pituitrin and Cell-Transformation--Neoplastic

Topics: Acromegaly; Adrenocorticotropic Hormone; Animals; Blood Glucose; Calcitonin; Carcinoma, Small Cell; Cell Transformation, Neoplastic; Chorionic Gonadotropin; Corticotropin-Releasing Hormone; Cushing Syndrome; Dexamethasone; Female; Galactorrhea; Gene Expression Regulation; Growth Hormone; Humans; Hypercalcemia; Lymphokines; Nerve Tissue Proteins; Paraneoplastic Endocrine Syndromes; Parathyroid Hormone; Peptide Biosynthesis; Pregnancy; Pro-Opiomelanocortin; Prolactin; Prostaglandins; Transforming Growth Factors; Vasopressins; Vitamin D

Topics: Animals; Biological Transport, Active; Blood Physiological Phenomena; Cations, Monovalent; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Fibroblasts; Growth Substances; Humans; Mitogens; Osmolar Concentration; Structure-Activity Relationship; Vasopressins

Topics: Animals; Brain Neoplasms; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Clone Cells; Culture Techniques; Humans; Hypothalamus; Mice; Neoplasms, Experimental; Neurons; Neurophysins; Rats; Simian virus 40; Thyrotropin-Releasing Hormone; Vasopressins

Arginine vasopressin (AVP), a vasoactive peptide hormone that binds to three G-protein coupled receptors (V1R, V2R, and V3R), has long been known to activate V1R and elicit mitogenesis in several cell types, including adrenal glomerulosa cells. However, in the mouse Y1 adrenocortical malignant cell line, AVP triggers not only a canonical mitogenic response but also novel RhoA-GTP-dependent mechanisms which downregulate cyclin D1, irreversibly inhibiting K-ras oncogene-driven proliferation. In Y1 cells, AVP blocks cyclin D1 expression, induces senescence-associated beta-galactosidase (SAbeta-Gal) and inhibits proliferation. However, ectopic expression of cyclin D1 renders Y1 cells resistant to both SAbeta-Gal induction and proliferation inhibition by AVP. In addition, ectopic expression of the dominant negative RhoAN19 mutant blocks RhoA activation, yielding Y1 cell sub-lines which are no longer susceptible to cyclin D1 downregulation, SAbeta-Gal induction, or proliferation inhibition by AVP. Furthermore, inhibiting RhoA with C3 exoenzyme protects Y1 cells from AVP proliferation inhibition and SAbeta-Gal induction. On the other hand, AVP treatment does not activate caspases 3 and 7, and the caspase inhibitor Ac-DEVD-CMK does not protect Y1 cells from proliferation inhibition by AVP, implying that AVP does not trigger apoptosis. These results underline a pivotal survival activity of cyclin D1 that protects K-ras oncogene-dependent malignant cells from senescence.

Topics: Adrenal Cortex Neoplasms; Animals; Arginine Vasopressin; Caspases; Cell Transformation, Neoplastic; Cellular Senescence; Cyclin D1; Down-Regulation; Genes, ras; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Mice; Mitogen-Activated Protein Kinase 3; Phosphorylation; Proto-Oncogene Proteins c-akt; rhoA GTP-Binding Protein; Vasopressins

Neuropeptides are often ectopically expressed by non-endocrine tumours. We used transgenic mice to assess the effect of ectopic expression of the neuropeptide, vasopressin, in mammary tumours induced by the transgenetic expression of an activated ras oncogene. Mice bearing a mouse mammary tumour virus-vasopressin (MMTV-VP) fusion transgene synthesise authentic VP in mammary ducts and alveoli. Bitransgenic mice bearing both MMTV-VP and MMTV-v-Ha-ras transgenes developed tumours that were histologically indistinguishable from those of single MMTV-v-Ha-ras animals. However, tumour onset was significantly delayed in the bitransgenic animals. These data provide evidence that an ectopic neuropeptide can slow the development of ras tumours in vivo.

Topics: Animals; Blotting, Northern; Cell Transformation, Neoplastic; Female; Genes, ras; Immunoenzyme Techniques; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Time Factors; Vasopressins

