Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0684249 (lung carcinoma)
 23,830 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR-complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down-modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.
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PMID:Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells. 751 43

Non-metastatic Lewis lung carcinoma cells (LLC-C8) become more motile when protein phosphatases (PP-1 and -2A) are inhibited by okadaic acid, attaining the same level of motility as metastatic LLC (LLC-LN7) variants. This stimulation of LLC-C8 motility was tempered when protein kinase A activity was inhibited. We examined whether the okadaic acid-stimulated LLC-C8 motility was associated with alterations in the cytoskeletal organization so that these non-metastatic cells acquire the rounded morphology and diffuse cytoskeletal organization previously described for metastatic LLC-LN7 cells. Non-metastatic LLC-C8 are typically adherent during culture, achieving a spread morphology. Treatment of non-metastatic LLC-C8 cells with okadaic acid resulted in a contraction of most of their extended processes, formation of spikes and membrane blebs within 10 min, and complete cell rounding within 20 min for most of the cells. While the overall level of F-actin was minimally affected by the okadaic acid, its uniform distribution shifted to localization toward the periphery of the rounded cells, often concentrating at a single focus. Immunofluorescent staining for vimentin showed a similar shift to the cell periphery and similar capping. After okadaic acid treatment, the filamentous network of microtubules in non-metastatic LLC-C8 cells disappeared and was replaced with a diffusely staining distribution of beta-tubulin. These results show that PP-1 and -2A maintain cytoskeletal organization and that inhibition of this control reduces cytoskeletal organization and increases tumor cell motility.
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PMID:Regulation of cytoskeletal organization in tumor cells by protein phosphatases-1 and -2A. 753 53

RS7-3G11 is a murine monoclonal antibody (MAb) raised against human non-small-cell lung carcinoma, and is under clinical evaluation. The epithelial/carcinoma antigen EGP-1, defined by RS7-3G11, was isolated and purified to homogeneity from a cervical carcinoma cell line, ME180. EGP-1 is a glycoprotein with an average molecular mass of 47.8 kDa. Metabolic labeling of the antigen with 32P-orthophosphate and subsequent immunoprecipitation with RS7-3G11 showed that it is a phosphoprotein. Phosphoamino acid analysis of the in vivo phosphorylated EGP-1 revealed that the phosphorylation is on serine. In vitro analysis with purified antigen demonstrated that protein kinase C, and not protein kinase A, is involved in phosphorylating the antigen in vitro. In vitro analysis indicated a stoichiometry of phosphorylation of 0.54 mole of phosphate per mole of EGP-1. Phosphoamino acid analysis and phosphopeptide mapping of the antigen phosphorylated in vitro by protein kinase C showed that phosphorylation occurred on a serine residue, specifically on serine 303, located in the cytoplasmic domain of EGP-1. Treatment of ME180 cells with phorbol ester increased the phosphorylation of EGP-1. The biological function of EGP-1 remains to be elucidated. In this report we elucidate an involvement of protein kinase C in phosphorylating EGP-1, which may signify a role for this antigen in signal transduction across the cell membrane.
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PMID:The epithelial/carcinoma antigen EGP-1, recognized by monoclonal antibody RS7-3G11, is phosphorylated on serine 303. 763 74

The involvement of protein kinase c (PKC) in the mechanism underlying the antimetastatic properties of triazenes was studied in C57BL/6 mice bearing Lewis lung carcinoma (3LL). In vivo and in vitro treatment with temozolomide, an in-vitro active analogue of dacarbazine, or calphostin c produced a concentration-dependent reduction of spontaneous and artificial metastases. Both agents reduced the ability of 3LL cells to adhere to endothelium. Diethylaminoethyl (DEAE)-sepharose chromatography of cell extracts revealed that incubation of 3LL cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a rapid translocation of protein kinase c activity from cytosol to the membrane fraction. Membrane PKC activity induced by TPA was reduced by 60% after treatment with temozolomide. Coincident with these changes, TPA induced phosphorylation of alpha-6 integrin, whereas temozolomide or calphostin c abolished the appearance of this phosphoprotein. These results suggest that temozolomide reduced metastatic potential by interfering with alpha-6 phosphorylation induced by PKC activation.
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PMID:Temozolomide reduces the metastatic potential of Lewis lung carcinoma (3LL) in mice: role of alpha-6 integrin phosphorylation. 764 49

