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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nucleosomal DNA fragmentation is detected in myoblasts only when apoptosis is induced under differentiating conditions. However, the molecular mechanisms and the DNase responsible for the differentiation-dependent apoptotic DNA laddering are poorly understood. Here we show that a Ca(2+)/Mg(2+)-dependent endonuclease, DNase gamma, is induced in C2C12 myoblasts during myogenic differentiation and catalyzes apoptotic DNA fragmentation in differentiating myoblasts. A Ca(2+)/Mg(2+)-dependent, Zn(2+)-sensitive endonuclease activity appears in C2C12 myoblasts during myogenic differentiation. The enzymatic properties of the inducible DNase were found to be quite similar to those of DNase I family of DNases. Reverse transcriptase-PCR analysis revealed that the induction of DNase gamma, a member of the DNase I family of DNases, is correlated with the appearance of inducible DNase activity. The induction of DNase gamma occurs simultaneously with myogenin induction but precedes the up-regulation of p21. A high level of DNase gamma expression was also detected in differentiated myotubes but not in skeletal muscle fibers in which DNase X is highly expressed. The role of DNase gamma in myoblast apoptosis was evaluated in the following experiments. Proliferating myoblasts acquire DNA ladder producing ability by the ectopic expression of DNase gamma, but not DNase X, suggesting that the expression level of DNase gamma is the determinant of the differentiation-dependent apoptotic DNA laddering observed in myoblasts. DNA fragmentation during differentiation-induced apoptosis is strongly suppressed by the antisense-mediated down-regulation of DNase gamma. Importantly, the extent of DNA laddering is well correlated with the level of endogenous DNase gamma activity. Our data demonstrate that DNase gamma is the endonuclease responsible for DNA fragmentation in apoptosis associated with myogenic differentiation.
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PMID:Involvement of DNase gamma in apoptosis associated with myogenic differentiation of C2C12 cells. 1205 Jan 66

Multiple drug resistance occurs when cells fail to respond to chemotherapy. Although it has been established that the drug efflux protein P-glycoprotein protects the brain from xenobiotics, the mechanisms involved in the regulation of expression of multiple drug resistance genes and proteins are not fully understood. Re-entry into the cell cycle and integrity of the p53 signaling pathway have been proposed as triggers of multiple drug resistance expression in tumor cells. Whether this regulation occurs in non-tumor CNS tissue is not known. Since multiple drug resistance overexpression has been reported in glia and blood vessels from epileptic brain, we investigated the level of expression of multidrug resistance protein, multidrug resistance-associated proteins and lung resistance protein in endothelial cells and astrocytes isolated from epileptic patients or studied in situ in surgical tissue samples by double label immunocytochemistry. Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that multiple drug resistance, multidrug resistance protein, and lung resistance protein are expressed in these cells. Given that lung resistance proteins have been reported to be preferentially expressed by tumors, we investigated expression of tumor suppressor genes in epileptic cortices. The pro-apoptotic proteins p53 and p21 could not be detected in "epileptic" astrocytes, while endothelial cells from the same samples readily expressed these proteins, as did normal brain astroglia and normal endothelial cells. Other apoptotic markers were also absent in epileptic glia. Our results suggest a possible link between loss of p53 function and expression of multiple drug resistance in non-tumor CNS cells.
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PMID:Relationship between expression of multiple drug resistance proteins and p53 tumor suppressor gene proteins in human brain astrocytes. 1456 21

A highly distinctive subset of renal neoplasms of children and young adults contains a t(6;11)(p21;q12), a translocation recently been shown to result in fusion of Alpha, a gene on 11q12, with the transcription factor gene TFEB on 6p21. To define the clinicopathologic spectrum of this nascent entity and to establish immunohistochemical (IHC) and molecular methods for the detection of the specific Alpha-TFEB fusion, we studied 7 renal neoplasms that showed the t(6;11) by cytogenetic or molecular analysis (patient age: range, 9-33 years; mean, 17 years). While all tumors were confined to the kidney, 3 tumors demonstrated vascular invasion. In limited follow-up, none has metastasized. We postulated that the Alpha-TFEB gene fusion may result in deregulated expression of TFEB protein that would be detectable by IHC. Using a polyclonal antibody to TFEB on formalin-fixed, paraffin-embedded tissue sections, we found that all 7 renal neoplasms with the t(6;11) demonstrated moderate (2 cases) or strong (5 cases) nuclear TFEB immunoreactivity. In contrast, none of 1089 other tumors (of 74 histologic types from 16 sites) labeled significantly for TFEB. Nuclear immunoreactivity for TFEB in normal tissues was extremely rare, limited to weak labeling of scattered benign lymphocytes. We also show that the Alpha-TFEB fusion RNAs are highly variable in size and structure, making detection by reverse-transcriptase polymerase chain reaction (RT-PCR) less reliable than for other gene fusions. Because Alpha is an intronless gene and therefore lacks splice signals, we hypothesized that DNA PCR and RT-PCR products would be identical, allowing for the use of more robust molecular assays based on genomic DNA. Indeed, in 2 cases with available frozen tissue, we showed the genomic Alpha-TFEB junction detected by DNA PCR to be identical to the Alpha-TFEB fusion mRNA detected by RT-PCR. In summary, renal neoplasms with the t(6;11) are a distinctive neoplastic entity with many similarities to the Xp11 translocation carcinomas, and together with the latter form a growing "MiTF/TFE family" of translocation carcinomas. Nuclear immunoreactivity for TFEB protein is a highly sensitive and specific diagnostic marker for these renal neoplasms. Finally, the special molecular features of the Alpha-TFEB gene fusion allow its molecular detection by DNA PCR as a robust alternative to RT-PCR in clinical tumor samples.
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PMID:Renal carcinomas with the t(6;11)(p21;q12): clinicopathologic features and demonstration of the specific alpha-TFEB gene fusion by immunohistochemistry, RT-PCR, and DNA PCR. 1564 81