REF52, a rat embryo cell line, and several transformed derivatives were used to examine the lipid-related events associated with agonist treatment (phorbol diesters, vasopressin, fetal bovine serum). Exposure of cells, prelabeled with [3H]glycerol, to TPA (12-O-tetradecanoylphorbol 13-acetate) resulted in 3-4-fold increase in the amount of intracellular diacyl[3H]glycerols as early as 10 min after treatment. Continued incubation (up to 60 min) revealed that the diacyl[3H]glycerol formed was under dynamic metabolic regulation as shown by the production of triacyl[3H]glycerols and free [3H]glycerol. Serum and vasopressin likewise induced the generation of intracellular diacyl[3H]glycerol, thereby illustrating that physiological agents provoke a similar reaction. In the three SV-40-transformed variants examined, the diacylglycerol generative-response to TPA, serum and vasopressin, was greatly diminished or totally absent. Experiments employing REF52 cells prelabeled with [3H]choline demonstrated that both TPA and vasopressin induce the hydrolysis of cellular choline-containing glycerophospholipids; this was measured by both a decrease in cell-associated phosphatidylcholine radioactivity and an increase in the production of water-soluble [3H]choline-containing metabolites in the culture medium. 92-97% of the tritium released to the medium was identified as [3H]choline. Vasopressin treatment of REF52 cells prelabeled with [3H]arachidonic acid elicited an increase of more than 11-fold in the amount of cellular diacyl[3H]glycerol and a concomitant release of arachidonic acid to the culture medium that was 12-fold higher than controls. These data demonstrate that tumor-promoting phorbol esters (agonists of protein kinase C), serum and vasopressin, increase the levels of cellular diacylglycerol by stimulating the hydrolysis of choline-containing glycerophospholipids. This agonist-directed mechanism is inoperable in transformed cells. Further, collateral with vasopressin-induced phosphatidylcholine hydrolysis, the cellular release of arachidonic acid occurs. The participation of these lipid-related responses in the signaling of agonist-directed events and their relation to cellular homeostasis is currently being explored.

Topics: Animals; Blood; Cell Line; Cell Transformation, Neoplastic; Culture Media; Diglycerides; Embryo, Mammalian; Glycerides; Kinetics; Phorbol Esters; Phosphatidylcholines; Rats; Simian virus 40; Tetradecanoylphorbol Acetate; Vasopressins

A simple enzymatic method for the quantitation of the mass of sn-1,2-diacylglycerol (DAG) present in crude lipid extracts was developed to assess the function of DAGs as intracellular "second messengers" of extracellular agents and of oncogene products. The assay employed Escherichia coli DAG kinase which constituted approximately 15% of the membrane protein of a plasmid-bearing strain and defined mixed micellar conditions to solubilize the DAG present and allow its quantitative conversion to [32P]phosphatidic acid. The assay was proportional with the amount of DAG added over the range of 25 pmol to 25 nmol. The rapid rise of DAG in platelets stimulated with thrombin (210% over basal) and in hepatocytes stimulated with vasopressin (230% over basal) was quantitated and the values agreed with previous measurements. The amounts of DAG in normal rat kidney (NRK) cells grown at 34 and 38 degrees C, respectively, were 0.47 and 0.61 nmol/100 nmol of phospholipid. In K-ras-transformed NRK cells grown at 34 or 38 degrees C, DAG levels were elevated 168 or 138%, respectively. When a temperature-sensitive K-ras NRK cell line was investigated, the amount of DAG present was elevated at the permissive but not at the restrictive temperature. These data are consistent with the K-ras protein functioning in transmembrane signalling by activating phospholipase C. Protein kinase C (Ca2+/phospholipid-dependent enzyme) activation by DAG may play an important role in cellular transformation.

Topics: Animals; Blood Platelets; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Diacylglycerol Kinase; Diglycerides; Escherichia coli; Glycerides; Humans; In Vitro Techniques; Kidney; Kinetics; Liver; Phosphotransferases; Rats; Vasopressins

Topics: Amphotericin B; Biological Transport, Active; Blood; Blood Platelets; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Fibroblasts; Growth Substances; Melitten; Monensin; Peptides; Platelet-Derived Growth Factor; Potassium; Rubidium; Sodium; Vasopressins

Hypothalamic cells taken from 14-day-old mouse embryos were cultured for 6 days and transformed with simian virus 40. After cloning, a homogeneous cell population was obtained. Its morphological, Ultrastructural, biochemical, and immunochemical properties were studied. These cells possess ultrastructural features of primitive neurosecretory cells. They synthesize (35)S-labeled protein components that have the molecular weight and isoelectric focusing behavior of, and display the same immunoreactivity as, neurophysin. In addition, a (35)S-labeled peptidic fraction with a molecular weight close to 1000 is synthesized and is radioimmunologically indistinguishable from vasopressin. Immunochemical staining shows that both neurophysin and vasopressin are localized in the cytoplasm. These observations strongly suggest that a clone of mouse hypothalamic neurosecretory cells has been obtained with the synthesizing capacities of secretory neurons of the magnocellular hypothalamic nuclei.

Topics: Animals; Antigens, Viral; Cell Line; Cell Transformation, Neoplastic; Chromatography, Gel; Clone Cells; Cricetinae; Cytoplasm; Fluorescent Antibody Technique; Hypothalamus; Isoelectric Focusing; Mice; Molecular Weight; Neurophysins; Neurosecretion; Oxytocin; Rabbits; Radioimmunoassay; Simian virus 40; Sulfur Radioisotopes; Vasopressins

Topics: Adrenocorticotropic Hormone; Cell Transformation, Neoplastic; Cushing Syndrome; Humans; Hypercalcemia; Hyponatremia; Neoplasms; Parathyroid Hormone; Precancerous Conditions; Vasopressins