Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time. This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor. No prelabeling of the cells is required, and many different cell types can be studied using this system. Under the pulsed-field conditions used, DNA in the range of 2 to 6 Mb enters the PFGE gel and forms an upper compression zone directly under each well. To quantify the DSBs after electrophoresis, the DNA was transferred to nylon membranes and hybridized with 32P-labeled chromosomal DNA. Phosphor screens were exposed to the membranes and scanned on a phosphor imager. The kinetics of induction and repair was determine by measuring the amount of DNA in the compression zones compared to the amount in the wells. EMT-6 cells were used to demonstrate this method. Induction of DSBs by doses of 0-7.5 Gy X rays was assayed using approximately 12,500 cells per dose and was shown to be linear. Double-strand breaks from 1 Gy were detected above background. To determine a lower limit of the number of cells that could be used to measure DSB repair, cells were embedded in agarose at decreasing concentrations per plug, exposed to 7.5 Gy X irradiation and allowed to repair at 37 degrees C for up to 60 min. DNA from approximately 12,500, 1,250 and 125 cells per time was loaded and subjected to PFGE. The average fast-repair half-time was 3 min and the slow-repair half-time was 35 min. The kinetics of DSB repair in glioblastoma multiforme cells was also determined using this system. Agarose plugs were prepared from a cell suspension, irradiated with 7.5 Gy X rays and allowed to repair for up to 90 min. DNA from approximately 1,250 tumor cells was electrophoresed and analyzed as described above for EMT-6 cells. For this particular tumor, approximately 75% of the induced DSBs were repaired after 90 min. Data presented show that this PFGE-based system is an extremely sensitive method for measuring DSB induction and repair after low doses of X rays using very few cells.
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PMID:An assay for quantifying DNA double-strand break repair that is suitable for small numbers of unlabeled cells. 898 67

Loss of the tumor suppressor MMAC1 has been shown to be involved in breast, prostate and brain cancer. Consistent with its identification as a tumor suppressor, expression of MMAC1 has been demonstrated to reduce cell proliferation, tumorigenicity, and motility as well as affect cell-cell and cell-matrix interactions of malignant human glioma cells. Subsequently, MMAC1 was shown to have lipid phosphatase activity towards PIP3 and protein phosphatase activity against focal adhesion kinase (FAK). The lipid phosphatase activity of MMAC1 results in decreased activation of the PIP3-dependent, anti-apoptotic kinase, AKT. It is thought that this inhibition of AKT culminates with reduced glioma cell proliferation. In contrast, MMAC1's effects on cell motility, cell - cell and cell - matrix interactions are thought to be due to its protein phosphatase activity towards FAK. However, recent studies suggest that the lipid phosphatase activity of MMAC1 correlates with its ability to be a tumor suppressor. The high rate of mutation of MMAC1 in late stage metastatic tumors suggests that effects of MMAC1 on motility, cell - cell and cell - matrix interactions are due to its tumor suppressor activity. Therefore the lipid phosphatase activity of MMAC1 may affect PIP3 dependent signaling pathways and result in reduced motility and altered cell - cell and cell - matrix interactions. We demonstrate here that expression of MMAC1 in human glioma cells reduced intracellular levels of inositol trisphosphate and inhibited extracellular Ca2+ influx, suggesting that MMAC1 affects the phospholipase C signaling pathway. In addition, we show that MMAC1 expression inhibits integrin-linked kinase activity. Furthermore, we show that these effects require the catalytic activity of MMAC1. Our data thus provide a link of MMAC1 to PIP3 dependent signaling pathways that regulate cell - matrix and cell - cell interactions as well as motility. Lastly, we demonstrate that AKT3, an isoform of AKT highly expressed in the brain, is also a target for MMAC1 repression. These data suggest an important role for AKT3 in glioblastoma multiforme. We therefore propose that repression of multiple PIP3 dependent signaling pathways may be required for MMAC1 to act as a tumor suppressor.
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PMID:The MMAC1 tumor suppressor phosphatase inhibits phospholipase C and integrin-linked kinase activity. 1064 97

