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:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Histone acetylation is emerging as a major regulatory mechanism thought to modulate gene expression by altering the accessibility of transcription factors to DNA. In this study, treatment of human tumor cells with the histone deacetylase inhibitor, trapoxin (TPX), resulted in selective changes in genes that control the cell cycle. TPX activated p21(waf1) transcription that led to elevated p21(waf1) protein levels in three human tumor cell lines without altering the protein levels of cdk2, cdk4, or cyclin B. In addition, TPX increased cyclin E transcription without increasing the levels of Rb, E2F, dihydrofolate reductase, or glyceraldehyde-3-phosphate dehydrogenase. The elevated levels of p21(waf1) protein led to decreased Rb phosphorylation and cdk2 activity. These effects resulted in G(1) and G(2) cell cycle arrest in H1299 human lung and MDA-MB-435 breast carcinoma cells and apoptosis in A549 lung carcinoma cells. Chromatin immunoprecipitation assays revealed that TPX increased the level of chromatin acetylation associated with histone H3 in the trapoxin-responsive region of the p21(waf1) promoter. This study demonstrates that inhibition of HDAC by TPX increases acetylation of H3-associated chromatin and alters gene expression with marked selectivity.
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PMID:Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. 1057 69

The presence of interleukin-1 (IL-1) and IL-2 mRNA in five human prostate cancer (PCa) cell lines and their effects on cellular proliferation and prostate-specific antigen (PSA) levels were examined. IL-1 was found in androgen-unresponsive PC-3 and DU-145 but not in the androgen-responsive LNCaP, MDA-PCA-2a and MDA-PCA-2b cell lines. IL-2 message was absent while that of GAPDH (positive control) was present in all five cell lines. IL-1 decreased while IL-2 increased PSA levels of near-confluent LNCaP cells after 24 h of treatment. IL-1 inhibited whereas IL-2 stimulated the growth of sub-confluent LNCaP cells (72 h). Neither cytokine affected the proliferation of DU-145 or PC-3 cells.
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PMID:Differences in the expression and effects of interleukin-1 and -2 on androgen-sensitive and -insensitive human prostate cancer cell lines. 1073 6

We developed a real-time one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method for the routine quantification of c-erbB-2 oncogene expression in breast cancer, using a 7700 ABI PRISM Sequence Detector System (Perkin Elmer-Applied Biosystems, Courtaboeuf, France). The real-time quantification of the polymerase chain reaction products is based on the TaqMan 5' nuclease assay. The optimal experimental conditions we determined were as follows: 6 mM MgCl2, 200 nM of fluorogenic probe, 200 nM of each primer, and 12.5 units MuLV reverse transcriptase. The GAPDH housekeeping gene was used for normalization of c-erbB-2 expression. In human breast cancer cell lines, the normalized expression of c-erbB-2 ranged from 8 x 10(-6) to 2,600 x 10(-6), the two highest values corresponding to the c-erbB-2 overexpressing cells MDA-MB-453 and SK-BR-3. In a series of 100 breast cancer samples, c-erbB-2 normalized expression was found to range from 0.4 x 10(-6) to 350 x 10(-6). A close correlation was observed between this real-time one-step quantitative RT-PCR method and both semiquantitative conventional RT-PCR (N = 22; r = 0.8543; P < .0001) and c-erbB-2 protein expression (p185) quantified by an enzyme immunoassay (EIA) (N = 27; r = 0.71; P < .0001). The current realtime RT-PCR assay is rapid, sensitive, and reproducible and appears particularly suitable to quantify gene expression in large series of samples.
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PMID:A real-time one-step reverse transcriptase-polymerase chain reaction method to quantify c-erbB-2 expression in human breast cancer. 1097 82

