Search PubMed⌕ Search

Biomedical subjects

M J Weber

Publications and source records attributed to M J Weber.

At least 19 recordsLinked to original sources
PubMed2006

Conditional expression of PTEN alters the androgen responsiveness of prostate cancer cells.

BACKGROUND: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is lost as a function of prostate tumor androgen dependence. While the transcriptional activity of the androgen receptor (AR) is inhibited by PTEN in androgen sensitive prostate cancer (CaP), the role of PTEN in androgen disease is unclear. METHODS: We developed a system where PTEN can be conditionally re-expressed at physiologic levels into a PTEN null metastatic human CaP cell line, C4-2, and androgen responsiveness examined. RESULTS: PTEN induction reduces cell growth and blocks the growth effect of synthetic androgen R1881. The anti-androgen Casodex enhances the growth-inhibitory action of PTEN and this effect is independent of Akt phosphorylation. Combined PTEN induction and Casodex, result in a further decrease in prostate specific antigen promoter activity compared to PTEN but not Casodex alone. CONCLUSIONS: PTEN induction confers androgen independent CaP cells enhanced responsiveness to the anti-proliferative effects of anti-androgens and this action may involve non-AR mediated effects.

Androgen Antagonists↗
PubMed2005

The serine/threonine protein kinase, p90 ribosomal S6 kinase, is an important regulator of prostate cancer cell proliferation.

An increase in the activity of mitogen-activated protein kinase (MAPK) has been correlated with the progression of prostate cancer to advanced disease in humans. The serine/threonine protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of MAPK but its role in prostate cancer has not previously been examined. Increasing RSK isoform 2 (RSK2) levels in the human prostate cancer line, LNCaP, enhanced prostate-specific antigen (PSA) expression, an important diagnostic marker for prostate cancer, whereas inhibiting RSK activity using a RSK-specific inhibitor, 3Ac-SL0101, decreased PSA expression. The RSK2 regulation of PSA expression occurred via a mechanism involving both RSK2 kinase activity and its ability to associate with the coactivator, p300. RNA interference of the androgen receptor (AR) showed that the AR was important in the RSK2-mediated increase in PSA expression. RSK levels are higher in approximately 50% of human prostate cancers compared with normal prostate tissue, which suggests that increased RSK levels may participate in the rise in PSA expression that occurs in prostate cancer. Furthermore, 3Ac-SL0101 inhibited proliferation of the LNCaP line and the androgen-independent human prostate cancer line, PC-3. These results suggest that proliferation of some prostate cancer cells is dependent on RSK activity and support the hypothesis that RSK may be an important chemotherapeutic target for prostate cancer.

Cell Growth Processes↗
PubMed2001

Neuronal promoter of human aromatic L-amino acid decarboxylase gene directs transgene expression to the adult floor plate and aminergic nuclei induced by the isthmus.

In order to analyze the regulatory sequences involved in the neuronal expression of aromatic L-amino acid decarboxylase (AADC), we have generated transgenic mice carrying the LacZ gene under the control of a 3.6-kb human aadc genomic fragment flanking the neuronal alternative first exon. A series of double labeling experiments were performed to compare the pattern of transgene expression to that of specific markers for catecholaminergic and serotonergic neurons. In the adult brain parenchyma, transgene expression was observed in the substantia nigra (SN), the ventral tegmental area (VTA) and the dorsal, medial and pontine raphe nuclei. A large degree of co-expression was observed with tyrosine-hydroxylase (TH) in the SN and VTA, and with serotonin (5-HT) in the dorsal raphe nucleus. Moreover, expression was observed in cells that were both TH- and 5-HT-negative, in particular in the ventral tegmental decussation and the dorsal tip of the VTA. Transgene expression was also observed in the walls of central cavities. Cells positive for both beta-gal and PSA-NCAM were localized in the ventral ependyma of the third and fourth ventricle, and of the central canal of the spinal cord, in what appears to be the adult floor plate. Transgene expressing, PSA-NCAM negative, cells located along the ventral midline of the spinal cord seemed to have migrated out of the ependyma. Our data thus reveal the complexity of aadc gene regulation. The present transgene provides a unique marker for monoaminergic nuclei induced by the isthmus and for the adult floor plate.

Animals↗
PubMed2001

Lu2SiO5 by single-crystal X-ray and neutron diffraction.

