guanosine-5--o-(3-thiotriphosphate) and Lung-Neoplasms

The two major forms of lung carcinoma, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), are clinically distinct, and are also differentiated by morphology and behavior in culture. SCLC cells have a greater metastatic potential than NSCLC cells in vivo, and exhibit a unique spherical morphology in culture due to their inability to adhere and spread on the substratum. Because the small GTPase RhoA affects metastatic properties and regulates cell morphology, we examined whether differences in RhoA expression and activity contribute to the distinct SCLC and NSCLC phenotypes. We found that the expression and GTPgammaS-dependent activation of RhoA are generally greater in SCLC cell lines (SCC-9, NCI-H69, NCI-H146, and NCI-H345) than in NSCLC cell lines (NCI-H23, NCI-H157, NCI-H520, and NCI-H522). The effects of inhibiting Rho-mediated signaling in these cells were investigated by transfecting the cells with cDNA coding for C3 exoenzyme, which ADP-ribosylates and inactivates Rho. Expression of C3 exoenzyme in SCLC cells induces cell-cell compaction, and causes NCI-H345 cells to adhere and spread on collagen IV. In contrast, expression of C3 exoenzyme in NSCLC cells does not induce detectable compaction, but induces cell spreading of NCI-H23 and NCI-H157 cells. Cell proliferation is diminished by Rho inactivation in the majority of the NSCLC cell lines, but not the SCLC cell lines. Expression of p21Cip1/WAF1 is also diminished by Rho inactivation in two of the SCLC cell lines, but is not significantly altered in the NSCLC lines. These results indicate that Rho-mediated signaling may regulate different events in SCLC and NSCLC cells, including adhesion of SCLC cells and proliferation of NSCLC cells.

Topics: Adenocarcinoma; Adenosine Diphosphate Ribose; ADP Ribose Transferases; Botulinum Toxins; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Cell Size; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA, Complementary; Gene Expression Regulation, Neoplastic; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Proteins; rhoA GTP-Binding Protein; Signal Transduction

Aberrant signal transduction pathways involved in the development of metastatic disease are poorly defined in both small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Neuropeptide-driven positive feedback loops stimulating cell proliferation are characteristic of SCLC. The activation of phospholipase C (PLC)-beta1 is an early and common response to stimulation of G protein-coupled receptors by these neuroendocrine growth factors. The importance of PLC-beta in neuropeptide signaling prompted us to compare PLC-beta isoform expression and activity in four independent SCLC cell lines and four independent NSCLC cell lines. We found that PLC-beta1 is more highly expressed in SCLC than in NSCLC, as indicated by Western blotting of cell lysates. All SCLC lines studied express PLC-beta1; only one of the NSCLC lines investigated showed detectable levels of the enzyme. NSCLC lines are significantly more sensitive to the antiproliferative effects of ET-18-OCH3 (edelfosine) compared with the SCLC lines, as indicated by [3H]thymidine uptake. The only SCLC cell line (NCI-H345) that is as sensitive as the NSCLC cell lines to ET-18-OCH3 also expresses uniquely low levels of PLC-beta1. The participation of PLC-beta1 in signaling by SCLC growth factor receptors is indicated by our finding that PLC-beta1 (but not PLC-beta3) coimnunoprecipitates with G(alpha)q/11 upon activation of neurotensin receptors; this association is inhibited by ET-18-OCH3. Ca2+ mobilization mediated by neurotensin receptors is also inhibited by ET-18-OCH3. The binding of GTPgammaS to G(alpha)q/11 upon treatment of SCLC cells with neurotensin is not inhibited by ET-18-OCH3. These findings indicate that ET-18-OCH3 does not interfere with G(alpha)q/11 activation but rather inhibits the association of G(alpha)q/11 with PLC-beta1. Our data suggest that PLC-beta is an important mediator of both SCLC and NSCLC proliferation. Differences in PLC-beta1 expression may be exploitable in the development of effective diagnostic and therapeutic tools.

Topics: Antineoplastic Agents; Calcium; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Drug Resistance, Neoplasm; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Isoenzymes; Lung Neoplasms; Phosphodiesterase Inhibitors; Phospholipase C beta; Phospholipid Ethers; Tumor Cells, Cultured; Type C Phospholipases

In [3H]inositol-labeled membranes prepared from Swiss mouse 3T3 and human small cell lung carcinoma cells, [Tyr4]-bombesin stimulated production of water-soluble inositol phosphates. The reaction was stimulated by guanosine 5'-O-[3-thiotriphosphate] and was specifically inhibited by both [Leu13-psi-CH2NHLeu14]-bombesin and the antibombesin antibody 2A11. [Tyr4]-bombesin-induced activation of phospholipase C is most apparent in Ca2(+)-depleted conditions (less than 1 microM[Ca2+]free). The kinetics of activation by ligand also demonstrate that [Tyr4]-bombesin-dependent phospholipase C activation is most apparent at [Mg2+]free of approximately 0.2 microM. At millimolar concentrations of [Mg2+]free, there is considerably less dependence on [Tyr4]-bombesin for activation of phospholipase C. ATP is not necessary for initial activation of phospholipase C, and beta, gamma-imidoadenosine-5'-triphosphate does not inhibit the reaction. These results demonstrate that in these cell types [Tyr4]-bombesin activates phospholipase C in conjunction with guanine nucleotides. Phospholipase C-coupled guanine nucleotide regulatory proteins would be appropriately considered as novel targets for the development of therapeutic strategies in small cell lung carcinoma.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Antibodies; Bombesin; Calcium; Carcinoma, Small Cell; Cell Line; Cell Membrane; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Kinetics; Lung Neoplasms; Magnesium; Mice; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases