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:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Because previous PCR-based methodologies for detection of minimal residual disease (MRD) in leukemia patients have been too cumbersome to allow for widespread clinical usefulness, we have employed a real-time quantitative PCR (RQ-PCR) system to develop an MRD assay for t(12;21). We initially determined the expression of the different alternatively spliced TEL-AML1 mRNAs found in t(12;21) breakpoint variants I and II. We then optimized PCR primers for the RQ-PCR system and, using the t(12;21)+ REH cell line in spiking experiments, found a linear detection of TEL-AML1 over at least five logs. Moreover, 1 malignant cell in a background of 1,000,000 normal cells could be detected. The expression of the GAPDH, ABL, and beta(2)-microglobulin (beta2M) housekeeping genes were then compared in normal donors and in leukemic patients, and the very stably expressed beta2M was selected as an internal reference gene, allowing us to compensate for variation in RNA quality and day-to-day variation. In 12 samples from t(12;21)-positive patients at diagnosis, the levels of the TEL-AML1 fusion transcripts were found to vary up to 14-fold after normalization to beta2M. Interestingly, in samples obtained from seven patients at diagnosis, during induction chemotherapy, or relapse, the level of TEL-AML1 in peripheral blood (PB) and bone marrow (BM) was found to differ only by threefold, suggesting that MRD may be evaluated in PB samples in most patients. We conclude that this assay could set new standards for t(12;21) MRD detection with its accuracy, its high throughput, and its short turnover time for samples. Genes Chromosomes Cancer 26:355-365, 1999.
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PMID:Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript. 1053 71

We have used a new single-step real-time reverse transcription polymerase chain reaction (RT-PCR) method to quantify BCR-ABL transcripts, thereby estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic transplants. In 402 samples from 172 patients, BCR-ABL expression was determined and normalized, using the GAPDH housekeeping gene product as an endogenous reference. In our real-time RT-PCR assay, serial dilutions of RNA of the K562 cell line remained positive down to 7.5 pg. The median normalized BCR-ABL amount differed significantly (P < 0.001) between the various disease stages and was 0.06% (range 0.001-1.55%), 3.2% (range 1.4-5.6%) and 21.5% (range 6.8 -827%) in 17 patients with a molecular relapse, in eight patients with a cytogenetic relapse and in 10 patients with a haematological relapse respectively.
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PMID:Estimating the relapse stage in chronic myeloid leukaemia patients after allogeneic stem cell transplantation by the amount of BCR-ABL fusion transcripts detected using a new real-time polymerase chain reaction method. 1144 4

Using a real-time RT-PCR method, we analyzed the expression of e1a2 BCR-ABL mRNA in bone marrow samples from 13 patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia (ALL) at different time points during chemotherapy and after bone marrow transplantation (BMT). The detection limit of the method, assessed using serial dilutions of ALL/MIK cells, was found to be 1:10(5), similar to what is observed for the conventional RT-nested PCR method. The e1a2 BCR-ABL values were normalized with respect to those of the housekeeping gene GAPDH. The decrease in the e1a2 BCR-ABL/GAPDH ratio after remission induction chemotherapy reflects well the response to chemotherapy and consequently correlates with the prognosis. Although molecular remission was achieved by chemotherapy alone, some patients relapsed, and the e1a2 BCR-ABL/GAPDH ratios in these cases progressively increased to the levels seen prior to hematological relapse. Long-term hematological complete remission (more than 30 months) could be achieved in cases in which a more than 4.0 log decrease in the e1a2 BCR-ABL/GAPDH ratio was obtained by chemotherapy alone, and BMT was then performed. In conclusion, real-time RT-PCR allows for an evaluation of the kinetics of e1a2 BCR-ABL/GAPDH expression during the various phases of chemotherapy or after BMT and may be effective for the indication and control of disease relapse in Ph-positive ALL patients.
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PMID:Quantification of minimal residual disease in patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia using a real-time RT-PCR assay. 1204 Apr 49

IL-6 has emerged as an important cytokine upregulated in states of insulin resistance such as type 2 diabetes. We evaluated the chronic effect of IL-6 on insulin signaling in 3T3-F442A and 3T3-L1 adipocytes. First, cells responded to a chronic treatment with IL-6 by initiating an autoactivation process that increased IL-6 secretion. Second, IL-6-treated adipocytes showed a decreased protein expression of IR-beta subunit and IRS-1 but also an inhibition of the insulin-induced activation of IR-beta, Akt/PKB, and ERK1/2. Moreover, IL-6 suppressed the insulin-induced lipogenesis and glucose transport consistent with a diminished expression of GLUT4. IL-6-treated adipocytes failed to maintain their adipocyte phenotype as shown by the downregulation of the adipogenic markers FAS, GAPDH, aP2, PPAR-gamma, and C/EBP-alpha. IL-6 also induced the expression of SOCS-3, a potential inhibitor of insulin signaling. Finally, the effects of IL-6 could be prevented by rosiglitazone, an insulin-sensitizing agent. Thus, IL-6 may play an important role in the set-up of insulin resistance in adipose cell.
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PMID:Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion and induced insulin resistance in adipocyte: prevention by rosiglitazone. 1459 24

