BackgroundB cell precursor acute lymphoblastic leukaemia (BCP-ALL) is the most common paediatric cancer. BCP-ALL blasts typically retain wild type p53, and are therefore assumed to rely on indirect measures to suppress transformation-induced p53 activity. We have recently demonstrated that the second messenger cyclic adenosine monophosphate (cAMP) through activation of protein kinase A (PKA) has the ability to inhibit DNA damage-induced p53 accumulation and thereby promote survival of the leukaemic blasts.Development of BCP-ALL in the bone marrow (BM) is supported by resident BM-derived mesenchymal stromal cells (MSCs). MSCs are known to produce prostaglandin E2 (PGE2) which upon binding to its receptors is able to elicit a cAMP response in target cells. We hypothesized that PGE2 produced by stromal cells in the BM microenvironment could stimulate cAMP production and PKA activation in BCP-ALL cells, thereby suppressing p53 accumulation and promoting survival of the malignant cells.MethodsPrimary BCP-ALL cells isolated from BM aspirates at diagnosis were cocultivated with BM-derived MSCs, and effects on DNA damage-induced p53 accumulation and cell death were monitored by SDS-PAGE/immunoblotting and flow cytometry-based methods, respectively. Effects of intervention of signalling along the PGE2-cAMP-PKA axis were assessed by inhibition of PGE2 production or PKA activity. Statistical significance was tested by Wilcoxon signed-rank test or paired samples t test.ResultsWe demonstrate that BM-derived MSCs produce PGE2 and protect primary BCP-ALL cells from p53 accumulation and apoptotic cell death. The MSC-mediated protection of DNA damage-mediated cell death is reversible upon inhibition of PGE2 synthesis or PKA activity. Furthermore our results indicate differences in the sensitivity to variations in p53 levels between common cytogenetic subgroups of BCP-ALL.ConclusionsOur findings support our hypothesis that BM-derived PGE2, through activation of cAMP-PKA signalling in BCP-ALL blasts, can inhibit the tumour suppressive activity of wild type p53, thereby promoting leukaemogenesis and protecting against therapy-induced leukaemic cell death. These novel findings identify the PGE2-cAMP-PKA signalling pathway as a possible target for pharmacological intervention with potential relevance for treatment of BCP-ALL.

Increasing contractility through stimulation of alpha 1-adrenoceptors in situ by the endogenous agonist may be an alternative way of inotropic support during heart failure and even more so during beta-adrenoceptor blockade.

BackgroundOral squamous cell carcinoma is an aggressive neoplasm with serious morbidity and mortality, which typically spreads through local invasive growth. Lysophosphatidic acid (LPA) is involved in a number of biological processes, and may have a role in cancer cell migration and invasiveness. LPA is present in most tissues and can activate cells through six different LPA receptors (LPAR1-6). Although LPA is predominantly promigratory, some of the receptors may have antimigratory effects in certain cells. The signalling mechanisms of LPA are not fully understood, and in oral carcinoma cells the specific receptors and pathways involved in LPA-stimulated migration are unknown.MethodsThe oral carcinoma cell lines E10, SCC-9, and D2 were investigated. Cell migration was studied in a scratch wound assay, and invasion was demonstrated in organotypic three dimensional co-cultures. Protein and mRNA expression of LPA receptors was studied with Western blotting and qRT-PCR. Activation of signalling proteins was examined with Western blotting and isoelectric focusing, and signalling mechanisms were further explored using pharmacological agents and siRNA directed at specific receptors and pathways.ResultsLPA stimulated cell migration in the two oral carcinoma cell lines E10 and SCC-9, but was slightly inhibitory in D2. The receptor expression profile and the effects of specific pharmacological antagonist and agonists indicated that LPA-stimulated cell migration was mediated through LPAR3 in E10 and SCC-9. Furthermore, in both these cell lines, the stimulation by LPA was dependent on PKC activity. However, while LPA induced transactivation of EGFR and the stimulated migration was blocked by EGFR inhibitors in E10 cells, LPA did not induce EGFR transactivation in SCC-9 cells. In D2 cells, LPA induced EGFR transactivation, but this was associated with slowing of a very high inherent migration rate in these cells.ConclusionThe results demonstrate LPA-stimulated migration in oral carcinoma cells through LPAR3, mediated further by PKC, which acts either in concert with or independently of EGFR transactivation.

BackgroundNeurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells.MethodsColon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting.ResultsNeurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells.ConclusionsWhile acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK and stimulation of DNA synthesis was PKC-dependent, whereas activation of the PI3K/Akt pathway was mediated by stimulation of metalloproteinases and subsequent transactivation of the EGFR. Thus, the data show that the signalling mechanisms mediating the effects of neurotensin involve multiple pathways and are cell-dependent.

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR-independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2-stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor-sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF-stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2-treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part G(i) protein-mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways.

Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high-and low-affinity EGFR. At 4°C EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37°C. Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligandinduced recruitment of EGFR to clathrin-coated pits.

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Several agents that act through G-protein-coupled receptors and also stimulate phosphoinositide-specific phospholipase C (PI-PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F2alpha, activated the ERK1 (p44mapk) and ERK2 (p42mapk) members of the mitogen-activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in-gel assays. All these agonists induced a peak activation (two to threefold increase in MBP-phosphorylation) at 3-5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI-PLC, implicating a Gq-dependent pathway, the elevations of the concentration of inositol (1,4,5)-trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF2alpha. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G-protein-coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that Gi-dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G-protein-coupled receptors as well as the EGF receptor.