The structure of dilutetium silicon pentaoxide, Lu2SiO5, has isolated ionic SiO4 tetrahedral units and non-Si-bonded O atoms in distorted OLu4 tetrahedra. The OLu4 tetrahedra form edge-sharing infinite chains and double O2Lu6 tetrahedra along the c axis. The edge-sharing chains are connected to the O2Lu6 double tetrahedra by isolated SiO4 units. The structure has been determined by neutron diffraction.

Crystallography, X-Ray↗
PubMed2001

NF-Y binding is required for transactivation of neuronal aromatic L-amino acid decarboxylase gene promoter by the POU-domain protein Brn-2.

We have previously characterized binding sites for the NF-Y transcription factor (-71/-52) and Brn-2 POU-domain protein (-92/-71) in the neuronal promoter of the human aromatic L-amino acid decarboxylase gene [Mol. Brain Res. 56 (1998) 227]. We have now explored the functional role of these binding sites in transfected SK-N-BE neuroblastoma cells. Mutations of the NF-Y site that abolish binding depressed expression of a luciferase reporter gene up to 25-fold. The overexpression of a dominant negative mutant of NF-YA subunit depressed expression by 60%. Promoter activity was increased by the overexpression of Brn-2. Mutations or deletion of the binding site of Brn-2 did not suppress transcriptional activation by overexpressed Brn-2, while promoters defective in NF-Y binding were not transactivated by Brn-2. A GST-pulldown experiment showed that recombinant human Brn-2 protein weakly interacts with recombinant NF-Y outside of DNA. Cooperative binding of recombinant NF-Y and GST--Brn-2 proteins on the neuronal promoter was evidenced by an electrophoretic mobility shift assay. The POU-domain of Brn-2 was sufficient for such interaction. The results thus suggest that the activation of the neuronal promoter of the aromatic L-amino acid decarboxylase gene requires a direct interaction between the ubiquitous NF-Y factor and a cell-specific POU-domain protein. The NF-Y, but not the Brn-2 binding site, is essential for the recruitment of the NF-Y/Brn-2 complex on the promoter.

Animals↗
PubMed2001

Tumor necrosis factor alpha induces BID cleavage and bypasses antiapoptotic signals in prostate cancer LNCaP cells.

Survival of cancer cells in response to therapy, immune response, or metastasis depends on interactions between pro- and antiapoptotic signals. Two major proapoptotic pathways have been described: (a) a death receptor pathway; and (b) a mitochondrial pathway. We reported previously that Akt and the epidermal growth factor (EGF) receptor send separate, redundant survival signals that act to inhibit the mitochondrial proapoptotic pathway in prostate cancer LNCaP cells. However, it was unclear at what level the pro- and antiapoptotic signals interact in these cells, and it was also unclear whether these signals would inhibit the death receptor pathway. We found that EGF can protect LNCaP cells from apoptosis induced by LY294002 but not from tumor necrosis factor a (TNF-alpha)-induced apoptosis. Furthermore, TNF-alpha induced apoptosis under conditions in which Akt was active. Treatment with TNF-alpha resulted in activation of caspase 8 and cleavage of BID, which in turn induced cytochrome c release and caspase 9-dependent activation of effector caspases. Thus, proapoptotic signals induced by both TNF-alpha and LY294002 converge on mitochondria and trigger cytochrome c release. Because EGF can inhibit cytochrome c release induced by LY294002 but not cytochrome c release induced by TNF-alpha, we suggest that the EGF survival mechanism operates on the mitochondrial pathway at a site upstream of cytochrome c release. The ability of TNF-alpha to bypass survival signals from activated EGF receptor and Akt in prostate cancer cells makes death receptor signaling a promising avenue for therapeutic intervention.

Apoptosis↗
PubMed2001

Biochemical and biological functions of the N-terminal, noncatalytic domain of extracellular signal-regulated kinase 2.

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 are important components in signal transduction pathways involved in many cellular processes, including cell differentiation and proliferation. These proteins consist of a central kinase domain flanked by short N- and C-terminal noncatalytic domains. While the regulation of ERK2 by sequences within the kinase domain has been extensively studied, little is known about the small regions outside of the kinase domain. We performed mutational analysis on the N-terminal, noncatalytic domain of ERK2 in an attempt to determine its role in ERK2 function and regulation. Deleting or mutating amino acids 19 to 25 (ERK2-Delta19-25) created an ERK2 molecule that could be phosphorylated in response to growth factor and serum stimulation in a MEK (mitogen-activated protein kinase kinase or ERK kinase)-dependent manner but had little kinase activity and was unable to bind to MEK in vivo. Since MEK acts as a cytoplasmic anchor for the ERKs, the lack of a MEK interaction resulted in the aberrant nuclear localization of ERK2-Delta19-25 mutants in serum-starved cells. Assaying these mutants for their ability to affect ERK signaling revealed that ERK2-Delta19-25 mutants acted in a dominant-negative manner to inhibit transcriptional signaling through endogenous ERKs to an Elk1-responsive promoter in transfected COS-1 cells. However, ERK2-Delta19-25 had no effect on the phosphorylation of RSK2, an ERK2 cytoplasmic substrate, whereas a nonactivatable ERK (T183A) that retained these sequences could inhibit RSK2 phosphorylation. These results suggest that the N-terminal domain of ERK2 profoundly affects ERK2 localization, MEK binding, kinase activity, and signaling and identify a novel dominant-negative mutant of ERK2 that can dissociate at least some transcriptional responses from cytoplasmic responses.

