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Peter J Roberts

Publications and source records attributed to Peter J Roberts.

At least 19 recordsLinked to original sources
PubMed2006

Mechanisms of glutamate receptor induced proliferation of astrocytes.

Astrocytes express mainly metabotropic glutamate receptor 3 and metabotropic glutamate receptor 5 receptor subtypes, which show opposing effects on cellular proliferation upon activation. In this study, we investigated the mechanisms by which activation of these receptors modulates astrocyte proliferation. Activation of metabotropic glutamate receptor 5 with (S)-3,5-dihydroxyphenylglycine increased phospholipase D activity in astrocytes as well as astrocyte proliferation. The 3,5-dihydroxyphenylglycine-induced proliferation was inhibited in the presence of the metabotropic glutamate receptor 5 antagonist (2-methyl-6-(phenylethynyl)pyridine), the protein kinase C inhibitor GF109203X, brefeldin A and 1-butanol. Activation of metabotropic glutamate receptor 3 with (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine-IV (DCG-IV) inhibited astrocyte proliferation without affecting metabotropic glutamate receptor 5-mediated phospholipase D activity. Metabotropic glutamate receptor 3 activation, however, only partially inhibited metabotropic glutamate receptor 5-mediated proliferation. In conclusion, metabotropic glutamate receptor 5 stimulates astrocyte proliferation via a protein kinase C-phospholipase D-phosphatidic acid-dependent pathway, whereas metabotropic glutamate receptor 3-mediated inhibition of astrocyte proliferation does not involve phospholipase D, and is independent of metabotropic glutamate receptor 5-mediated effects.

Animals↗
PubMed2006

Molecular correlates of imatinib resistance in gastrointestinal stromal tumors.

PURPOSE: Gastrointestinal stromal tumors (GISTs) commonly harbor oncogenic mutations of the KIT or platelet-derived growth factor alpha (PDGFRA) kinases, which are targets for imatinib. In clinical studies, 75% to 90% of patients with advanced GISTs experience clinical benefit from imatinib. However, imatinib resistance is an increasing clinical problem. PATIENTS AND METHODS: One hundred forty-seven patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib. Specimens from pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. Secondary kinase mutations of KIT or PDGFRA that were identified in imatinib-resistant GISTs were biochemically profiled for imatinib sensitivity. RESULTS: Molecular studies were performed using specimens from 10 patients with primary and 33 patients with secondary resistance. Imatinib-resistant tumors had levels of activated KIT that were similar to or greater than those typically found in untreated GISTs. Secondary kinase mutations were rare in GISTs with primary resistance but frequently found in GISTs with secondary resistance (10% v 67%; P = .002). Evidence for clonal evolution and/or polyclonal secondary kinase mutations was seen in three (18.8%) of 16 patients. Secondary kinase mutations were nonrandomly distributed and were associated with decreased imatinib sensitivity compared with typical KIT exon 11 mutations. Using RNAi technology, we demonstrated that imatinib-resistant GIST cells remain dependent on KIT kinase activity for activation of critical downstream signaling pathways. CONCLUSION: Different molecular mechanisms are responsible for primary and secondary imatinib resistance in GISTs. These findings have implications for future approaches to the growing problem of imatinib resistance in patients with advanced GISTs.

Antineoplastic Agents↗
PubMed2005

Synthesis and pharmacology of willardiine derivatives acting as antagonists of kainate receptors.

The natural product willardiine (8) is an AMPA receptor agonist while 5-iodowillardiine (10) is a selective kainate receptor agonist. In an attempt to produce antagonists of kainate and AMPA receptors analogues of willardiine with substituents at the N3 position of the uracil ring were synthesized. The N3-4-carboxybenzyl substituted analogue (38c) was found to be equipotent at AMPA and GLUK5-containing kainate receptors in the neonatal rat spinal cord. The N3-2-carboxybenzyl substituted analogue (38a) proved to be a potent and selective GLUK5 subunit containing kainate receptor antagonist when tested on native rat and human recombinant AMPA and kainate receptor subtypes. The GLUK5 kainate receptor antagonist activity was found to reside in the S enantiomer (44a) whereas the R enantiomer (44b) was almost inactive. 5-Iodo substitution of the uracil ring of 44a gave 45, which was found to have enhanced potency and selectivity for GLUK5.