The tumor suppressor PTEN is one of the most commonly inactivated genes in human cancer. Glioblastoma multiforme cells harboring mutant PTEN have abnormally high levels of 3' phosphoinositides and elevated protein kinase B activity. Expression of wild-type PTEN in glioma cells, containing endogenous mutant PTEN, reduces 3' phosphoinositides levels, inhibits PKB activity, and induces G1 cell cycle arrest. We investigated the mechanism of the PTEN-induced growth arrest in glioma cell lines. Expression of PTEN is associated with increased expression of p27Kip1, decreased expression of cyclins A and D3, inhibition of cdk2 activity, and dephosphorylation of pRb. Inactivation of p53, by the human papilloma virus E6 oncoprotein, does not prevent PTEN-induced G1 arrest, implying that p53 is not required for G1 arrest. In contrast, p27Kip1 antisense oligonucleotides abrogated the growth arrest induced by PTEN. Furthermore, blocking p27Kip1 expression prevented the PTEN-induced reduction of cyclin-dependent kinase 2 activity, indicating that p27Kip1 functions upstream of cyclin-dependent kinase 2 in the PTEN regulatory cascade. These results implicate p27Kip1 as a critical mediator of PTEN-induced G1 arrest.
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PMID:p27Kip1 is required for PTEN-induced G1 growth arrest. 1128 Jul 73

We have used a new method of genomic microarray to investigate amplification of oncogenes throughout the genome of glioblastoma multiforme (GBM). Array-based comparative genomic hybridization (array CGH) allows for simultaneous examination of 58 oncogenes/amplicons that are commonly amplified in various human cancers. Amplification of multiple oncogenes in human cancers can be rapidly determined in a single experiment. Tumor DNA and normal control DNA were labeled by nick translation with green- and red-tagged nucleotides, respectively. Instead of hybridizing to normal metaphase chromosomes in conventional comparative genomic hybridization (CGH), the probes of the mixed fluorescent labeled DNA were applied to genomic array templates comprised of P1, PAC, and BAC clones of 58 target oncogenes. The baseline for measuring deviations was established by performing a series of independent array CGH using test and reference DNA made from normal individuals. In the present study, we examined fourteen GBMs (seven cell lines and seven tumours) with CGH and array CGH to reveal the particular oncogenes associated with this cancer. High-level amplifications were identified on the oncogenes/amplicons CDK4, GLI, MYCN, MYC, MDM2, and PDGFRA. The highest frequencies of gains were detected on PIK3CA (64.3%), EGFR (57.1%), CSE1L (57.1%), NRAS (50%), MYCN (42.9%), FGR (35.7%), ESR (35.7%), PGY1 (35.7%), and D17S167 (35.7%). These genes are suggested to be involved in the GBM tumorigenesis.
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PMID:Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization. 1135 Oct 43

The biodistributions of carborane-containing copper porphyrins, CuTCP and CuTCPH, have been studied previously in mice bearing subcutaneously implanted mammary carcinomas. We now report biodistributions of those porphyrins in Fischer 344 rats bearing intracranial and/or multiple subcutaneous isogeneic 9L gliosarcomas (9LGS). The porphyrin was given either by i.v. infusion or by multiple i.p. injections. When 190 mg CuTCPH/kg body weight was given to the rats by i.v. infusion, median tissue boron concentrations (microg/g) 3 days after the end of infusion were: 64 in subcutaneous tumor, 13 in intracranial tumor, 1 in blood and 3 in brain. When 450 mg CuTCPH/kg body weight was given to the rats by serial i.p. injections, the median concentrations (microg B/g) 4 days after the last injection were: 117 in subcutaneous tumor, 50 in intracranial tumor, 4 in blood, and 4 in brain. CuTCPH biodistribution was also studied in xenografts of the human malignant gliomas U87 and U373, and of the murine EMT-6 mammary carcinoma and the rat 9LGS, each grown subcutaneously in mice with severe combined immunodeficiency (SCIDs). In SCIDs, median boron concentrations (microg/g) 2 days after the last s.c. injection of a total of 190 mg CuTCPH/kg body weight were: 251 in U373, 33 in U87, <0.6 in blood and <0.5 in brain. Because there were such high boron levels in the U373, and because xenografted U373 is similar to spontaneous intracerebral human glioblastoma multiforme (GBM) microscopically, CuTCPH could prove useful as a boron carrier for boron neutron-capture therapy (BNCT) of GBM and of other human malignant gliomas.
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PMID:Biodistribution of copper carboranyltetraphenylporphyrins in rodents bearing an isogeneic or human neoplasm. 1150 10