Metastatic Lewis lung carcinoma (LLC-LN7) cells have increased protein kinase A (PKA) activity and are more invasive in vitro than are non-metastatic (LLC-C8) cells. To determine whether PKA mediates the in vitro invasiveness and in vivo metastatic capabilities of these tumor cells, the LLC variants were stably transfected to over-express the C alpha subunit of PKA, and thus to have increased PKA activity, or to express a mutant cAMP-resistant PKA R1 alpha subunit which blocks PKA activation. Wild-type LLC-LN7 tumor cells were invasive in vitro and in vivo, recurred after tumor excision and metastasized to the lungs. However, they lost these properties after transfection to express the mutant R1 alpha that blocks PKA activation. The non-invasive, non-recurring and non-metastatic LLC-C8 cells gained the capacity to invade, to recur following tumor excision and to metastasize when transfected to express the PKA C alpha subunit.
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PMID:Regulation of Lewis lung carcinoma invasion and metastasis by protein kinase A. 770 21

Granulocyte-macrophage colony-stimulating factor (GM-CSF) that is produced by metastatic Lewis lung carcinoma (LLC-LN7) cells functions as an autocrine stimulator of tumor-cell motility through protein kinase A (PKA) signal transduction. This GM-CSF-mediated enhancement of LLC-LN7 cell motility coincides with a reduction in the level of polymerized F-actin. In contrast, non-metastatic LLC-C8 tumor cells, which have a diminished level of PKA signaling, do not produce GM-CSF and do not respond to exogenous GM-CSF, since they remain non-motile and retain a high content of filamentous actin. The capacity of PKA to regulate the cytoskeletal organization of tumor cells was further studied with the use of LLC variants that had been stably transfected to over-express the C alpha subunit of PKA (CEV cells) or to express a mutant cAMP-resistant PKA RI alpha subunit resulting in a defective PKA (REV cells). When compared with wild-type metastatic LLC-LN7 cells, in which the F-actin staining was too diffuse to be clearly visualized microscopically, the PKA-defective REV-LN7 transfectants had an increased level of F-actin. In comparison with the wild-type non-metastatic LLC-C8 cells, which had a high content of F-actin, the CEV-C8 transfectants that over-expressed PKA activity had a reduced level of F-actin. The reduced polymerization of actin in these CEV-C8 transfectants was accompanied by reduced levels of the intermediate filament protein vimentin and a shift in the distribution both of F-actin and of vimentin to the periphery of the cells. These results show reduced cytoskeletal organization in metastatic LLC-LN7 cells as compared with that of non-metastatic LLC-C8 cells, and indicate that elevation of PKA activity, either by autologous GM-CSF or by genetic manipulation, diminishes cytoskeletal organization.
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PMID:Activation of the protein kinase a signal transduction pathway by granulocyte-macrophage colony-stimulating factor or by genetic manipulation reduces cytoskeletal organization in Lewis lung carcinoma variants. 831 33

Elevators of cAMP, such as prostaglandin E2 (PGE2), activate protein kinase A (PKA) and induce PKA-stimulated motility and metastasis by metastatic Lewis lung carcinoma cells (LLC-LN7). Non-metastatic LLC (LLC-C8) are unresponsive to cAMP elevation even though they are not deficient in the PKA enzymes. To determine whether this PKA unresponsiveness might be due to increased dephosphorylation by serine/threonine protein phosphatases (PP-1/2A) within non-metastatic LLC-C8, the effects of the PP-1/2A inhibitor okadaic acid on the migration and invasion by non-metastatic LLC-C8 cells was measured. Okadaic acid stimulated motility of non-metastatic LLC-C8 cells to a level that was comparable to that of metastatic LLC-LN7 cells. PGE2 further increased the motility of the non-metastatic LLC-C8 cells when okadaic acid was present, although not in the absence of okadaic acid. The stimulation of motility by okadaic acid was diminished when PKA activity was inhibited. Dose-response studies with concentrations of okadaic acid that selectively inhibited PP-2A or both PP-2A and PP-1 showed a progressive increase in migration of non-metastatic LLC-C8 cells, suggesting that both PP-1 and PP-2A limit their motility. By contrast, metastatic LLC-LN7 cells were more motile than were non-metastatic LLC-C8 cells, but this motility was only marginally affected by okadaic acid. Comparisons of the levels of PP-1/2A enzyme activities in the LLC variants showed more activity in non-metastatic LLC-C8 than in metastatic LLC-LN7 cells. The identity of the PP whose activity was increased in the non-metastatic LLC-C8 was assessed by using okadaic acid, which selectively inhibits PP-2A activity at low concentrations and PP-1 and PP-2A at high concentrations, and calyculin A, which inhibits PP-2A at a similar concentration to that affected by okadaic acid but is more potent at inhibiting PP-1. The inhibition of PP activities by okadaic acid and by calyculin A showed a pattern which suggested the presence both of PP-1 and of PP-2A in non-metastatic LLC-C8 cells, but the presence of PP-1 and a reduction in PP-2A in metastatic LLC-LN7 cells. The sum of these data suggests that PKA-stimulated motility is restricted both by PP-1 and by PP-2A in non-metastatic LLC, and that a deficiency in this restriction results in increased migration and invasion.
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PMID:Protein phosphatases limit tumor motility. 839 79