Alanine↗
PubMed2005

Analysis of mGluR1a constitutive internalization using a pulse-chase enzyme-linked immuno-sorbant assay (ELISA).

The surface expression of G protein-coupled receptors is regulated by internalization. For many receptors, a constitutive level of internalization in the absence of agonist has been reported. The constitutive internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse-chase ELISA method that allows the investigation of mGluR1a constitutive internalization. When investigated by pulse-chase ELISA, the constitutive internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse-chase labelling procedure. The application of the pulse-chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive internalization. Interestingly, only DMII underwent significant constitutive internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse-chase ELISA appears to be an efficient tool to analyze the constitutive internalization of different mGluR1 splice variants.

Alanine Transaminase↗
PubMed2005

The significance of tumor markers for proliferation and apoptosis in predicting survival in colorectal cancer.

PURPOSE: Clinicopathologic staging is even today the best prognostic factor in both colon and rectal cancers. There is still considerable variation in survival within the stages. To find other prognostic indicators we investigated six biologic markers associated with apoptosis and cell proliferation. METHODS: Formalin-fixed, paraffin-embedded tissue samples of 363 patients with primary colon or rectal cancer of Dukes Stages A to D were chosen for immunohistochemical staining of five tumor markers: bcl-2, p53, Ki-67, cyclin D1, and carcinoembryonic antigen. Also, the number of apoptotic cells was studied by the terminal deoxynucleotidyl transferase-mediated D: -UTP nick end labeling method in 347 cases. The study was done on specially prepared tissue arrays. RESULTS: In rectal cancer, patients with a Ki-67 labeling index of 5 percent or higher had a better prognosis than those with a lower index. Also, positive cytoplasmic p53 expression predicted a favorable outcome in rectal cancer. In colon cancer, positive nuclear staining of cyclin D1 reflected better survival. Weak and moderate staining of carcinoembryonic antigen correlated with better prognosis than strong staining, but negative staining predicted poor outcome. High apoptotic index of 100 or higher correlated with poor prognosis in colon cancer. However, in rectal cancer, the trend was the opposite. Bcl-2 staining tended to be more intense in samples of patients living 5 years or longer compared with those with worse prognosis. CONCLUSIONS: Colon cancer and rectal cancer seem to have different biologic behavior, at least with respect to apoptosis, cytoplasmic p53 expression, and perhaps Ki-67 and carcinoembryonic antigen. Further studies are needed to clarify the significance of these factors.

Aged↗
PubMed2004

The influence of training level and surgical experience on survival in colorectal cancer.

BACKGROUND AND AIMS: The effect of surgical training level, experience, and operation volume on complications and survival in colorectal cancer during a 10-year period in a medium-volume university hospital was retrospectively studied. PATIENTS AND METHODS: Four hundred and fifty-six patients were resected for primary colorectal adenocarcinoma during the 10-year period of 1981-1990, and of these, 387 patients underwent resection with curative intent. The surgeons were divided into three groups according to training level and volume: group 1, surgeons in training and other surgeons operating annually on only 1-4 patients; group 2, surgeons specializing in gastrointestinal surgery (average annual volume 4-13 operations); group 3, specialists in gastrointestinal surgery (average annual volume 3-8 operations). Postoperative morbidity and mortality rates, as well as long-term survival rates, were analysed, and comparisons were made between the patients in the three groups. RESULTS: There were no statistically significant differences between the three groups in postoperative morbidity or mortality. Cancer-specific 5-year survival rate of all patients was 57%, and that of those resected in the aforementioned three groups was 51%, 63%, and 55%, respectively, P=0.087. The 5-year survival rates for colon cancer were 59% (total), 52%, 69%, and 58%, respectively, P=0.067, and for rectal cancer were 51% (total), 42%, 53%, and 52%, respectively, P=0.585. CONCLUSION: There were no significant differences in the rates of postoperative mortality, morbidity, and long-term overall survival between the volume groups. However, in patients with colon cancer, there was a trend for better survival for those operated on by the surgeons specializing in gastrointestinal surgery, and in rectal cancer patients, a tendency of fewer local recurrences in those operated on by the specialist surgeons.

