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)

The Wilms tumor suppressor gene (WT1) is mutated in a number of cases of Wilms' tumor as well as in mesothelioma and leukemia. It encodes a transcription factor derived from any one of four alternate transcripts. WT1 has a restricted pattern of expression within the body and within the hemopoietic system its expression is limited to primitive leukemias and a number of leukemic cell lines. Given the overexpression of WT1 in leukemias, we have addressed the question of whether this gene is expressed within the normal hemopoietic system. Mononuclear bone marrow (BM) cells obtained from normal donors were separated by fluorescence-activated cell sorting (FACS) into "primitive" (CD34+) and "mature" (CD34-) cell populations. Total RNA extracted from these cells was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using primers based on the WT1 sequence, to examine the expression of this gene within the hemopoietic system. Phenotypic purity of cells was guaranteed by performing single-cell sorting followed by RT-PCR to define the precise cellular phenotypes that express WT1. Expression of WT1 was detected in cells bearing the CD34+ phenotype but not in those cells lacking expression of CD34. In addition, single-cell analysis revealed that expression of WT1 occurred in the candidate stem cell-containing population of hemopoietic cells which have the phenotype CD34+ CD38-. Moreover, the single-cell RT-PCR analysis also demonstrated that differential expression of alternate transcripts of WT1 occurs between hemopoietic progenitor cells with the same phenotype. In conclusion, expression of WT1 is limited to early progenitors of the blood system, which suggests that this gene plays a critical role in hemopoietic development.
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PMID:Expression of the Wilms' tumor gene (WT1) in normal hemopoiesis. 940 89

Mesothelial cells are of mesenchymal origin, although they also have epithelial characteristics. Such cells obtained from benign effusions are not terminally differentiated and can be kept in short-term cultures. These cultures grow with an either epithelial or fibroblast-like phenotype, a pattern which is stable through the early passages. Several factors have been associated with mesothelial differentiation. The Wilms' tumour susceptibility gene 1 (WT1) is expressed during transitions of mesenchyme to epithelial tissues, as in the embryonic kidney, and it has been suggested as a marker for the mesothelial lineage. The proteoglycans (PGs) and hyaluronan are also differentially synthesised by epithelial and fibroblastic malignant mesothelioma cells and the cell surface PGs seem to indicate phenotypic differentiation even in epithelial tumours. To investigate how the epithelial and fibroblast-like differentiation of benign mesothelial cells was correlated to WT1, PGs and hyaluronan synthase, we studied their expression by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analyses. The expressions of these genes were all associated with a variation in phenotypic differentiation. Cell lines with epithelial morphology expressed more mRNA coding for WT1 and cell surface PGs than did the fibroblastic ones, the difference being greatest for syndecan-4 and glypican. The increase in WT1-associated mRNA was about as great as that of syndecans. Fibroblast-like cells, on the other hand, expressed substantially more of the matrix PGs versican and biglycan, while decorin expression was detected in only trace amounts in both morphological phenotypes. Hyaluronan synthase varied individually between the cell lines, although epithelial cells often expressed higher levels. The results indicate that the regulation of mesothelial differentiation is multifactorial and also involves WT1 and several PGs.
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PMID:Expression of genes coding for proteoglycans and Wilms' tumour susceptibility gene 1 (WT1) by variously differentiated benign human mesothelial cells. 1055 May 42

Differentiating desmoplastic small round cell tumor (DSRCT) from another similar small round cell tumor of childhood, the Ewing sarcoma/primitive neuroectodermal tumor (EWS/PNET), can be difficult because morphologic and immunohistochemical features overlap. We studied the predictive value of immunohistochemistry with an antibody to the C-terminal region of the Wilms tumor (WT1) protein for differentiating DSRCT from EWS/PNET in 24 malignant small round cell tumors that had been previously diagnosed as DSRCT or EWS/PNET by standard methods. We performed reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in cases with available tissue as a confirmatory measure: 6 of 13 DSRCTs were informative by RT-PCR, and 6 of 6 showed an EWS-WT1 fusion; all 13 DSRCTs showed strong, definitive nuclear staining with the WT1 antibody. All 11 EWS/PNETs were WT1 antibody negative; 7 of 11 cases classified as EWS/PNET were informative by RT-PCR, and 7 of 7 showed an EWS-FLI-1 fusion. For cases in which the morphologic and immunohistochemical features are consistent with a diagnosis of DSRCT, WT1 antibody staining predicts the EWS-WT1 translocation with high sensitivity and specificity and is, therefore, useful for differentiating DSRCT from EWS/PNET when genetic information is unavailable.
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PMID:WT1 staining reliably differentiates desmoplastic small round cell tumor from Ewing sarcoma/primitive neuroectodermal tumor. An immunohistochemical and molecular diagnostic study. 1098 34

