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)

Fluoromisonidazole labeled with H-3 or F-18 has been tested as a quantitative probe for hypoxic cells in vitro and in rodent and spontaneous dog tumors in vivo. In V-79, EMT-6(UW), RIF-1, and canine osteosarcoma cells in vitro, the binding of 50 microM [H-3]Fluoromisonidazole was 50% inhibited by 1000-2000 ppm O2, relative to binding under anoxic conditions. After a 3 hr incubation with labeled drug, the anoxic/oxic binding ratios ranged from 12 to 27 for the four cell types. Retention of [H-3]fluoromisonidazole 4 hr after injection was greater in large KHT tumors (400-600 mm3) with an estimated hypoxic fraction greater than 30%, than in smaller tumors (50-200 mm3) with an estimated hypoxic fraction of 7-12%. RIF-1 tumors, with an estimated hypoxic fraction of 1.5%, retained the least label, with tumor: blood ratios ranging from 1.7 to 1.9. Spontaneous dog osteosarcomas were imaged with a time of flight positron emission tomograph for up to 5 hr following injection of [F-18] fluoromisonidazole. Analysis of regions of interest in images allowed creation of dynamic tissue time activity curves and calculation of tissue uptake in cpm/gram. These values were compared to radioactivity in plasma. In all cases, retention in some tumor regions exceeded that in plasma and in normal tissue, such as muscle or brain, by 3 to 5 hr post injection. Uptake of fluoromisonidazole in tumors was heterogeneous, with ratios of maximum to minimum uptake as high as 4 in different regions of interest in the same tumor. Tumor:plasma values ranged from 0.28 to 2.02. The oxygen dependency of fluoromisonidazole retention was similar in a variety of cell types and was 50% inhibited by O2 levels in the transition between full radiobiological hypoxia and partial sensitization. The quantitative regional imaging of [F-18] fluoromisonidazole in spontaneous canine tumors at varying times post-injection lays the basis for imaging and modeling of oxygen-dependent drug retention in different regions of human neoplasms.
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PMID:Radiolabelled fluoromisonidazole as an imaging agent for tumor hypoxia. 280 61

Oncastatin M (OSM) is one member of the leukemia inhibitory factor/interleukin-6 family of cytokines that has been shown to be a growth regulatory molecule. In osteoblastic cultures, OSM causes marked phenotypic changes and the enhanced secretion of interleukin-6. In this study, we have shown that stimulation of murine and human osteoblastic cultures and a human osteosarcoma cell line with OSM resulted in the tyrosine phosphorylation of a number of cellular proteins including members of both the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) family of signaling proteins. The JAKs, a family of intracellular kinases, and the STATs, a family of transcription factors, have both previously been shown to be tyrosine phosphorylated and activated in response to various cytokines, interferons, and growth factors in cells of non-skeletal origin. Using three different sources of cells of the osteoblast lineage, we demonstrate that OSM induces a rapid but transient tyrosine phosphorylation of the three JAK family members tested, JAK1, JAK2 and Tyk2. In addition, two members of the STAT family, Stat1alpha and Stat3, are tyrosine phosphorylated in osteoblastic cells in culture in response to OSM. OSM activation of this pathway in cells of the osteoblast lineage will result in the transcription of specific genes that ultimately may be associated with osteoblast function.
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PMID:Activation of the JAK-STAT signal transduction pathway by oncostatin-M cultured human and mouse osteoblastic cells. 862 84

GH induces phosphorylation of a number of cellular proteins, of which several have now been identified, such as mitogen-activated protein kinase, insulin receptor substrate-1, and members of the JAK kinase and STAT families of proteins. However, other phosphorylated proteins remain unidentified. Growth factors and cytokines, including epidermal growth factor, insulin, pp60v-scr, and angiotensin II, induce a rapid phosphorylation of annexin I, a 35-kDa member of the annexin family of Ca2+ and phospholipid-binding proteins. The osteoblast-like rat osteosarcoma cell-line UMR-106.01, in which GH acts as a mitogen via a high affinity GH receptor, was used as a model for GH-induced protein phosphorylation. It is demonstrated by immunoblotting and immunoprecipitation techniques that GH induces the phosphorylation of annexin I on tyrosine residues. This phosphorylation is dose and time dependent. Induction of annexin I phosphorylation is delayed compared with that of JAK2. These results identify annexin I as a protein that becomes tyrosine phosphorylated under the influence of GH and show that phosphorylation of annexin I is a general phenomenon that follows activation of a cell by hormones or cytokines.
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PMID:Growth hormone induces tyrosine phosphorylation of annexin I in rat osteosarcoma cells. 882 96

Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) are important growth factors for postnatal longitudinal bone growth. Although many effects of GH on bone growth are mediated by IGF-1, GH can directly influence bone cells. Limited knowledge exists regarding specific intracellular signaling pathways and genes activated by GH in bone cells. GH is known to activate several intracellular signaling pathways, among them the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. GH mainly activates JAK2 and both isoforms of STAT5, A and B. STAT5 gene deletion experiments have shown the importance of these transcription factors for growth. To understand the molecular mechanism(s) behind this, different experimental models are needed. The UMR 106 cell line is a rat clonal osteosarcoma cell line with osteoblast-like phenotypic properties, one is the endogenous expression of GH receptor (GHR). The present study focused on whether these cells express a functional GH-responsive JAK2/STAT5 pathway. Analysis of cell extracts by immunoprecipitation and Western blot showed that physiological concentrations of GH activated JAK2. Western blot analysis of nuclear extracts from GH-stimulated UMR 106 cells showed that physiological concentrations of GH induced nuclear translocation of both STAT5 isoforms, but with STAT5A being predominant. Both isoforms displayed similar nuclear turnover after GH stimulation of cells. Gel electrophoretic mobility shift assay (GEMSA) of nuclear extract revealed that both STAT5A and STAT5B obtained DNA-binding capacity after GH stimulation. Thus, we have shown, for the first time, the expression and GH-induced activation of JAK2 and STAT5A/B in UMR 106 osteoblast-like cells. This study also shows that this cell line is a suitable experimental model to study unique GH effects in osteoblasts mediated by STAT5.
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PMID:Growth hormone-regulated intracellular signaling in UMR 106 osteosarcoma cells. 1109 11

Rho, a member of the small GTP-binding proteins, and one of its downstream effectors ROCK (Rho-associated coiled-coil forming protein kinase) play an important role in the invasion of tumor cells. Lysophosphatidic acid (LPA) activates Rho and ROCK and promotes the organization of stress fibers and focal adhesions. However, the effect of LPA on tumor cell invasion is still controversial. In the present study, human osteosarcoma cells treated with a high concentration of LPA (high LPA) showed considerable formation of stress fibers and focal adhesions compared to the cells treated with a low concentration of LPA (low LPA). C3 (inhibitor of Rho) or Y27632 (an inhibitor of ROCK) inhibited the effects of LPA, indicating that LPA activates the Rho-ROCK pathway in the cells. In addition, Rho activation assay showed that the activation level of Rho can be altered by changing the concentration of LPA. Low LPA stimulated the motility and invasion of the cells, while high LPA reduced both. The disruption of extracellular matrix (ECM) by matrix metalloproteinase 2 (MMP2) is also critical for tumor cell invasion. MMP2 is activated by membranous type-1 MMP (MT1-MMP) and type-2 tissue inhibitor of MMP (TIMP2). High LPA suppressed the activation of MMP2 through down-regulation of MT1-MMP and TIMP2. C3 and Y27632 reversed the suppression of the activation of MMP2 and expression of MT1-MMP and TIMP2, suggesting the involvement of the Rho-ROCK pathway in ECM degradation. Tyrosine phosphorylation of focal adhesion kinase (FAK) was also required for the invasion of tumor cells to occur. Low LPA enhanced the tyrosine phosphorylation of FAK whereas high LPA reduced it. In conclusion, we suggest that Rho has a dual effect on the invasion of osteosarcoma cells by modulating the motility, the ability to degrade ECM and tyrosine phosphorylation of FAK.
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PMID:Small GTP-binding protein, Rho, both increased and decreased cellular motility, activation of matrix metalloproteinase 2 and invasion of human osteosarcoma cells. 1134 66

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.
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PMID:Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to Apo2L/TRAIL-induced apoptosis. 1199 38