Aged↗
PubMed2004

Desensitization and internalization of metabotropic glutamate receptor 1a following activation of heterologous Gq/11-coupled receptors.

In this study we characterized the heterologous desensitization and internalization of the metabotropic glutamate receptor 1 (mGluR1) splice variants mGluR1a and mGluR1b following activation of endogenous G(q/11)-coupled receptors in HEK293 cells. Agonist activation of M1 muscarinic acetylcholine or P2Y1 purinergic receptors triggered the PKC- and CaMKII-dependent internalization of mGluR1a. In co-immunoprecipitation studies, both glutamate and carbachol increased the association of GRK2 with mGluR1a. Co-addition of the protein kinase C (PKC) inhibitor GF109203X and the Ca(2+) calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 blocked the ability of glutamate and carbachol to increase the association of GRK2 with mGluR1a. Glutamate also increased the association of GRK2 with mGluR1b, whereas carbachol did not. However, unlike mGluR1a, glutamate-stimulated association of GRK2 with mGluR1b was not reduced by PKC/CaMKII inhibition. Pretreatment of cells expressing mGluR1a or mGluR1b with carbachol rapidly desensitized subsequent glutamate-stimulated inositol phosphate accumulation. The carbachol-induced heterologous desensitization and internalization of mGluR1a was blocked by LY367385, an mGluR1a antagonist with inverse agonist activity. Furthermore, LY367385 blocked the ability of carbachol to increase the association of GRK2 with mGluR1a. On the other hand, LY367385 had no effect on the carbachol-induced desensitization and internalization of the nonconstitutively active mGluR1b splice variant. These results demonstrate that the internalization of mGluR1a, triggered homologously by glutamate or heterologously by carbachol, is PKC/CaMKII-, GRK2-, arrestin-, and clathrin-dependent and that PKC/CaMKII activation appears to be necessary for GRK2 to associate with mGluR1a. Furthermore, the heterologous desensitization of mGluR1a is dependent upon the splice variant being in an active conformation.

Arrestins↗
PubMed2004

Novel metabotropic glutamate receptor negatively coupled to adenylyl cyclase in cultured rat cerebellar astrocytes.

Several excitatory amino acid ligands were found potently to inhibit forskolin-stimulated cAMP accumulation in rat cultured cerebellar astrocytes: L-cysteine sulfinic acid (L-CSA) = L-aspartate > L-glutamate >/= the glutamate uptake inhibitor, L-PDC. This property did not reflect activation of conventional glutamate receptors, since the selective ionotropic glutamate receptor agonists NMDA, AMPA, and kainate, as well as several mGlu receptor agonists [(1S,3R)-ACPD, (S)-DHPG, DCG-IV, L-AP4, L-quisqualate, and L-CCG-I], were without activity. In addition, the mGlu receptor antagonists, L-AP3, (S)-4CPG, Eglu, LY341495, (RS)-CPPG, and (S)-MCPG failed to reverse 30 microM glutamate-mediated inhibitory responses. L-PDC-mediated inhibition was abolished by the addition of the enzyme glutamate-pyruvate transaminase. This finding suggests that the effect of L-PDC is indirect and that it is mediated through endogenously released L-glutamate. Interestingly, L-glutamate-mediated inhibitory responses were resistant to pertussis toxin, suggesting that G(i)/G(o) type G proteins were not involved. However, inhibition of protein kinase C (PKC, either via the selective PKC inhibitor GF109203X or chronic PMA treatment) augmented glutamate-mediated inhibitory responses. Although mGlu3 receptors (which are negatively coupled to adenylyl cyclase) are expressed in astrocyte populations, in our study Western blot analysis indicated that this receptor type was not expressed in cerebellar astrocytes. We therefore suggest that cerebellar astrocytes express a novel mGlu receptor, which is negatively coupled to adenylyl cyclase, and possesses an atypical pharmacological profile.

