guanosine-triphosphate and Urinary-Bladder-Neoplasms

Heterotrimeric G-proteins (Gαβγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gβγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.

Topics: Amino Acid Motifs; Animals; Bioluminescence Resonance Energy Transfer Techniques; Biosensing Techniques; Cells, Cultured; GTP-Binding Protein alpha Subunits, Gq-G11; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; HEK293 Cells; HeLa Cells; Heterotrimeric GTP-Binding Proteins; Humans; Mice; Mice, Inbred C57BL; Mutation; Neoplasms; Neurons; Receptors, G-Protein-Coupled; Signal Transduction; Urinary Bladder Neoplasms

Topics: Antineoplastic Agents; Carcinoma; Cell Division; Cell Movement; Chemoprevention; Computer-Aided Design; Coumarins; Drug Design; Guanine; Guanosine Triphosphate; Humans; Molecular Structure; Point Mutation; Protein Binding; Proto-Oncogene Proteins p21(ras); Structure-Activity Relationship; Tumor Cells, Cultured; Umbelliferones; Urinary Bladder Neoplasms

The effects of 7-hydroxycoumarin, genistein and quercetin on two ras-oncogene-driven tumour cells (rat breast adenocarcinoma and human bladder carcinoma) were investigated using cellular (proliferation and migration) and molecular targets (p21ras GTPase activity and intracellular amount of p21ras protein). All three compounds inhibited the growth of both cell lines. Genistein was the most effective substance. Furthermore, 7-hydroxycoumarin and genistein affected the motile machinery of both cell lines because major fractions of the cells were slowed down or stopped locomotion. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), a well-known tumour promoter, increased the locomotion behaviour of the cells; the time of migration, the velocity and the distance of migration increased under the control of PMA. 7-Hydroxycoumarin decreased the relative amount of intracellular p21ras, and concomitantly a PMA-induced decrease of p21ras GTPase activity could be partially antagonized by 7-hydroxycoumarin. Because of the low toxicity and the mode of action evaluated, it is likely that the best role for these substances may be adjuvant therapy of some malignancies following surgery. Profiles directed to migration and proliferation inhibition make these drugs exceptional candidates for chemopreventive strategies in tumours diagnosed as having increased ras oncogene levels.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cell Division; Cell Movement; Collagen; Culture Media; Genes, ras; Genistein; Guanosine Triphosphate; Humans; Indicators and Reagents; Isoflavones; Mammary Neoplasms, Experimental; Oxidation-Reduction; Proto-Oncogene Proteins p21(ras); Quercetin; Rats; Tetrazolium Salts; Tumor Cells, Cultured; Umbelliferones; Urinary Bladder Neoplasms

Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.

Topics: Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Line, Transformed; Enzyme Inhibitors; Genes, ras; Guanosine Triphosphate; Humans; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Point Mutation; Protein Binding; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; Reference Values; Rifabutin; Transformation, Genetic; Tumor Cells, Cultured; Urinary Bladder Neoplasms