Serum amyloid A (SAA) is known to be a precursor of amyloid A (AA) protein in AA (secondary) amyloidosis and SAA1 to be mainly involved in AA amyloidosis. We established an SAA isoform real-time quantitative RT-PCR assay and found that beta-2 microglobulin is more stable as an internal control than GAPDH and beta-actin for our system. Either IL-6 and IL-1beta or IL-6 and TNFalpha, but not IL-1beta and TNFalpha, induced the synergistic induction of SAA1 and SAA2 genes. Anti-IL-6 receptor monoclonal antibody completely inhibited the synergistic induction of SAA1 and SAA2 during triple stimulation with IL-6, IL-1beta, and TNFalpha, but, IL-1 receptor antagonist or anti-TNFalpha monoclonal antibody was only partially inhibited in HepG2, Hep3B, and PLC/PRF/5 cells. Although the SAA1 promoter has no STAT3 consensus sequence, the JAK2 inhibitor-AG490 reduced SAA1 gene expression to 30%, suggesting the involvement of STAT3. We were able to demonstrate that IL-6 plays a critical role in the synergistic induction of human SAA gene when stimulated with proinflammatory cytokines.
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PMID:IL-6 plays a critical role in the synergistic induction of human serum amyloid A (SAA) gene when stimulated with proinflammatory cytokines as analyzed with an SAA isoform real-time quantitative RT-PCR assay system. 1473 13

The present study investigates the molecular details of how arsenic trioxide inhibits preadipocyte differentiation and examines the role of Akt/PKB in regulation of differentiation and apoptosis. Continual exposure of arsenic trioxide, at the clinic achievable dosage that does not induce apoptosis, suppressed 3T3-L1 cell differentiation into fat cells by inhibiting the expression of PPARgamma and C/EBPalpha and disrupting the interaction between PPARgamma and RXRalpha, which determines the programming of the adipogenic genes. Interestingly, if we treated the cells for 12 or 24 h and then withdrew arsenic trioxide, the cells were able to differentiate to the comparable levels of untreated cells as assayed by the activity of GAPDH, the biochemical marker of preadipocyte differentiation. Long term treatment blocked the differentiation and the activity of GAPDH could not recover to the comparable levels of untreated cells. Continual exposure of arsenic trioxide caused accumulation in G2/M phase and the accumulation of p21. We found that arsenic trioxide induced the expression and the phosphorylation of Akt/PKB and it inhibited the interaction between Akt/PKB and PPARgamma . Akt/PKB inhibitor appears to block the arsenic trioxide suppression of differentiation. Our results suggested that Akt/PKB may play a role in suppression of apoptosis and negatively regulate preadipocyte differentiation.
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PMID:The role of Akt on arsenic trioxide suppression of 3T3-L1 preadipocyte differentiation. 1591 24

It is known that the extracellular matrix (ECM) is able to signal to cells and thereby direct or modulate the transcription of certain mRNAs. This signaling plays an important role in tumor invasion and metastasis, wound healing, remodeling of the ECM and cell differentiation. There are several mechanisms whereby the ECM signals cells to change their metabolism: (1) receptor molecules binding to specific domains in the ECM, (2) direct phagocytosis of the ECM molecules or domains into the cell, (3) structural changes of the ECM domains. We report the effect of an ECM containing either mutant or normal Fbn1 on the transcription levels of several collagen mRNAs. Tsk/Tsk, Tsk/+ and +/+ mouse embryonic fibroblast cell lines were used. Tsk/Tsk cells produce only mutated fibrillin-1 which arises from mRNA containing an in-frame duplication of exons 17-40. To test the effect of the ECM containing mutant Fbn1, cells of the Tsk/Tsk, Tsk/+ and the wild-type (+/+) genotype were each grown on an ECM produced by either Tsk/Tsk, Tsk/+ cells or by wild-type cells (+/+). The embryonic cells were genotyped by Northern analyses for Fbn1 and grown to confluence. The cultures were then harvested and the cells removed, leaving the matrix in the flasks. Matrices produced from Tsk/Tsk, Tsk/+ and from +/+ cells were reseeded with Tsk/Tsk cells, Tsk/+ cells or +/+ cells. The cells were plated at a confluent concentration and incubated on the matrices for 48 h, after which total RNA was harvested and cDNA generated. Real-time PCR using cDNA or Northern analyses using RNA were performed for Fbn1 and Types I, III and V collagens. The PCR and Northern results were normalized using beta-actin and GAPDH, respectively. The Northern analyses showed that the steady state levels of mRNA for Col1a1 were depressed in both Tsk/Tsk and +/+ cells when grown on the matrix produced by Tsk/Tsk cells. Real-time PCR was then performed with primers specific for Col1a2, Col3a1, Col5a1 and Col5a2. The results showed that cells with the Tsk/Tsk, Tsk/+, and +/+ genotype all had lower steady-state levels of the above 4 collagen mRNAs when grown on the matrix produced by homozygous Tsk/Tsk cells or the matrix produced by heterozygous Tsk/+ cells compared with those grown on a matrix produced by +/+ cells. We hypothesize that the mutated Fbn1 molecules with many additional EGF-calcium binding regions and TGF-beta binding domains may (1) change the homeostasis of the ECM by binding additional growth factors and/or (2) present a radically different ECM 3-dimensional architecture. Either or both of these changes could signal the cell to produce less collagen.
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PMID:Extracellular matrix containing mutated fibrillin-1 (Fbn1) down regulates Col1a1, Col1a2, Col3a1, Col5a1, and Col5a2 mRNA levels in Tsk/+ and Tsk/Tsk embryonic fibroblasts. 1658 19

