptase-polymerase chain reaction (RT-PCR) was assessed after consolidation and patients who were PCR- were randomized for different maintenances. One hundred and seven children were eligible and evaluable for induction: 103 (96%) achieved a hematologically complete remission. Overt ATRA syndrome was observed in 2 patients and pseudotumor cerebri was observed in 10 patients. Ninety-four patients were evaluable for RT-PCR analysis at the end of consolidation: 91 (97%) proved PCR+ and 3 PCR-. The overall survival and event-free survival (EFS) are 89% (95% confidence interval [c.i.]: 83%-95%) and 76% (c.i.: 65%-85%), respectively, at more than 10 years. A white blood cell (WBC) count at diagnosis of greater than 10 x 10(9)/L had a significant impact on EFS (59% vs 83% at 10 years). These results highlight the efficacy and feasibility of the AIDA protocol in the pediatric APL population.
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PMID:GIMEMA-AIEOPAIDA protocol for the treatment of newly diagnosed acute promyelocytic leukemia (APL) in children. 1567 59

CMKLR1 (chemoattractant-like receptor 1) is a G-protein-coupled receptor implicated in cartilage and bone development and is expressed in organs like the parathyroid gland, brain, and lung. The receptor is also expressed in dendritic cells and in macrophages where it acts as a co-receptor for entry of HIV/SIV isolates into human CD4(+) cells. Recently, a protein named "chemerin" (also known as TIG2) was isolated from human inflammatory fluids and hemofiltrate and found to be the endogenous ligand for CMKLR1. We have previously described the genomic organization of the cmklr1 gene and characterized its promoter in mouse neuroblastoma NB4 1A3 cells. In the present study we identify a second transcript, cmklr1b, in mouse microglia BV2 cells. Cmklr1b is transcribed from an alternative promoter with a transcription start site located 6780 bp downstream of the previously identified exon 1 (cmklr1a). The cmklr1b promoter lacks a TATA box but contains two CCAAT boxes in opposite directions. 5' Deletion analysis of the promoter region in BV2 cells using a luciferase reporter gene assay indicates two regions, between 623-755 bp and 56-125 bp upstream of transcription start site, to be important for promoter function. The proximal promoter region includes both CCAAT boxes, and site-directed mutagenesis separately within these elements revealed that only the forward CCAAT element was important for transcription. Although the forward CCAAT element is essential for transcription electrophoretic mobility shift and super-shift assays demonstrated that both CCAAT elements actually bind nuclear proteins from BV2 cells and identified the binding factor as NFY. Real-time reverse transcriptase-PCR experiments of cmklr1b expression in all-trans retinoic acid (ATRA)- stimulated BV2 cells showed strong up-regulation of receptor transcript. Luciferase reporter gene assay of the promoter in ATRA-stimulated BV2 cells confirmed that transcriptional activity of the cmklr1b promoter is increased by ATRA. However, deletion analysis could not identify an ATRA-responsive element within the promoter region suggesting that gene activation is likely to occur through alternative mechanisms. The results emphasise a possible role of cmklr1 in bone modelling.
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PMID:The mouse chemerin receptor gene, mcmklr1, utilizes alternative promoters for transcription and is regulated by all-trans retinoic acid. 1579 32

Brazilian propolis obtained from honeybee hives was extracted with water or ethanol. Cell growth-inhibitory activities of these propolis extracts were found in HL-60 human myeloid leukemia cells. The extracts-induced apoptosis in the cells, which was characterized by morphological and nucleosomal DNA fragmentation analysis. The apoptosis was mainly attributed to the induction of granulocytic differentiation, which was evaluated by nitro blue tetrazolium (NBT) reducing assays and cytofluorometric analysis for the expression of cell surface marker CD11b. DNA microarray analysis was performed to examine the gene expression profiles in the propolis-treated HL-60 cells accompanied with granulocytic differentiation, which were compared with those in all-trans retinoic acid-treated cells. Several genes were up- or down-regulated. Two genes encoding S100 calcium binding protein A9 and ferritin, heavy polypeptide 1 were up-regulated, which were also confirmed by semi-quantitative reverse transcriptase-PCR (RT-PCR). Propolis-induced growth inhibition in HL-60 cells was, at least in part, due to differentiation with gene expression profiles, which are similar to those induced by all-trans retinoic acid.
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PMID:Effects of propolis on cell growth and gene expression in HL-60 cells. 1584 13