It has been proposed that a lack of apoptosis plays an important role in neuroblastoma (NB) progression. We therefore screened cDNA array filters, including 198 apoptotic genes, in order to identify mRNA transcripts that are differentially expressed in tumours with unfavourable versus favourable biology. Twenty-one genes were analysed further using real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). Significantly lower levels of DNCL1 (PIN; P(c)(corrected) = 0.0054) and NTRK1 (TrkA; P(c) = 0.039) were found in NB tumours with unfavourable biology. In addition, BID, BCL2, APAF1, CASP2, CASP3 and CASP9 were found to be preferentially expressed in tumours with favourable biology, whereas CDKN1A (p21), IL2RA, and MCL1, were found to be preferentially expressed in NB tumours with unfavourable biology. In conclusion, mRNA levels of transcripts encoding pro-apoptotic mediators of the mitochondrial apoptotic pathway were found to be expressed to a lower extent in tumours with unfavourable biology. Our data also suggest that the mitochondrial pathway is suppressed in advanced stages of NB tumours, due to an imbalance between anti-apoptotic and pro-apoptotic mediators which is a finding that may have therapeutic significance.
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PMID:Imbalance of the mitochondrial pro- and anti-apoptotic mediators in neuroblastoma tumours with unfavourable biology. 1573 69

Systemic sclerosis (SSc) is a connective tissue disorder characterized by excessive deposition of extracellular matrix in the affected skin as well as various internal organs, vascular injury and immune abnormality; however, the etiology of SSc remains still unknown. We previously established an experimental mouse model for scleroderma by repeated local injections of bleomycin, a DNA damaging agent. In this study, we examined the induction of apoptosis and the expression of p53, p21 (Waf1/Cip1), and proliferating cell nuclear antigen (PCNA) in the lesional skin following bleomycin exposure in this model. Dermal sclerosis was induced by alternate day's injections of bleomycin for 4 weeks. TUNEL assay showed that apoptotic cells began to appear at 1 week after bleomycin exposure, and were prominently detected at 3-4 weeks. Immunohistochemical examination showed increased expression of p53 and p21 mainly in the infiltrating mononuclear cells at 2 weeks after bleomycin treatment. Bleomycin treatment markedly enhanced PCNA expression at 1-2 weeks, mainly in mesenchyme, as compared with control phosphate buffered saline treatment. Reverse transcriptase-polymerase chain reaction analysis showed that the expression of p53 and p21 mRNA was concurrently upregulated at 1-2 weeks after bleomycin treatment. Taken together, coordinate increased levels of p53 and p21 preceded the maximal induction of apoptosis and dermal sclerosis. Our findings suggest that apoptotic processes are involved in the pathophysiology of bleomycin-induced scleroderma, which may be mediated, in part, by the upregulation of p53 and p21.
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PMID:Increased expression of p53 and p21 (Waf1/Cip1) in the lesional skin of bleomycin-induced scleroderma. 1580 28

ABL1 amplification, due to a cryptic episomal translocation NUP214/ABL1, is a novel finding in T-cell acute lymphoblastic leukemia (ALL). Here we report on the incidence and clinical features of this genetic defect in a series of 30 consecutive adult T-cell ALL patients. Multiple copies of the ABL1 gene were detected in two patients (6.6%), one with the karyotype 46,XY,t(1;3)(p36;p21),del(6)(q23)/46,XY and the other without analyzable metaphases. Metaphase/interphase fluorescence in situ hybridization (FISH) detected multiple uncountable signals corresponding to ABL1 in mitotic cells and nuclei from both patients. In one patient, no signals corresponded with the 9p21 chromosomal region, which contains the p16INK4a gene, and in the other one signal was observed. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) demonstrated that in these patients ABL1 gene expression was 14- and 18-fold greater than in normal controls, and returned to normal levels only when complete remission was achieved. We reached the following conclusions: (1) FISH is the only technique that promptly identifies T-cell ALL patients with ABL1 amplification, (2) quick identification with FISH is fundamental in the clinic because this T-cell ALL subset is imatinib sensitive but may become resistant due to development of additional mutations, and (3) ABL1 quantitative RT-PCR may be easily applied to monitor minimal residual disease.
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PMID:ABL1 amplification in T-cell acute lymphoblastic leukemia. 1621 63