Amino Acid Sequence↗
PubMed2000

Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway.

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.

Alkyl and Aryl Transferases↗
PubMed2000

Urokinase-type plasminogen activator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells.

Urokinase-type plasminogen activator (uPA) stimulates MCF-7 cell migration by binding to the UPA receptor and activating the Ras-extracellular signal-regulated kinase (Ras-ERK) signaling pathway. Studies presented here show that soluble uPA receptor and a peptide derived from the linker region between domains 1 and 2 of the uPA receptor also stimulate cellular migration via a mitogen-activated protein kinase/ERK kinase (MEK)-dependent pathway. Signaling proteins that function upstream of Ras in uPA- stimulated cells remain undefined. To address this problem, we transfected MCF-7 cells to express the noncatalytic carboxylterminal domain of focal adhesion kinase (FAK), FAK(Y397F), kinase-defective c-Src, or Shc FFF, all of which express dominant-negative activity. In each case, ERK phosphorylation and cellular migration in response to uPA were blocked. Both activities were rescued by co-transfecting the cells to express constitutively active MEK1, indicating that FAK, c-Src, and Shc are upstream of MEK. Shc was tyrosine-phosphorylated in uPA-treated cells. The level of phosphorylated Shc was increased within 1 min and remained increased for at least 30 min. Sos co-immunoprecipitated with Shc in cells that were treated with uPA for 1-2.5 min, probably reflecting the formation of Shc-Grb2/Sos complex; however, by 10 min, co-immunoprecipitation of Sos with Shc was no longer observed. Rapid dissociation of Sos from Shc represents a possible mechanism for the transient phosphorylation of ERK in uPA-treated MCF-7 cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals.

Mitogen-activated protein kinase (MAPK) cascades are activated by diverse extracellular signals and participate in the regulation of an array of cellular programs. In this study, we investigated the roles of MAPKs in the induction of phase II detoxifying enzymes by chemicals. Treatment of human hepatoma (HepG2) and murine hepatoma (Hepa1c1c7) cells with tert-butylhydroquinone (tBHQ) or sulforaphane (SUL), two potent phase II enzyme inducers, stimulated the activity of extracellular signal-regulated protein kinase 2 (ERK2) but not c-Jun N-terminal kinase 1. tBHQ and SUL also activated MAPK kinase. Inhibition of MAPK kinase with its inhibitor, PD98059, abolished ERK2 activation and impaired the induction of quinone reductase, a phase II detoxifying enzyme, and antioxidant response element (ARE)-linked reporter gene by tBHQ and SUL. Overexpression of a dominant-negative mutant of ERK2 also attenuated tBHQ and SUL induction of ARE reporter gene activity. Interestingly, although expression of Ras and its mutant forms showed distinct effects on basal ARE reporter gene activity, they did not affect the activation of reporter gene by the inducers. Furthermore, a dominant-negative mutant of Ras had little effect on ERK2 activation by tBHQ and SUL, implicating a Ras-independent mechanism. Indeed, both tBHQ and SUL were able to stimulate Raf-1 kinase activity in vivo as well as in vitro. Thus, our results indicate that the induction of ARE-dependent phase II detoxifying enzymes is mediated by a MAPK pathway, which may involve direct activation of Raf-1 by the inducers.

Animals↗
PubMed1999

c-Raf-mediated inhibition of epidermal growth factor-stimulated cell migration.