Glutathione S-transferases (GSTs) are a family of detoxification isozymes that protect cells by conjugating GSH to a variety of toxic compounds, and they may also play a role in the regulation of both cellular proliferation and apoptosis. We have previously shown that human GST P1-1, which is the most widely distributed extrahepatic isozyme, could be inactivated by the catechol estrogen metabolite 4-hydroxyequilenin (4-OHEN) in vitro [Chang, M., Shin, Y. G., van Breemen, R. B., Blond, S. Y., and Bolton, J. L. (2001) Biochemistry 40, 4811-4820]. In the present study, we found that 4-OHEN and another catechol estrogen, 4,17beta-hydroxyequilenin (4,17beta-OHEN), significantly decreased GSH levels and the activity of GST within minutes in both estrogen receptor (ER) negative (MDA-MB-231) and ER positive (S30) human breast cancer cells. In addition, 4-OHEN caused significant decreases in GST activity in nontransformed human breast epithelial cells (MCF-10A) but not in the human hepatoma HepG2 cells, which lack GST P1-1. We also showed that GSH partially protected the inactivation of GST P1-1 by 4-OHEN in vitro, and depletion of cellular GSH enhanced the 4-OHEN-induced inhibition of GST activity. In addition, 4-OHEN GSH conjugates contributed about 27% of the inactivation of GST P1-1 by 4-OEHN in vitro. Our in vitro kinetic inhibition experiments with 4-OHEN showed that GST P1-1 had a lower K(i) value (20.8 microM) compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 52.4 microM), P450 reductase (PR, 77.4 microM), pyruvate kinase (PK, 159 microM), glutathione reductase (GR, 230 microM), superoxide dismutase (SOD, 448 microM), catalase (562 microM), GST M1-1 (620 microM), thioredoxin reductase (TR, 694 microM), and glutathione peroxidase (GPX, 1410 microM). In contrast to the significant inhibition of total GST activity in these human breast cancer cells, 4-OHEN only slightly inhibited the cellular GAPDH activity, and other cellular enzymes including PR, PK, GR, SOD, catalase, TR, and GPX were resistant to 4-OHEN-induced inhibition. These data suggest that GST P1-1 may be a preferred protein target for equine catechol estrogens in vivo.
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PMID:Inhibition of cellular enzymes by equine catechol estrogens in human breast cancer cells: specificity for glutathione S-transferase P1-1. 1211 4

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST-XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at -80 degrees C for at least 60 days. M30-Apoptosense plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at -80 degrees C, while at 37 degrees C it had a half-life of 80-100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.
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PMID:Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP. 1568 40

Eukaryotic initiation factor 4E (eIF4E) binds to the 5' m(7)G cap of mRNAs and is a focal point of regulation of initiation of mRNA translation. High levels of expression of eIF4E in many epithelial cancers, including breast, head and neck, colon, and bladder, correlate with increased tissue invasion and metastasis. To further examine the role of eIF4E in the biology of cancer cells, variants of eIF4E with impaired 5' cap binding function were expressed in MDA-MB-435 carcinoma cells. Cell lines overexpressing variants of eIF4E had impaired growth properties and exhibited a different morphology compared to cells expressing similar amounts of exogenous wild-type eIF4E or control cells. Cells expressing variant eIF4E did not form foci in culture and produced smaller colonies in soft agar compared to cells expressing wild-type eIF4E. In addition, analysis of polyribosomes for vascular endothelial growth factor (VEGF) mRNA demonstrated a shift from translationally active to inactive fractions in variant eIF4E cells, while GAPDH mRNA did not. The long G-C rich 5' untranslated region of VEGF mRNA is a feature of other mRNAs encoding growth regulating proteins that are predicted to have their translation enhanced by increases in eIF4E; whereas mRNA with shorter and less structured 5' UTRs, like that of GAPDH, are predicted to be largely unaffected. These data suggest that targeting the 5' cap-binding domain of eIF4E may be a viable option to slow cancer cell growth and alter the malignant phenotype.
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PMID:Eukaryotic initiation factor 4E variants alter the morphology, proliferation, and colony-formation properties of MDA-MB-435 cancer cells. 1709 71

An estrogen receptor-negative variant of the MCF-7 breast cancer cell line, TMX2-28, was used as a model in which to study breast cancer cell invasion. Using a reconstituted basement membrane (Matrigel) assay to evaluate cell invasion, we determined that TMX2-28 cells are more invasive than MCF-7 cells and that the invasiveness of TMX2-28 is similar to that of the aggressive MDA-MB-231 breast cancer cell line. TMX2-28 cells displayed a rounded, epithelial cell-like morphology, suggesting an amoeboid mode of cell invasion, in contrast to the mesenchymal mode of invasion characteristic of spindle-shaped, fibroblast-like MDA-MB-231 cells. Using real-time reverse transcription-PCR, we found that mitogen-inducible gene 2 (MIG2) is expressed at a 17-fold higher level in TMX2-28 cells than in nonaggressive MCF-7 cells and that MIG2 mRNA levels are low in the nontumorigenic human mammary epithelial cell line, 184. We determined that MIG2 plays a role in cell invasion by using small interfering RNA (siRNA) to suppress the expression of MIG2 mRNA levels in TMX2-28 cells. TMX2-28 cell invasion was reduced by 48% when the cells were transfected with siRNAs targeting MIG2, relative to cells transfected with siRNAs against glyceraldehyde-3-phosphate dehydrogenase. Finally, MIG2 expression was evaluated in reductive mammoplasty and breast tumor tissue. Although all 21 normal tissues from reduction mammoplasty showed immunoreactivity for MIG2, ranging from weak (62%) to strong (24%), only half of the 34 formalin-fixed breast tumors showed immunoreactivity for MIG2. Of these 17 positive cases, 10 were considered to overexpress MIG2 (moderate to strong staining). Examination of 30 frozen breast tumors supported the finding that MIG2 is overexpressed in a subset of breast cancers. We suggest that MIG2's normal regulation and function are disrupted in breast cancer.
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PMID:Use of an aggressive MCF-7 cell line variant, TMX2-28, to study cell invasion in breast cancer. 1718 81

