Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin

There is an urgent need to develop non-invasive pharmacodynamic endpoints for the evaluation of new molecular therapeutics that inhibit signal transduction. We hypothesised that, when labelled appropriately, changes in choline kinetics could be used to assess geldanamycin pharmacodynamics, which involves inhibition of the HSP90 molecular chaperone→Raf1→Mitogenic Extracellular Kinase→Extracellular Signal-Regulated Kinase 1 and 2 signal transduction pathway. Towards identifying a potential pharmacodynamic marker response, we have studied radiolabelled choline metabolism in HT29 human colon carcinoma cells following treatment with geldanamycin. We studied the effects of geldanamycin, on net cellular accumulation of (methyl-14C)choline and (methyl-14C)phosphocholine production. In parallel experiments, the effects of geldanamycin on extracellular signal-regulated kinase 1 and 2 phosphorylation and cell viability were also assessed. Additional validation studies were carried out with the mitogenic extracellular kinase inhibitor U0126 as a positive control; a cyclin-dependent kinase-2 inhibitor roscovitine and the phosphatidylinositol 3-kinase inhibitor LY294002 as negative controls. Hemicholinium-3, an inhibitor of choline transport and choline kinase activity was included as an additional control. In exponentially growing HT29 cells, geldanamycin inhibited extracellular signal-regulated kinase 1 and 2 phosphorylation in a concentration- and time-dependent manner. These changes were associated with a reduction in (methyl-14C)choline uptake, (methyl-14C) phosphocholine production and cell viability. Brief exposure to U0126, suppressed phosphocholine production to the same extent as Hemicholinium-3. In contrast to geldanamycin and U0126, which act upstream of extracellular signal-regulated kinase 1 and 2, roscovitine and LY294002 failed to suppress phosphocholine production. Our results suggest that when labelled with carbon-11 isotope, (methyl-11C)choline may be a useful pharmacodynamic marker for the non-invasive evaluation of geldanamycin analogues

Downstream signalling and specific inhibition of c-MET/HGF pathway in small cell lung cancer: implications for tumour invasion

The c-MET receptor can be overexpressed, amplified, or mutated in solid tumours including small cell lung cancer (SCLC). In c-MET-overexpressing SCLC cell line NCI-H69, hepatocyte growth factor (HGF) dramatically induced c-MET phosphorylation at phosphoepitopes pY1230/1234/1235 (catalytic tyrosine kinase), pY1003 (juxtamembrane), and also of paxillin at pY31 (CRKL-binding site). We utilised a global proteomics phosphoantibody array approach to identify further c-MET/HGF signal transduction intermediates in SCLC. Strong HGF induction of specific phosphorylation sites in phosphoproteins involved in c-MET/HGF signal transduction was detected, namely adducin-α [S724], adducin-γ [S662], CREB [S133], ERK1 [T185/Y187], ERK1/2 [T202/Y204], ERK2 [T185/Y187], MAPKK (MEK) 1/2 [S221/S225], MAPKK (MEK) 3/6 [S189/S207], RB [S612], RB1 [S780], JNK [T183/Y185], STAT3 [S727], focal adhesion kinase (FAK) [Y576/S722/S910], p38α-MAPK [T180/Y182], and AKT1[S473] and [T308]. Conversely, inhibition of phosphorylation by HGF in protein kinase C (PKC), protein kinase R (PKR), and also CDK1 was identified. Phosphoantibody-based immunohistochemical analysis of SCLC tumour tissue and microarray established the role of c-MET in SCLC biology. This supports a role of c-MET activation in tumour invasive front in the tumour progression and invasion involving FAK and AKT downstream. The c-MET serves as an attractive therapeutic target in SCLC, as shown through small interfering RNA (siRNA) and selective prototype c-MET inhibitor SU11274, inhibiting the phosphorylation of c-MET itself and its downstream molecules such as AKT, S6 kinase, and ERK1/2. Investigation of mechanisms of invasion and, ultimately, metastasis in SCLC would be very useful with these signal transduction molecules