Epidermal growth factor stimulates migration of a number of cell types, yet the signaling pathways that regulate epidermal growth factor-stimulated migration are poorly defined. In this report, we employ a transient transfection migration assay to assess the role of components of the Ras-mitogen-activated protein (MAP) kinase signaling pathway in epidermal growth factor-stimulated chemotaxis of rat embryo fibroblasts. Expression of dominant negative Ras blocks epidermal growth factor-mediated chemotaxis, while constitutively active Ras has no effect on chemokinesis or chemotaxis. PD98059 and U0126, inhibitors of MAP kinase kinase (MEK) activity, decreased epidermal growth factor-stimulated migration, while kinase-defective MEK1, an inhibitor of MAP kinase activation, enhanced migration. To understand the paradoxical effects of these molecules on epidermal growth factor-induced migration, we examined the role of c-Raf on migration. Expression of either wild type c-Raf or the catalytic domain of c-Raf effectively inhibited epidermal growth factor-stimulated cell migration. We suggest that, whereas Ras activity is necessary to promote epidermal growth factor-stimulated migration, sustained activation of c-Raf may be important in down-regulating migratory signaling pathways triggered by epidermal growth factor receptor activation. Further, activation of c-Raf upon inhibition of the MEK-MAP kinase pathway may contribute to the inhibition of cell migration observed with pharmacological MEK inhibitors.

Animals↗
PubMed1999

Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

Antibodies↗
PubMed1999

Antiapoptotic signaling in LNCaP prostate cancer cells: a survival signaling pathway independent of phosphatidylinositol 3'-kinase and Akt/protein kinase B.

Constitutive activation of the phosphatidylinositol 3'-kinase (PI3 kinase)-Akt/protein kinase B (PKB) "survival signaling" pathway is a likely mechanism by which many cancers become refractory to cytotoxic therapy. In LNCaP prostate cancer cells, the PTEN phosphoinositide phosphatase is inactivated, leading to constitutive activation of Akt/PKB and resistance to apoptosis. However, apoptosis and inactivation of Akt/PKB can be induced in these cells by treatment with PI3 kinase inhibitors. Surprisingly, androgen, epidermal growth factor, or serum can protect these cells from apoptosis, even in the presence of PI3 kinase inhibitors and without activation of Akt/PKB, indicating the activity of a novel, Akt/PKB-independent survival pathway. This pathway blocks apoptosis at a level prior to caspase 3 activation and release of cytochrome c from mitochondria.

Apoptosis↗
PubMed1999

A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways.

Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms.

Animals↗
PubMed1999

Activation of mitogen-activated protein kinase associated with prostate cancer progression.

Using an antibody specific for dually phosphorylated extracellular-regulated kinases 1 and 2, we have examined 82 primary and metastatic prostate tumor specimens for the presence of activated mitogen-activated protein (MAP) kinase. Nonneoplastic prostate tissue showed little or no staining with activated MAP kinase antiserum. In prostate tumors, the level of activated MAP kinase increased with increasing Gleason score and tumor stage. In a separate analysis, tumor samples from two patients showed no activation of MAP kinase before androgen ablation therapy; however, following androgen ablation treatment, high levels of activated MAP kinase were detected in the recurrent tumors. Collectively, these data suggest an increase in the activation of the MAP kinase signal transduction pathway as prostate cancer progresses to a more advanced and androgen-independent disease.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Elevation of cyclic adenosine 3',5'-monophosphate potentiates activation of mitogen-activated protein kinase by growth factors in LNCaP prostate cancer cells.

Prostate cells are simultaneously exposed to a variety of peptide growth factors and neuropeptides that elevate cAMP. Both the growth factors and cAMP have large effects on the growth, differentiation, and movement of many cell types. Because mitogen-activated protein kinase (MAPK) is central to these effects, we analyzed the ways in which these agonists interact in regulating MAPK in prostate cancer cells. We show that, in LNCaP prostate cancer cells, elevation of intracellular cAMP can potentiate the ability of epidermal growth factor (EGF), interleukin 6, and serum to activate MAPK and that this potentiation depends on protein kinase A and Rap1. The response to cAMP is different in the androgen-independent prostate cancer cell line PC-3, where elevation of cAMP slightly inhibits MAPK activation by EGF. We also show that treatment of LNCaP with the calcium ionophore A23187 or the phorbol ester phorbol 12-myristate 13-acetate activates MAPK, but the activation of MAPK by these agonists is inhibited rather than potentiated by increasing cAMP. Finally, we show that phorbol 12-myristate 13-acetate and interleukin 6 can potentiate the signaling activity of EGF. We conclude that neuroendocrine factors that elevate cAMP sensitize LNCaP prostate cancer cells to signaling by peptide growth factors and that low levels of mixtures of growth factors can activate intracellular signaling to a greater degree than would be predicted from the activity of the individual agonists.

Blood Proteins↗