Micro-RNAs are small noncoding RNAs, which diminish the stability and/or translation of mRNAs. This study examined whether miR-206, previously shown to be elevated in estrogen receptor (ER)alpha-negative breast cancer, regulates the expression of ERalpha. Two putative miR-206 sites, (hERalpha1 and hERalpha2), were found in silico within the 3'-untranslated region of human ERalpha mRNA. Transfection of MCF-7 cells with pre-miR-206 or 2'-O-methyl antagomiR-206 specifically decreased or increased, respectively, ERalpha mRNA levels. Overexpression of pre-miR-206 reduced ERalpha and beta-actin protein levels, with no effect on ERbeta, E-cadherin, or glyceraldehyde-3-phosphate dehydrogenase. Reporter constructs containing the hERalpha1 or hERalpha2 binding sites inserted into the 3'-untranslated region of the luciferase mRNA conferred a 1.6- and 2.2-fold repression of luciferase activity, respectively, in HeLa cells. Both miR-206 sites responded accordingly to exogenous hsa-pre-miR-206 and 2'-O-methyl antagomiR-206, and both sites were rendered inactive by mutations that disrupted hybridization to the 5'-seed of miR-206. A C-->T single nucleotide polymorphism in the hERalpha1 site increased repression of luciferase activity to approximately 3.3-fold in HeLa cells. MiR-206 levels were higher in ERalpha-negative MB-MDA-231 cells than ERalpha-positive MCF-7 cells, but only the ERalpha1 site mediated significantly more repression in reporter constructs. MiR-206 expression was strongly inhibited by ERalpha agonists, but not by an ERbeta agonist or progesterone, indicating a mutually inhibitory feedback loop. These findings provide the first evidence for the posttranscriptional regulation of ERalpha by a micro-RNA in the context of breast cancer.
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PMID:The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-alpha (ERalpha) and represses ERalpha messenger RNA and protein expression in breast cancer cell lines. 1731 70

Liposomes are a useful means of delivering molecular targeting agents such as small interfering RNA (siRNA) to downregulate specific pathways important in cancer growth and progression. The ability to non-invasively image these carriers is important to ascertain their delivery within the tumor. As cyclooxygenase-2 (COX-2) is an important therapeutic target in cancer, we investigated loading COX-2-specific siRNA into cationic liposomes containing MR contrast agents for imaging delivery in cancer cells and tumors. COX-2 and GAPDH siRNA, as well as Magnevist or Feridex, were loaded directly into the liposomes. These lipoplexes were used for cell transfection of the poorly differentiated and highly metastatic breast cancer cell line MDA-MB-231. PEGylated liposomes loaded with Feridex and fluorescently labeled COX-2 siRNA were used for in vivo delivery of lipoplexes in MDA-MB-231 breast cancer xenografts in female SCID mice. Transient transfection assays demonstrated potent and specific downregulation of the COX-2 protein in cells in culture. Tail vein injections of PEGylated COX-2 lipoplexes resulted in intratumoral delivery of siRNA. Biodistribution studies showed significant localization in the lung, liver and kidney at 24 h. These data demonstrate the feasibility of liposomal-mediated delivery of COX-2-specific siRNA to downregulate COX-2 in cancer cells, and multi-modality imaging of the delivery of specific siRNA in tumors.
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PMID:Imaging of cationic multifunctional liposome-mediated delivery of COX-2 siRNA. 1892 99

To quantify gene expression levels, appropriate controls have to be used to adjust for experimental variation. Endogenous control genes are widely used as they are stably expressed independent of cell cycle and experimental conditions, however, they can be altered upon drug treatment. DNA methylation is widely studied in chemotherapy drug resistance and the DNA methylation inhibitor decitabine showed promising results reversing drug resistance in cancer. We aimed to investigate the effect of different decitabine concentrations on the expression of selected endogenous control genes (GAPDH, 18S rRNA, PPIA, RPL13A, OAZ1) in two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) compared to untreated cells. In MCF-7 cells, 18S rRNA remained stable, however, GAPDH, PPIA and OAZ1 gene expression was increased after treatment. RPL13A was stably expressed at 8 muM decitabine but was increased at lower drug concentrations. In MDA-MB-231 cells, GAPDH levels remained relatively stable following decitabine treatment and so was PPIA expression at low decitabine concentrations. Decitabine increased 18S rRNA, RPL13A and OAZ1 gene expression. In this study, we observed cell line specific effects of decitabine and suggest that 18S rRNA is most suitable to use in MCF-7 cells, while GAPDH is recommended to use in MDA-MB-231 cells during decitabine treatment.
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PMID:Effects of decitabine on the expression of selected endogenous control genes in human breast cancer cells. 1983 53


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