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) by metastatic Lewis lung carcinoma cells (LLC-LN7) was previously shown to contribute to the maintenance of phenotypic characteristics associated with an increased capacity to metastasize. In the present study, pre-incubation of LLC-LN7 cells with neutralizing anti-GM-CSF antibodies diminished the capacity of the tumor cells to form experimental metastases after i.v. inoculation, while pre-incubation with recombinant GM-CSF (rGM-CSF) increased formation of metastases. In the presence of rGM-CSF, the LLC-LN7 cells exhibited an increased capacity to migrate, invade through a reconstituted basement membrane, and adhere to lung tissue. Studies to identify the signal transduction pathway through which GM-CSF enhanced the in vitro metastatic properties of the LLC-LN7 tumor cells implicated protein kinase A (PKA). Signaling through PKA was suggested by the demonstration that the stimulation of tumor-cell motility by GM-CSF was blocked in the presence of the adenylate cyclase inhibitor nicotinic acid, or the PKA inhibitors A3 or KT5720. In addition, the role of PKA as a signaling mechanism for GM-CSF was assessed by using REV-LN7 cells, which are LLC-LN7 cells that have been stably transfected with an expression vector encoding a mutant PKA RI alpha subunit and which, in turn, express a cAMP-resistant PKA. Adherence and invasion by the PKA-defective REV-LN7 cells were not stimulated by rGM-CSF, contrasting with the stimulation observed for wild-type LLC-LN7 cells. These data suggest that rGM-CSF can further enhance the in vitro metastatic characteristics of LLC-LN7 tumor cells and that this is dependent on signal transduction through PKA.
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PMID:Granulocyte-macrophage colony-stimulating factor stimulates the metastatic properties of Lewis lung carcinoma cells through a protein kinase A signal-transduction pathway. 843 41

Tumor cell adhesion to and migration through the extracellular matrix (ECM) can influence their capacity to disseminate. Since prior studies with Lewis lung carcinoma (LLC) tumors had shown metastatic clones to have more protein kinase A (PKA) activity than nonmetastatic clones, the present study assessed if PKA regulates the interaction between tumor and the ECM, and how this may be associated with the metastatic capacity of the tumor cells. This was accomplished with the use of metastatic (LLC-LN7) and nonmetastatic (LLC-C8) variants that had been stably transfected to overexpress the PKA Calpha subunit or to have blocked PKA activity. Cells with increased PKA activity were less adherent to vitronectin, laminin, and collagen I, and could more readily migrate through these ECM components than could transfectants with reduced PKA activity. PKA did not regulate adhesion to or migration through fibronectin, and did not appear to be associated with changes in expression of surface integrins. In addition to modulating tumor adhesion and migration in vitro, PKA activation caused an increased formation of metastases from s.c. tumors, but did not regulate formation of experimental metastases by i.v. injected tumor cells. These results suggest that PKA signaling is important for modulating the tumor-ECM interaction and can facilitate tumor transit from the primary tumor site.
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PMID:Protein kinase A regulates Lewis lung carcinoma adherence to extracellular matrix components and spontaneous metastasis. 867 86

Cell cycle progression requires activation of different cyclin-dependent kinases (CDKs) which are positively regulated by cyclins and negatively regulated by CDK inhibitors. Growth inhibition of the Calu-1 lung carcinoma cells induced with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, is associated with G2/M arrest and induction of expression of a novel, faster-migrating form of p21(WAF1/CIP1/SDI1) (p21) protein, an inhibitor of cyclin-dependent kinases. This faster-migrating p21 protein was also expressed in TPA-treated A549 lung carcinoma cells which also exhibited G2/M arrest but not in TPA-treated U937 leukemia cells, which only expressed a slower-migrating form of p21 protein. However, reverse transcriptase-polymerase chain reaction and Southern analysis demonstrated no evidence of novel splice in TPA-treated Calu-1 cells. On the other hand, immunoblotting analysis demonstrated that the faster-migrating p21 protein could be detected only by peptide antibody directed against the N terminus but not the C terminus, suggestive of truncation of the latter or protein modification that results in the loss of the C-terminal epitope. Correlation of G2/M arrest with expression of the faster-migrating p21 protein suggests that this novel form of p21 protein may be a mediator of G2/M arrest and growth inhibition.
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PMID:Novel form of p21(WAF1/CIP1/SDI1) protein in phorbol ester-induced G2/M arrest. 893 83


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