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved among various species. Mammalian TSC-22 is a potential tumor suppressor gene. It translocates into nuclei and suppresses cell division upon antiproliferative stimuli. In human colon carcinoma cells, TSC-22 inhibits cell growth by upregulating expression of the p21 gene, a cyclin-dependent kinase (Cdk) inhibitor. We previously showed that the Xenopus laevis homologue of the TSC-22 gene (XTSC-22) is required for cell movement during gastrulation through cell cycle regulation. In this report, we investigated the molecular mechanism of the antiproliferative effect of XTSC-22. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis suggested that XTSC-22 did not affect the expression levels of the p21 family of Cdk inhibitors or other cell cycle regulators. Analysis of deletion mutants of XTSC-22 revealed that nuclear localization of the N-terminal TSC-box is necessary for cell cycle inhibition by XTSC-22. Further experiments suggested that p27Xic1, a key Cdk inhibitor in Xenopus, interacts with XTSC-22. Because p27Xic1 is a cell cycle inhibitor with a nuclear localization signal, it is possible that XTSC-22 suppresses cell division by translocating into the nucleus with p27Xic1, where it may potentiate the intranuclear action of p27Xic1.
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PMID:TSC-box is essential for the nuclear localization and antiproliferative effect of XTSC-22. 1739 98

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies.
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PMID:p53 activation by knockdown technologies. 1753 Sep 25

To determine whether adenovirus-mediated wild-type p53 transfer after radiotherapy could radiosensitize non-small-cell lung cancer (NSCLC) cells to subclinical-dose carbon-ion beam (C-beam), H1299 cells were exposed to a C-beam or gamma-ray and then infected with 5 MOI of AdCMV-p53 or GFP (C-beam or gamma-ray with p53 or GFP). Cell cycle was detected by flow cytometric analysis. The apoptosis was examined by a fluorescent microscope with DAPI staining. DNA fragmentation was monitored by the TUNEL assay. P53 mRNA was detected by reverse-transcriptase polymerase chain reaction. The expression of p53, MDM(2), and p21 was monitored by Western blot. Survival fractions were determined by colony-forming assay. The percentages of G(1)-phase cells in C-beam with p53 increased by 8.2%-16.0%, 5.2%-7.0%, and 5.8%-18.9%, respectively, compared with C-beam only, gamma-ray with p53, or p53 only. The accumulation of G(2)-phase cells in C-beam with p53 increased by 5.7%-8.9% and 8.8%-14.8%, compared with those in gamma-ray with p53 or p53 only, respectively. The percentage of apoptosis for C-beam with p53 increased by 7.4%-19.1%, 5.8%-11.7%, and 5.2 %-19.2%, respectively, compared with C-beam only, gamma-ray with p53, or p53 only. The level of p53 mRNA in C-beam with p53 was significantly higher than that in p53 only. The expression level of p53 and p21 in C-beam with p53 was significantly higher than that in both C-beam with GFP and p53 only. The survival fractions for C-beam with p53 were significantly less than those for the other groups (p < 0.05). The data suggested that AdCMV-p53 transfer could more efficiently radiosensitize H1299 cells to subclinical-dose C-beam irradiation through the restoration of p53 function.
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PMID:Adenovirus-mediated wild-type p53 transfer radiosensitizes H1299 cells to subclinical-dose carbon-ion irradiation through the restoration of p53 function. 1924 48

Cerebral amyloid angiopathy (CAA) caused by amyloid beta (Abeta) deposition around brain microvessels results in vascular degenerative changes. Antiangiogenic Abeta properties are known to contribute to the compromised cerebrovascular architecture. Here we hypothesize that Abeta peptides impair angiogenesis by causing endothelial cells to enter senescence at an early stage of vascular development. Wild-type (WT) Abeta and its mutated variant E22Q peptide, endowed with marked vascular tropism, were used in this study. In vivo, in zebrafish embryos, the WT or E22Q peptides reduced embryo survival with an IC(50) of 6.1 and 4.7 microM, respectively. The 2.5 microM concentration, showing minimal toxicity, was chosen. Alkaline phosphatase staining revealed disorganized vessel patterning, narrowing, and reduced branching of vessels. Beta-galactosidase staining and the cyclin-dependent kinase inhibitor p21 expression, indicative of senescence, were increased. In vitro, WT and E22Q reduced endothelial cell survival with an IC(50) of 12.3 and 8.8 microM, respectively. The 5 microM concentration, devoid of acute effects on the endothelium, was applied chronically to long-term cultured human umbilical vein endothelial cells (HUVECs). We observed reduced cumulative population doubling, which coincided with beta-galactosidase accumulation, down-regulation of telomerase reverse-transcriptase mRNA expression, decreased telomerase activity, and p21 activation. Senescent HUVECs showed marked angiogenesis impairment, as Abeta treatment reduced tube sprouting. The endothelial injuries caused by the E22Q peptide were much more aggressive than those induced by the WT peptide. Premature Abeta-induced senescence of the endothelium, producing progressive alterations of microvessel morphology and functions, may represent one of the underlying mechanisms for sporadic or heritable CAA.
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PMID:Abeta peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. 2020 41


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