Adenylyl Cyclases↗
PubMed2004

Agonist-independent internalization of metabotropic glutamate receptor 1a is arrestin- and clathrin-dependent and is suppressed by receptor inverse agonists.

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.

Animals↗
PubMed2004

Activation of cyclic AMP-dependent protein kinase inhibits the desensitization and internalization of metabotropic glutamate receptors 1a and 1b.

In this study, we characterized the effects of activation of cyclic AMP-dependent protein kinase (PKA) on the internalization and functional coupling of the metabotropic glutamate receptor (mGluR1) splice variants mGluR1a and mGluR1b. Using an enzyme-linked immunosorbent assay technique to assess receptor internalization, we found that the glutamate-induced internalization of mGluR1a or mGluR1b transiently expressed in human embryonic kidney (HEK) 293 cells was inhibited by coactivation of endogenous beta2-adrenoceptors with isoprenaline or by direct activation of adenylyl cyclase with forskolin. The PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked the effects of both isoprenaline and forskolin. The heterologous internalization of the mGluR1 splice variants triggered by carbachol was also inhibited by isoprenaline and forskolin in a PKA-sensitive fashion, whereas the constitutive (agonist-independent) internalization of mGluR1a was inhibited only modestly by PKA activation. Using inositol phosphate (IP) accumulation in cells prelabeled with [3H]inositol to assess receptor coupling, PKA activation increased basal IP accumulation in mGluR1a receptor-expressing cells and also increased glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells, but only at short times of glutamate addition. Furthermore, PKA activation completely blocked the carbachol-induced heterologous desensitization of glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells. In coimmunoprecipitation experiments, the ability of glutamate to increase association of GRK2 and arrestin-2 with mGluR1a and mGluR1b was inhibited by PKA activation with forskolin. Together, these results indicate that PKA activation inhibits the agonist-induced internalization and desensitization of mGluR1a and mGluR1b, probably by reducing their interaction with GRK2 and nonvisual arrestins.

Arrestins↗
PubMed2003

Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor.

PURPOSE: Most gastrointestinal stromal tumors (GISTs) express constitutively activated mutant isoforms of KIT or kinase platelet-derived growth factor receptor alpha (PDGFRA) that are potential therapeutic targets for imatinib mesylate. The relationship between mutations in these kinases and clinical response to imatinib was examined in a group of patients with advanced GIST. PATIENTS AND METHODS: GISTs from 127 patients enrolled onto a phase II clinical study of imatinib were examined for mutations of KIT or PDGFRA. Mutation types were correlated with clinical outcome. RESULTS: Activating mutations of KIT or PDGFRA were found in 112 (88.2%) and six (4.7%) GISTs, respectively. Most KIT mutations involved exon 9 (n = 23) or exon 11 (n = 85). All KIT mutant isoforms, but only a subset of PDGFRA mutant isoforms, were sensitive to imatinib, in vitro. In patients with GISTs harboring exon 11 KIT mutations, the partial response rate (PR) was 83.5%, whereas patients with tumors containing an exon 9 KIT mutation or no detectable mutation of KIT or PDGFRA had PR rates of 47.8% (P =.0006) and 0.0% (P <.0001), respectively. Patients whose tumors contained exon 11 KIT mutations had a longer event-free and overall survival than those whose tumors expressed either exon 9 KIT mutations or had no detectable kinase mutation. CONCLUSION: Activating mutations of KIT or PDGFRA are found in the vast majority of GISTs, and the mutational status of these oncoproteins is predictive of clinical response to imatinib. PDGFRA mutations can explain response and sensitivity to imatinib in some GISTs lacking KIT mutations.