The measurement of Wilms' tumor gene (WT1) mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) is useful in detecting minimal residual disease (MRD) in leukemia patients. In the present study, we quantified the level of WT1 mRNA in the peripheral blood and bone marrow of patients with acute myelocytic leukemia (AML) at initial onset, remission and recurrence by the use of nucleic acid sequence based amplification (NASBA), and then ascertained the clinical usefulness of this method. At initial onset, the level of WT1 mRNA in the peripheral blood was above 10(3) copies/microgram and that in the bone marrow was above 10(4) copies/microgram. The level of WT1 mRNA was decreased in cases where therapy resulted in complete remission, but it was abnormally high in recurring cases. In AML (M3) patients, the relationship between the level of WT1 mRNA and the expression of the PML-retinoic acid alpha receptor (RAR alpha) gene, assessed by fluorescence in situ hybridization (FISH), was investigated. When leukemia was in remission hematologically, the PML-RAR alpha gene was negative and the level of WT1 mRNA decreased. These findings suggest that the quantification of WT1 gene expression by competitive NASBA is useful in assessing therapeutic effects and detecting MRD.
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PMID:Quantification of WT1 mRNA by competitive NASBA in AML patients. 1150 93

The Wilms tumor gene (WT1) is expressed in blasts of patients with acute leukemia, irrespective of lineage, and WT1 nuclear protein is detectable in the majority of such blasts. Only very few physiologic hematopoietic progenitors express WT1, but the WT1 expression level of these progenitors and that of leukemic blasts are comparable. Although not specific for acute hematologic malignant diseases, continuous WT1 expression in almost all leukemic blasts strikingly contrasts to its rather transient expression in very few physiologic hematopoietic progenitors. Quantitative and semiquantitative WT1 reverse transcriptase polymerase chain reaction (RT-PCR) protocols have limitations in discriminating physiologic from pathologic overall WT1 expression levels in mononuclear cell preparations. Because of these limitations, reports conflict on the usefulness of long-term monitoring of WT1 expression in patients with acute leukemia. Real-time quantitative WT1 RT-PCR protocols, however, have been developed and tested in small series of patients with acute leukemia. Such protocols hold promise to enable evaluation of the individual treatment response (short-term monitoring) and early diagnosis of imminent relapse through the detection and long-term monitoring of minimal residual disease in patients with acute leukemia. These protocols also should facilitate the notoriously difficult distinction between eosinophilic leukemia and hypereosinophilic syndromes. Data on WT1 expression in leukemic blasts and their physiologic counterparts are discussed in light of clinical relevance.
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PMID:Wilms tumor gene (WT1) expression as a panleukemic marker. 1221 7

We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending on WT1 expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitating WT1 transcripts. Results of intraassay and interassay variability tests demonstrated that the real-time WT1 assay had high reproducibility. WT1 expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r = 0.998). Furthermore, a strong correlation was observed among WT1 transcript values normalized with 3 different control genes (beta-actin, ABL, and glyceraldehyde-3-phosphate dehydrogenase) and between relative WT1 transcript values with WT1 expression in K562 cells as the reference and absolute WT1 transcript copy numbers per microgram RNA. When WT1 expression and minor bcr-abl expression were concurrently monitored in 2 patients with bcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of a WT1 assay.
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PMID:Monitoring minimal residual disease in leukemia using real-time quantitative polymerase chain reaction for Wilms tumor gene (WT1). 1468 94