Enhanced expression of tenascin-C (TN-C) at the invasive edges of glioblastoma multiforme in close association with vascular sprouts, suggests a role for TN-C in microvascular cell migration. To test this hypothesis, we studied the migration of endothelial cells in vitro. In an aggregate migration assay, bovine retinal endothelial cells (BRECs) and human umbilical vein endothelial cells spread and migrated similarly on TN-C or fibronectin (FN). In contrast, U251 MG glioma cells migrated less on TN-C than on FN. Morphological features of U251 MG glioma cells on TN-C included poor cell spreading and short processes. In contrast, on FN, U251 MG glioma cells spread and exhibited long radial processes. Using a transmembrane migration assay, we observed that BREC adhesion was similar on TN-C or FN, whereas U251 MG glioma cells adhered better to FN than to TN-C. In addition, BRECs migrated more across the membrane toward regions coated with TN-C than FN, and conversely, U251 MG glioma cells migrated more toward FN than TN-C. Migration of endothelial and glioma cells toward TN-C or FN occurred in a dose-dependent manner and was strongly dependent on cell adhesion. In this assay, ultrastructural study revealed the migrating phenotype of the endothelial cells through the micropores of the membrane and their spread morphology on TN-C. Moreover, in situ hybridization revealed specific expression of TN-C in migrating microvascular cells in a cerebral microvascular ring assay. Finally in a phosphorylation assay, TN-C enhanced focal adhesion kinase phosphorylation of BRECs, but not of U251 MG glioma cells, and FN enhanced focal adhesion kinase phosphorylation of both BRECs and U251 MG cells. The expression of TN-C by migrating endothelial cells and the promotion of endothelial cell adhesion and migration by TN-C suggest a potential role for TN-C in pathological angiogenesis.
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PMID:Tenascin-C promotes microvascular cell migration and phosphorylation of focal adhesion kinase. 1198 Jun 65

The presence of telomerase activity in a glioma may be a predictor of its malignant potential. Activation of telomerase is regulated at the transcriptional level of the human telomerase reverse transcriptase (hTERT). Here, we evaluated whether the amount of hTERT mRNA provides a molecular marker of glioma malignancy that would have clinical utility. We used a real-time RT-PCR to assess the number of hTERT transcripts in primary tumour samples derived from 70 glioma patients. Results were standardised by quantifying the number of ABL transcripts as internal control and expressed as hTERT/ABL ratio. The percentage of patients with detectable hTERT mRNA markedly increased with enhanced malignancy: low-grade gliomas expressed hTERT in one out of 14 cases (7.1%), anaplastic gliomas in four out of 13 cases (30.8%) and glioblastoma multiforme (GBM) tumours in 30 out of 43 cases (69.8%). The mean hTERT/ABL ratio was significantly higher in GBMs than in non-GBMs. Subdividing hTERT/ABL ratios as low (< pr = 25%) and high (>25%), we found that the overall survival among hTERT-positive GBMs was significantly worse in high hTERT expressors than in low hTERT expressors (P=0.0082). We conclude that the amount of hTERT mRNA may represent a diagnostic and prognostic indicator for GBM patients.
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PMID:Clinical implications of quantitative real-time RT-PCR analysis of hTERT gene expression in human gliomas. 1259 64

The phosphoinositide 3-kinase (PI3-kinase) signaling pathway is frequently aberrantly activated in glioblastoma multiforme (GM) by mutation or loss of the 3' phospholipid phosphatase PTEN. PTEN abnormalities result in inappropriate signaling to downstream molecules including protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR). PI3-kinase activation increases resistance to radiation-induced cell death; conversely, PI3-kinase inhibition enhances the sensitivity of tumors to radiation. The effects of LY294002, a biochemical inhibitor of PI3-kinase, on the response to radiation were examined in the PTEN mutant glioma cell line U251 MG. Low doses of LY294002 sensitized U251 MG to clinically relevant doses of radiation. In contrast to LY294002, rapamycin, an inhibitor of mTOR, did not result in radiosensitization. We demonstrate that among multiple known targets of LY294002, PI3-kinase is the most likely molecule responsible for LY294002-induced radiosensitization. Furthermore, using a myristoylated PKB/Akt construct, we identified PKB/Akt as the downstream molecule that mediates the synergistic cytotoxicity between LY294002 and radiation. Thus PI3-kinase dysregulation may contribute to the notable radioresistance of GM tumors and inhibition of PKB/Akt offers an excellent target to enhance radiosensitivity.
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PMID:PKB/Akt mediates radiosensitization by the signaling inhibitor LY294002 in human malignant gliomas. 1573 8

Glioblastoma multiforme is extraordinarily aggressive due to the propensity of cells to migrate away from the tumor core into the surrounding normal brain. In this report, we investigated the role of proline-rich tyrosine kinase 2 (Pyk2) and FAK with regard to influencing glioma cell phenotypes. Expression of Pyk2 stimulated glioma cell migration, whereas expression of FAK inhibited glioma cell migration and stimulated cell cycle progression. Pyk2 autophosphorylation was necessary, but not sufficient, to stimulate cellular migration. The N-terminal domain of Pyk2 is required for stimulation of migration as an N-terminally deleted variant of Pyk2 failed to stimulate migration, whereas expression of an autonomous Pyk2 N-terminal domain inhibited cell migration. Substitution of the C-terminal domain of Pyk2 with the corresponding domain of FAK stimulated cell migration as effectively as wild-type Pyk2; however, substitution of the N-terminal domain of Pyk2 with that of FAK inhibited cell migration, substantiating that the N-terminal domain of Pyk2 was required to stimulate migration. Silencing of Pyk2 expression by RNA interference significantly inhibited glioma migration. Cell migration was restored on re-expression of Pyk2, but expression of FAK in Pyk2 knockdown cells failed to restore migration. We conclude that Pyk2 plays a central role in the migratory behavior of glioblastomas.
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PMID:The tyrosine kinase pyk2 promotes migration and invasion of glioma cells. 1596 96

Epidermal growth factor receptors (EGFRs) are amplified and overexpressed in many different human cancers, a phenomenon generally associated with poor prognosis. Inhibitors of the tyrosine kinase activity associated with this receptor have been approved for the treatment of chemotherapy-refractory nonsmall cell lung cancer, and are in clinical trials for additional tumor types. While these inhibitors, gefitinib and erlotinib, display limited response rates when assessed in cohorts that include all patients, there are subgroups, defined by patient and tumor characteristics, that preferentially respond to these agents. We recently performed an analysis of tumors obtained from a Phase I trial of erlotinib in patients with glioblastoma multiforme (GBM), the most common malignant brain tumor in adults. We showed that patients whose tumors exhibited overexpression and amplification of EGFR responded better than patients who had normal levels of this gene and protein. We also demonstrated that the phosphorylation state of PKB/Akt was an important determinant for response, with low phospho-PKB/Akt levels predicting good response to erlotinib. We discuss these findings in the context of recent molecular analyses of the placebo-controlled Phase III trials that led to approval of EGFR inhibitors. These data underscore the importance of placebo-controlled trials to distinguish between prognostic indicators of disease progression more generally and predictive markers of response to therapy. Ultimately the goal of these studies is to allow selection of patients who will preferentially respond to EGFR inhibitors.
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PMID:Biomarkers to predict response to epidermal growth factor receptor inhibitors. 1617 70


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