We describe a patient with acute promyelocytic leukemia (APL) and the karyotype 46,XX,i(17)(q10) with PML-RARA fusion gene detected by fluorescence in situ hybridization (FISH) and nested reverse transcriptase-polymerase chain reaction (RT-PCR). FISH using dual-color translocation probes for PML (promyelocytic leukemia) and RARA (retinoic acid receptor-alpha) showed fusion signal for PML-RARA on both arms of i(17q). The patient attained complete remission (CR) with all-trans retinoic acid treatment and became PML-RARA negative. One year later, while PML-RARA negative on FISH and RT-PCR, the patient presented with thrombocytopenia. Bone marrow examination suggested an acute monoblastic leukemia (AML-M5a) including the karyotype 46,XX,t(8;16) (p11.2;p13.3),inv(11)(p15q22 approximately q23)[11]/47,idem,+i(8)(q10)[9]. She is currently in CR. The occurrence of therapy related acute leukemia after successful therapy for APL is an emerging problem.
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PMID:Acute promyelocytic leukemia with PML-RARA fusion on i(17q) and therapy-related acute myeloid leukemia. 1589 84

The development of osteoarthritis (OA) has recently been implicated as a result of immune-mediated damage of chondrocytes and their supporting matrixes. Pro-inflammatory cytokines like interleukin (IL)-1 and tumor necrosis factor alpha (TNF-alpha) play pivotal roles in immunopathogenesis of OA. Because vitamins preserving anti-oxidative effects are suggested to provide protection in OA patients from joint damage, in the present study, we examined the effects and mechanisms of all-trans retinoic acid (t-RA) in suppressing pro-inflammatory cytokine-induced matrix metalloproteinases (MMPs) production in human chondrocytes. Chondrocytes were prepared from cartilage specimens of OA patients receiving total hip or total knee replacement. The protein concentration was measured by ELISA, the mRNA expression by reverse transcriptase-polymerase chain reaction, the protein expression by Western blotting, the transcription factor DNA-binding activity by electrophoretic mobility shift assay and the protein kinase activity by kinase assay. We showed that both MMP-1 and MMP-13 mRNA expression, protein production and enzyme activity induced by either IL-1 or TNF-alpha were suppressed by t-RA or different retinoid derivatives. The molecular investigation revealed that the t-RA-mediated suppression was likely through blocking p38 kinase and c-Jun N-terminal kinase-activator protein-1 signaling pathways. In contrast, t-RA had no effect on extracellular signal-regulated kinase activity, nuclear factor (kappa)B (NF-(kappa)B) DNA-binding activity and I(kappa)B(alpha) degradation. Furthermore, we showed that t-RA could reduce IL-1-induced TNF-alpha production in chondrocytes. Our results suggest that vitamin A may protect OA patients from pro-inflammatory cytokine-mediated damage of chondrocytes and their supporting matrixes.
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PMID:Retinoic acid blocks pro-inflammatory cytokine-induced matrix metalloproteinase production by down-regulating JNK-AP-1 signaling in human chondrocytes. 1594 54

Vascular endothelial growth factor (VEGF), a central mediator of angiogenesis, not only plays an important role in the pathogenesis of leukemia, but also is an independent prognostic factor in patients with hematologic malignancies, like those in solid tumors. However, the importance of VEGF during differentiation or apoptosis of leukemia cells remains to be elucidated. In order to assess the alternation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of NB4 acute promyelocytic leukemia cell line, and a competitor DNA fragment, VEGF gene competative template (T-VEGFDelta) was constructed by using gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) method was developed. A standard curve was obtained by co-amplification of serial dilutions of the target nulecules with constant amount of competitive template and this curve was used to detect molecular number of target gene in measuring sample. The surface expression of CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. The results showed that cQRT-PCR was a sensitive, reliable tool for analysis of VEGF gene expression with a detectable range from 1 x 10(4) to 2 x 10(5) molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3 x 10(5), 12.6 x 10(5), 3.6 x 10(5), and less than 1.0 x 10(5)/microg total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. This rapid down-regulation of VEGF gene expression, during ATRA-induced NB4 cell differentiation, was accompanied by the up-regulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR method was successtully constructed, confirming that ATRA efficiently repressed VEGF, at the same time, the ATRA might exert an antileukemic effect, other than induction of differentiation via inhibition of angiogenesis.
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PMID:[Quantitative analysis of gene expression for vascular endothelial growth factor and its application]. 1612 31

The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.
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PMID:The detection of differentiation-inducing chemicals by using green fluorescent protein expression in genetically engineered teratocarcinoma cells. 1618 Sep 84


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