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Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.

ABSTRACT: Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G(s)-dependent receptors for isoproterenol and prostaglandin E(2). Whereas two ligands, uridine 5 '-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G(q)/calcium and G(i), the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G(s)-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P(2) receptor and the heterotrimeric G(13) protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.

SUBMITTER: Jiang LI

PROVIDER: S-EPMC2526465 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.

Jiang Lily I LI Collins Julie J Davis Richard R Lin Keng-Mean KM DeCamp Dianne D Roach Tamara T Hsueh Robert R Rebres Robert A RA Ross Elliott M EM Taussig Ronald R Fraser Iain I Sternweis Paul C PC

The Journal of biological chemistry 20070205 14

Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for m ...[more]

PMID: 17283075

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Project description:Resistance to chemotherapy is common in gastroesophageal cancer. Mechanisms of resistance are incompletely characterised and there are no predictive biomarkers in clinical practice for cytotoxic drugs. We used new cell line models to characterise novel chemotherapy resistance mechanisms and validated them in tumour specimens to identify new targets and biomarkers for gastroesophageal cancer.Cell lines were selected for resistance to oxaliplatin, cisplatin and docetaxel and gene expression examined using Affymetrix Exon 1.0 ST arrays. Leads were validated by qRT-PCR and HPLC of tumour metabolites. Protein expression and pharmacological inhibition of lead target SPHK1 was evaluated in independent cell lines, and by immunohistochemistry in gastroesophageal cancer patients.Genes with differential expression in drug resistant cell lines compared to the parental cell line they were derived from, were identified for each drug resistant cell line. Biological pathway analysis of these gene lists, identified over-represented pathways, and only 3 pathways - lysosome, sphingolipid metabolism and p53 signalling- were identified as over-represented in these lists for all three cytotoxic drugs investigated. The majority of genes differentially expressed in chemoresistant cell lines from these pathways, were involved in metabolism of glycosphingolipids and sphingolipids in lysosomal compartments suggesting that sphingolipids might be important mediators of cytotoxic drug resistance in gastroeosphageal cancers . On further investigation, we found that drug resistance (IC50) was correlated with increased sphingosine kinase 1(SPHK1) mRNA and also with decreased sphingosine-1-phosphate lysase 1(SGPL1) mRNA. SPHK1 and SGPL1 gene expression were inversely correlated. SPHK1:SGPL1 ratio correlated with increased cellular sphingosine-1-phosphate (S1P), and S1P correlated with drug resistance (IC50). High SPHK1 protein correlated with resistance to cisplatin (IC50) in an independent gastric cancer cell line panel and with survival of patients treated with chemotherapy prior to surgery but not in patients treated with surgery alone. Safingol a SPHK1 inhibitor, was cytotoxic as a single agent and acted synergistically with cisplatin in gastric cancer cell lines.Agents that inhibit SPHK1 or S1P could overcome cytotoxic drug resistance in gastroesophageal cancer. There are several agents in early phase human trials including Safingol that could be combined with chemotherapy or used in patients progressing after chemotherapy.

Dataset Information

Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.

Publications

Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.

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