Institutional shared resources and translational cancer research

The development and maintenance of adequate shared infrastructures is considered a major goal for academic centers promoting translational research programs. Among infrastructures favoring translational research, centralized facilities characterized by shared, multidisciplinary use of expensive laboratory instrumentation, or by complex computer hardware and software and/or by high professional skills are necessary to maintain or improve institutional scientific competitiveness. The success or failure of a shared resource program also depends on the choice of appropriate institutional policies and requires an effective institutional governance regarding decisions on staffing, existence and composition of advisory committees, policies and of defined mechanisms of reporting, budgeting and financial support of each resource. Shared Resources represent a widely diffused model to sustain cancer research; in fact, web sites from an impressive number of research Institutes and Universities in the U.S. contain pages dedicated to the SR that have been established in each Center, making a complete view of the situation impossible. However, a nation-wide overview of how Cancer Centers develop SR programs is available on the web site for NCI-designated Cancer Centers in the U.S., while in Europe, information is available for individual Cancer centers. This article will briefly summarize the institutional policies, the organizational needs, the characteristics, scientific aims, and future developments of SRs necessary to develop effective translational research programs in oncology

Insulin-like growth factor-I induces the phosphorylation and nuclear exclusion of forkhead transcription factors in human neuroblastoma cells

Akt-mediated phosphorylation of forkhead transcription factors is linked to growth factor-stimulated cell survival. We investigated whether the survival activity of insulin-like growth factor-I (IGF-I) in SH-SY5Y human neuroblastoma (NBL) cells is associated with phosphorylation and/or localization changes in forkhead proteins. IGF-I induced phosphorylation of Erks (p42/p44), FKHR (FOXO1a) (Ser 253), FKHRL1 (FOXO3a) (Ser 256), and Akt (Ser 473). PI3-K inhibitor, LY294002, reduced IGF-I-stimulated phosphorylation of FKHR, FKHRL1, and Akt, but did not affect Erk phosphorylation. Using a GFP-FKHR construct, FKHR imported into the nucleus during growth factor withdrawal-induced apoptosis. In addition, IGF-I rescue from serum withdrawal-induced apoptosis is associated with a rapid export of GFP-FKHR into the cytoplasm. Leptomycin B, an inhibitor of Crm1-mediated nuclear export, decreased the level of FKHRL1 phosphorylation in the presence of IGF-I in vector and FKHR overexpressing cells, but had no effect on the phosphorylation status of FKHR. In addition, leptomycin B prevented IGF-I stimulated nuclear export of GFP-FKHR. These studies show IGF-I phosphorylation of FKHR and FKHRL1 via a PI3-K-dependent pathway in NBL cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44353/1/10495_2005_Article_429.pd

Profiling of protein kinases in the neoplastic transformation of human ovarian surface epithelium

Objective. The aim of this study was to study the pattern of protein kinase expression in a culture model of epithelial ovarian carcinogenesis. Methods. Cultures of normal human ovarian surface epithelium (OSE), OSE from women with BRCA1 mutations, a cell culture model of preneoplastic (SV40 T-antigen-immortalized, nontumorigenic) and neoplastic (SV40-E-cadherin transfected, tumorigenic) OSE, and three ovarian cancer cell lines were used to represent OSE phenotypes of different genetic backgrounds and at different, progressive stages of neoplastic transformation. The protein kinase network signaling was studied by Western blotting, simultaneously using multiple antibodies for specific protein kinases. Results. High levels of cGMP-dependent protein kinase were found in normal and preneoplastic OSE, but were absent in neoplastic OSE. In contrast, expression of MEK6 was detected exclusively in neoplastic OSE. The expressions of casein kinase II (CK2), p38 mitogen-activated protein kinase (MAPK), cyclin-dependent kinase, and the phosphatidylinositol 3-kinase (PI3K) effectors Akt2 and p70 S6 kinase (S6K) were several-fold higher in neoplastic OSE than in normal OSE, whereas the expressions of the MAPKs extracellular signal-regulated kinases ERK1 and -2 were unchanged. Importantly, constitutive phosphorylation of Akt2 and p70 S6K, as found in neoplastic OSE, was also observed in overtly normal OSE from women with predisposing BRCA1 gene mutations. Conclusions. These data demonstrate that different repertoires of downstream signaling proteins, particularly those of the MEK6-p38 MAPK-CK2 pathway and the PI3K pathway, are correlated with phenotypic manifestations of a cell culture model of OSE at progressive stages in the development of ovarian cancer. Changes in PI3K effectors are already found in overtly normal OSE from women with BRCA1 mutations. © 2001 Academic Press.link_to_subscribed_fulltex