The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that is vital during development of several organs including metanephric kidneys. Despite the critical regulatory role of WT1, the pathways and mechanisms by which WT1 orchestrates development remain elusive. To identify WT1 target genes, we performed a genome-wide expression profiling analysis in cells expressing inducible WT1. We identified a number of direct WT1 target genes, including the epidermal growth factor (EGF)-family ligands epiregulin and HB-EGF, the chemokine CX3CL1, and the transcription factors SLUG and JUNB. The target genes were validated using quantitative reverse transcriptase-polymerase chain reaction, small interfering RNA knockdowns, chromatin immunoprecipitation, and luciferase reporter analyses. Immunohistochemistry of fetal kidneys confirmed that a number of the WT1 target genes had overlapping expression patterns with the highly restricted spatiotemporal expression of WT1. Finally, using an in vitro embryonic kidney culture assay, we found that the addition of recombinant epiregulin, amphiregulin, CX3CL1, and interleukin-11 significantly enhanced ureteric bud branching morphogenesis. Our genome-wide screen implicates WT1 in the transcriptional regulation of the EGF-family of growth factors as well as the CX3CL1 chemokine during nephrogenesis.
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PMID:Identification of novel Wilms' tumor suppressor gene target genes implicated in kidney development. 1743 Aug 90

Wilms' tumor gene 1 (WT1) is overexpressed in various hematological malignancies and has been proposed as a target for minimal residual disease (MRD) detection and for immunotherapy. Although WT1 is known as a key molecule for tumor cell proliferation, the expression pattern of WT1 in leukemic cells in dependency of proliferation has not yet been investigated. Furthermore, WT1 expression was mostly studied by reverse transcriptase PCR and the expression of WT1 protein has not been extensively studied. Here, we analyzed WT1 protein expression in the human myeloid leukemia cell lines K562 and HL-60 by indirect immunofluorescence and flow cytometry. Both cell lines exhibited varying nuclear WT1 immunoreactivity pointing to a cell cycle-dependent and/or proliferation-dependent WT1 expression. In rapidly proliferating cells high levels of WT1 protein were detected by flow cytometry. A reduced proliferation rate was associated with a low WT1 protein expression and an accumulation of cells in G(0)/G(1) phase. During G(0)/G(1) phase cells expressed WT1 at a lower level than in S or G(2)/M phase. Moreover, WT1 expression was diminished in all cell cycle phases in slowly proliferating cells. We conclude that WT1 protein expression is dependent on the cell cycle phase as well as on the proliferation rate. This finding might be relevant for MRD studies and immunotherapeutic strategies targeting WT1.
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PMID:WT1 protein expression in slowly proliferating myeloid leukemic cell lines is scarce throughout the cell cycle with a minimum in G0/G1 phase. 1845 71

A standardized, sensitive and universal method for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) is still pending. Although hyperexpression of Wilms' tumor (WT1) gene transcript has been frequently proposed as an MRD marker in AML, wide comparability of the various methods used for evaluating WT1 expression has not been given. We established and standardized a multicenter approach for quantifying WT1 expression by quantitative reverse transcriptase PCR (qRT-PCR), on the basis of a primer/probe set combination at exons 6 and 7. In a series of quality-control rounds, we analyzed 69 childhood AML samples and 47 normal bone marrow (BM) samples from 4 participating centers. Differences in the individual WT1 expressions levels ranged within <0.5 log of the mean in 82% of the cases. In AML samples, the median WT1/1E+04 Abelson (ABL) expression was 3.5E+03 compared with that of 2.3E+01 in healthy BM samples. As 11.5% of childhood AML samples in this cohort harbored WT1 mutations in exon 7, the effect of mutations on WT1 expression has been investigated, showing that mutated cases expressed significantly higher WT1 levels than wild-type cases. Hence, our approach showed high reproducibility and applicability, even in patients with WT1 mutations; therefore, it can be widely used for the quantitation of WT1 expression in future clinical trials.
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PMID:Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: a European multicenter study. 1932 6

Recent studies have shown that high BAALC expression predicts an adverse prognosis and may define an important risk factor in acute myeloid leukemia patients with normal karyotype. We performed, using real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR), the molecular analysis of BAALC gene as a possible minimal residual disease (MRD) marker in 45 patients with newly diagnosed acute leukemia. BAALC transcript levels in 32 patients with CD34 expressed in leukemic blasts were 2-3 logs higher than background levels, and the copy number was reduced in patients achieving hematological remission. Comparative monitoring of MRD by RQ-PCR for the Wilms' tumor gene 1(WT1) or specific translocation markers demonstrated that BAALC had similar kinetics as WT1, AML1/ETO and minor BCR/ABL, but not PML/RARA. Quantitation of BAALC gene expression made it possible to assess MRD in patients with CD34-positive acute leukemia. To our knowledge, this is the first report concerning the use of BAALC mRNA expression for MRD monitoring.
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PMID:Molecular monitoring of BAALC expression in patients with CD34-positive acute leukemia. 2037 83


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