Animals↗
PubMed2003

Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization.

To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.

Amino Acid Sequence↗
PubMed2003

Disease-dependent adhesion of lactic acid bacteria to the human intestinal mucosa.

Their adhesion to the intestinal mucosa is considered one of the main reasons for the beneficial health effects of specific lactic acid bacteria (LAB). However, the influence of disease on the mucosal adhesion is largely unknown. Adhesion of selected LAB to resected colonic tissue and mucus was determined in patients with three major intestinal diseases (i.e., diverticulitis, rectal carcinoma, and inflammatory bowel disease) and compared to healthy control tissue. All strains were observed to adhere better to immobilized mucus than to whole intestinal tissue. Two strains (Lactobacillus rhamnosus strain GG and L. reuteri) were found to exhibit disease-specific adhesion to intestinal tissue. All tested strains, with the exception of L. rhamnosus strain GG, displayed disease-specific adhesion to intestinal mucus. These results suggest that strains with optimal binding characteristics for a particular intestinal disease can be selected.

Adult↗
PubMed2003

Surgical research.

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Biomedical Research↗
PubMed2002

Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors.

BACKGROUND: Constitutive activation of KIT receptor tyrosine kinase is critical in the pathogenesis of gastrointestinal stromal tumors. Imatinib mesylate, a selective tyrosine kinase inhibitor, has been shown in preclinical models and preliminary clinical studies to have activity against such tumors. METHODS: We conducted an open-label, randomized, multicenter trial to evaluate the activity of imatinib in patients with advanced gastrointestinal stromal tumor. We assessed antitumor response and the safety and tolerability of the drug. Pharmacokinetics were assessed in a subgroup of patients. RESULTS: A total of 147 patients were randomly assigned to receive 400 mg or 600 mg of imatinib daily. Overall, 79 patients (53.7 percent) had a partial response, 41 patients (27.9 percent) had stable disease, and for technical reasons, response could not be evaluated in 7 patients (4.8 percent). No patient had a complete response to the treatment. The median duration of response had not been reached after a median follow-up of 24 weeks after the onset of response. Early resistance to imatinib was noted in 20 patients (13.6 percent). Therapy was well tolerated, although mild-to-moderate edema, diarrhea, and fatigue were common. Gastrointestinal or intraabdominal hemorrhage occurred in approximately 5 percent of patients. There were no significant differences in toxic effects or response between the two doses. Imatinib was well absorbed, with pharmacokinetics similar to those reported in patients with chronic myeloid leukemia. CONCLUSIONS: Imatinib induced a sustained objective response in more than half of patients with an advanced unresectable or metastatic gastrointestinal stromal tumor. Inhibition of the KIT signal-transduction pathway is a promising treatment for advanced gastrointestinal stromal tumors, which resist conventional chemotherapy.

Adolescent↗
PubMed2002

Clinical presentation of gastrointestinal stromal tumors and treatment of operable disease.

Gastrointestinal stromal tumors (GISTs) are generally found in the stomach or small intestine and less commonly in the colon, rectum or an intra-abdominal site. The patients symptoms on presentation are most commonly gastrointestinal bleeding. Surgery remains the standard treatment for nonmetastatic GISTs, but rates of disease recurrence are significant--5% in primary disease and 90% in locally advanced disease. Five-year survival following surgical resection varies between 35% and 65% on the basis of several published studies. Clinical knowledge of the prognosis of patients with GISTs remains rather limited--small tumor size, low-grade mitotic index and stomach location are factors associated with a more favorable prognosis.

Gastrointestinal Neoplasms↗