guanosine-5--o-(3-thiotriphosphate) and Carcinoma--Squamous-Cell

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

Parathyroid hormone-related protein (PTHrP) has been identified as a causative factor in the pathogenesis of humoral hypercalcemia of malignancy (HHM). The regulation and mechanisms of PTHrP secretion in most normal and malignant cells are unknown. PTHrP secretion, mRNA expression, and transcription were measured in neoplastic human squamous carcinoma cells (A253) and normal human foreskin keratinocytes (NHFK) by radioimmunoassay, RNase protection assay, and transient transfections of the 5'-flanking region of human PTHrP in a luciferase expression vector. Mechanisms of PTHrP secretion were investigated using chemicals (monensin, colchicine, cytochalasin B, guanosine 5'-[gamma-thio]triphosphate (GTPgammaS)) that interfere with or facilitate intracellular transport. Monensin inhibited PTHrP secretion in both NHFK and A253 cells. Ultrastructurally, monensin caused dilatation of rough endoplasmic reticulum and the formation of numerous cytoplasmic secretory vacuoles in both cell lines. Colchicine decreased PTHrP production in NHFK cells and stimulated PTHrP production and mRNA levels in A253 cells. Colchicine also stimulated transcription of the PTHrP-luciferase reporter gene. Cytochalasin B stimulated PTHrP secretion and mRNA expression in A253 cells, but had no effect in NHFK cells. GTPgammaS had no effect on PTHrP secretion in either cell line. It was concluded that PTHrP secretion is dependent on the constitutive movement of secretory vesicles to the cytoplasmic membrane and regulation of PTHrP secretion and mRNA expression are altered in squamous carcinoma cells compared to normal human keratinocytes in vitro.

Topics: Actin Cytoskeleton; Biological Transport; Carcinoma, Squamous Cell; Cells, Cultured; Colchicine; Cytochalasin B; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Keratinocytes; Microtubules; Monensin; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Proteins; RNA, Messenger; Skin; Transcription, Genetic

Previous study shows volume-sensitive chloride currents are induced by hypotonicity in human cervical cancer cell lines, but not in normal cervical epithelium. To ascertain whether the preferential activation of these channels in cancer cell lines could be similarly and directly detected in cervical cancer tissues, we studied volume-sensitive chloride channels on the primary culture cells of invasive cervical carcinoma using the whole-cell patch-clamp technique. The process of regulatory volume decrease (RVD) was also studied using electronic cell sizing to measure cell volume. Results demonstrate that, in these cultured cells, RVD was mediated in part by chloride loss through the volume-sensitive Cl- channels. A small background current with a slope conductance of 0.32 +/- 0.07 nS/pF at +30 mV (n=60 cells from 10 different samples) was observed. Hypotonicity induced a fast activating and outward rectifying current which was reversed at about 0 mV, and the slope conductance at +30 mV was increased by 10-fold to 3.62 +/- 0.62 nS/pF. These effects were readily reversed by returning the cells to isotonic medium. Moreover, DIDS, NPPB, and 1,9-dideoxyforskolin, reversibly abolished the volume-sensitive Cl- currents. The EC50 required for the inhibitory effect of DIDS, NPPB and 1,9-dideoxyforskolin was 150, 120, and 50 microM, respectively. Volume-sensitive Cl- channels were ubiquitously expressed in cultured cells from 10 samples of different cancer stages, histopathologic types, and state of HPV DNA positivity. Interestingly, similar outward rectifying chloride currents were activated by intracellular 300 microM GTP gamma S. It is proposed that this Cl- conductance may play an important role leading to RVD in human cervical cancer.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Base Sequence; Carcinoma, Squamous Cell; Cell Membrane; Cesium; Chloride Channels; Chlorides; DNA Primers; DNA, Viral; Female; Genome, Viral; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kinetics; Membrane Potentials; Molecular Sequence Data; Neoplasm Staging; Papillomaviridae; Patch-Clamp Techniques; Polymerase Chain Reaction; Tumor Cells, Cultured; Uterine Cervical Neoplasms

The biochemical requirements for epidermal growth factor (EGF) and transferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxidase (HRP)-conjugated EGF and gold-labeled antitransferrin (Tfn) receptor antibodies were colocalized during endocytosis in vitro. The sequestration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTP gamma S, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (clathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, genistein. The biochemical differences detected between EGF and Tfn sequestration most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Carcinoma, Squamous Cell; Cell Membrane Permeability; Coated Pits, Cell-Membrane; Cytosol; Egtazic Acid; Endocytosis; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kinetics; Ligands; Microscopy, Immunoelectron; Receptors, Transferrin; Tumor Cells, Cultured

The growth-inhibiting peptide hormone somatostatin stimulates phosphotyrosine phosphatase activity in the human pancreatic cell line MIA PaCa-2. This hormonal activation was mediated by a pertussis toxin-sensitive guanosine 5'-triphosphate-binding protein (G protein) in the membranes of these cells. Activation of this G protein by somatostatin stimulated the dephosphorylation of exogenous epidermal growth factor receptor prepared from A-431 cells in vitro. This pathway may mediate the antineoplastic action of somatostatin in these cells and in human tumors and could represent a general mechanism of G protein coupling that is utilized by normal cells in the hormonal control of cell growth.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Enzyme Activation; ErbB Receptors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Kinetics; Pancreatic Neoplasms; Peptides; Pertussis Toxin; Phosphorylation; Protein Kinases; Protein Tyrosine Phosphatases; Somatostatin; Thionucleotides; Virulence Factors, Bordetella

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.

Topics: Animals; Binding Sites; Calcium; Carcinoma, Squamous Cell; Cell Membrane; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Isoproterenol; Kinetics; Liver; Male; Pertussis Toxin; Phosphorylation; Protein-Tyrosine Kinases; Rats; Rats, Inbred Strains; Virulence Factors, Bordetella

Topics: Alkaline Phosphatase; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Female; Giant Cells; Glycolipids; Glycosylphosphatidylinositols; Growth Substances; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Insulin; Isoenzymes; Kinetics; Phosphatidylinositols; Placenta; Pregnancy; Thionucleotides; Trophoblasts