Growth hormone (GH) and 1alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) are regulators of bone growth and bone metabolism. In target cells, GH activates several signaling pathways, among them the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. GH mainly activates JAK2 and STAT5a and b. The effects of 1,25-(OH)(2)D(3) are mediated via a nuclear receptor, the vitamin D receptor, which, when bound by 1,25-(OH)(2)D(3), activates the transcription of target genes. In earlier studies (Morel, G., Chavassieux, P., Barenton, B., Dubois, P. M., Meunier, P. J., and Boivin, G. (1993) Cell Tissue Res. 273, 279-286) synergistic interaction between 1,25-(OH)(2)D(3) and GH regarding expression of osteoblastic markers has been described. The UMR 106 cell line is a rat osteosarcoma cell line with osteoblast-like properties. We have recently shown (Morales, O., Lindgren, U., and Haldosen, L. A. (2000) J. Bone Miner. Res. 15, 2284-2290) that UMR 106 cells express a GH-responsive JAK2/STAT5 signaling system. These cells also express the vitamin D receptor and respond to 1,25-(OH)(2)D(3). In the present study we have investigated whether 1,25-(OH)(2)D(3) influences GH signaling via the JAK2/STAT5 pathway in UMR 106 cells. We found that 1,25-(OH)(2)D(3) prolonged GH signaling via the JAK2/STAT5 pathway. Pretreatment of cells with 1,25-(OH)(2)D(3) was also necessary in order to detect GH-induced STAT5 transcriptional response. Furthermore, the pretreatment of cells with 1,25-(OH)(2)D(3) rendered to the cells the capacity to respond to repetitive GH-stimulation. In UMR 106 cells, GH induced the expression of the JAK/STAT negative regulatory proteins SOCS-3 and CIS. Interestingly, pretreatment with 1,25-(OH)(2)D(3) inhibited GH-induced expression of these proteins. From these results we propose that 1,25-(OH)(2)D(3) has an inhibitory effect on negative regulatory pathways acting on JAK2 and/or STAT5 in UMR 106 cells and that this, in all or partly, explains the effects of 1,25-(OH)(2)D(3) on GH-signaling via the JAK/STAT pathway.
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PMID:1Alpha,25-dihydroxyvitamin D3 inhibits GH-induced expression of SOCS-3 and CIS and prolongs growth hormone signaling via the Janus kinase (JAK2)/signal transducers and activators of transcription (STAT5) system in osteoblast-like cells. 1210 79

Signal transduction downstream HGF receptor (MET) activation involves multiple pathways that account for mitogenesis, motility and morphogenesis in a cell type-dependent fashion. MET receptor is aberrantly expressed in almost 100% of human osteosarcomas. We analyzed the effect of the MET receptor activation in five human osteosarcoma cell lines evaluating the levels of HGF-dependent activation of MAPK and PKB/AKT as biochemical readouts of mitogenic and invasive responses, respectively. All the cell lines tested expressed high levels of the MET proto-oncogene. Four cell lines showed activation of the MAPK cascade upon HGF stimulation, suggesting that this growth factor serves a common proliferative function in osteosarcomas. Two lines showed activation of PKB/AKT that is known to be involved in migration mediated by HGF receptor. Accordingly, cell lines where MAPK cascade was activated responded to HGF with increased proliferation, while induction and inhibition of PKB/AKT activity corresponded to acquisition or block of the invasive-motile response to HGF, respectively. Both the HGF dependent responses were reverted by the specific MET inhibitor K252a. These data show that HGF activates both the mitogen and motogen machinery in osteosarcoma cells and suggest that HGF might promote their malignant behavior by concomitant activation of different pathways and biological functions.
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PMID:Role of the MET/HGF receptor in proliferation and invasive behavior of osteosarcoma. 1270 13

The aim of this study was to investigate the cytotoxic activity of the third-generation nitrogen-containing bisphosphonate zoledronic acid (ZOL) as a single agent, and in combination with clinically relevant anticancer drugs, in a panel of human osteogenic sarcoma cell lines (HOS, BTK-143, MG-63, SJSA-1, G-292, and SAOS2). We found that ZOL, when used alone, reduced cell number in a dose- and time-dependent manner, due either to cell cycle arrest in S-phase or to the induction of apoptosis. In the sensitive HOS, BTK-143, and G-292 cell lines, genomic DNA fragmentation and morphological changes characteristic of apoptosis were evident, and cells became nonadherent. Induction of apoptosis in osteosarcoma cells by ZOL was associated with caspase activation. However, coaddition of the broad-spectrum caspase inhibitors, z-VAD-fmk, Boc-D-fmk, or the caspase-3-specific inhibitor z-DEVD fmk, failed to protect these cells from ZOL-induced apoptosis. Our data support a ZOL-specific induction of cell apoptosis that involves cell detachment (anoikis), and in which caspase activation occurs secondarily to, and is redundant as a mediator of cell death. The addition of geranylgeraniol, an intermediate of the mevalonate pathway, suppressed the ZOL-induced apoptosis, suggesting that the cytotoxic effects of ZOL in osteosarcoma cells were mediated by the mevalonate pathway. While treatment of osteosarcoma cells with the chemotherapeutic agents doxorubicin or etoposide decreased cell viability, combination of these agents with ZOL did not significantly augment apoptosis in any of the cell lines tested. These observations suggest that ZOL has direct effects on the proliferation and survival of osteosarcoma cells in vitro, which has implications for future therapy of osteosarcoma.
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PMID:Induction of cell death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis. 1449 55

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.
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PMID:Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques. 1458 36


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