Tumour-specific chromosomal rearrangements are known to create chimaeric products with the ability to generate many human cancers. hTAF(II)68-TEC (where hTAF(II)68 is human TATA-binding protein-associated factor II 68 and TEC is translocated in extraskeletal chondrosarcoma) is such a fusion product, resulting from a t(9;17) chromosomal translocation found in extraskeletal myxoid chondrosarcomas, where the hTAF(II)68 NTD (N-terminal domain) is fused to TEC protein. To identify proteins that control hTAF(II)68-TEC function, we used affinity chromatography on immobilized hTAF(II)68 (NTD) and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS and isolated a novel hTAF(II)68-TEC-interacting protein, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). GAPDH is a glycolytic enzyme that is also involved in the early steps of apoptosis, nuclear tRNA export, DNA replication, DNA repair and transcription. hTAF(II)68-TEC and GAPDH were co-immunoprecipitated from cell extracts, and glutathione S-transferase pull-down assays revealed that the C-terminus of hTAF(II)68 (NTD) was required for interaction with GAPDH. In addition, three independent regions of GAPDH (amino acids 1-66, 67-160 and 160-248) were involved in binding to hTAF(II)68 (NTD). hTAF(II)68-TEC-dependent transcription was enhanced by GAPDH, but not by a GAPDH mutant defective in hTAF(II)68-TEC binding. Moreover, a fusion of GAPDH with the GAL4 DNA-binding domain increased the promoter activity of a reporter containing GAL4 DNA-binding sites, demonstrating the presence of a transactivation domain(s) in GAPDH. The results of the present study suggest that the transactivation potential of the hTAF(II)68-TEC oncogene product is positively modulated by GAPDH.
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PMID:Regulation of oncogenic transcription factor hTAF(II)68-TEC activity by human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 1730 60

Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To address this issue, six candidate housekeeping genes including several commonly used ones (ACTB, GAPDH, 18S rRNA, TBP, SDHA and YWHAZ) were investigated for their expression stability in placentae obtained from pregnancies complicated by idiopathic FGR (n=25) and gestation-matched control pregnancies (n=25). Real-time PCR was performed using pre-validated gene expression assay kits. The geNorm program was used for gene stability measure (M) for the entire housekeeping genes in all control and FGR-affected placental samples. Results showed that GAPDH and 18S rRNA were most stable, with an average expression stability of M=0.441 and 0.443, respectively, followed by YWHAZ (M=0.472). SDHA, ACTB and TBP were the least stable housekeeping genes (M=0.495, 0.548 and 1.737, respectively). We recommend geometric averaging of two or more housekeeping genes to determine relative gene expression levels between FGR-affected and control placentae.
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PMID:GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction. 1868 3

The somatic mutation JAK2 V617F is associated with BCR-ABL1-negative myeloproliferative neoplasms. Detection of this mutation aids diagnosis of these neoplasms, and quantification of JAK2 V617F may provide a method to monitor response to therapy. For these reasons, we designed a clinical assay that uses allele-specific PCR and real-time detection with hydrolysis probes for the quantification of JAK2 V617F, wild-type JAK2, and GAPDH transcripts. Mutant and wild-type JAK2 were quantified by using external plasmid standards that contain the relevant JAK2 V617F or JAK2 sequence, respectively. We tested 55 peripheral blood specimens from patients with suspected myeloproliferative neoplasms and 55 peripheral blood specimens from patients not known to have myeloproliferative neoplasms. Low-level, nonspecific amplification was detected in reactions containing a high copy number of plasmid standards and in specimens from patients not known to have myeloproliferative neoplasms, necessitating the use of a laboratory-established mutant to wild-type cutoff. The limit of detection established by using cell line dilutions is 0.1%, and this method identified three JAK2 V617F-positive patients who were not detected by a less sensitive method. The assay characteristics and our initial evaluation indicate this method can be used for the detection and quantification of JAK2 V617F, which should be useful for diagnosis of myeloproliferative neoplasms and potentially for monitoring minimal residual disease in future trials of therapies targeted to myeloproliferative neoplasms.
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PMID:Design and evaluation of a real-time PCR assay for quantification of JAK2 V617F and wild-type JAK2 transcript levels in the clinical laboratory. 1995 96


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