pituitrin and Cell-Transformation--Viral

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

Immortalization of cell lines with specific functions is important for examination of organ-specific functions in vitro. We established a kidney tubule cell line (TKC2) exhibiting a specific physiological response to hormone from the primary culture of kidneys of adult transgenic mice harboring temperature-sensitive SV40 large T-antigen gene. TKC2 cells showed temperature-sensitive growth in culture and exhibited characters of distal tubule cells such as dome formation at confluent culture and stimulation of cAMP synthesis by arginine vasopressin. These phenotypes are maintained after long passages in culture and may provide a new experimental system for studying renal physiology.

Topics: Animals; Antigens, Viral, Tumor; Cell Line, Transformed; Cell Transformation, Viral; Cyclic AMP; Genes, Viral; Kidney Tubules; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Simian virus 40; Stimulation, Chemical; Temperature; Vasopressins

Transformed cells differ from normal cells in that they fail to respond to normal signals for regulation of growth and differentiation. This disordered signal transduction probably contributes to maintenance of the transformed phenotype. Several lines of evidence suggest that changes in the Ca2(+)- and phospholipid-dependent protein kinase, protein kinase C (PKC), may be important for transformation. To determine the role of PKC in transformation, we compared the levels and subcellular distribution of total phorbol ester receptors and PKC in normal and SV40-transformed rat embryo fibroblasts (REF52 cells). We also used our alpha-PKC (Type 3)-specific monoclonal antibodies to compare alpha-PKC content and regulation. We found no differences in quantity or subcellular distribution of PKC in 100,000 x g soluble and pelletable fractions. Downmodulation, which represents a feedback loop for limiting PKC activity, occurs to the same extent in both cell types. A major difference between the normal and transformed cells was revealed by immunofluorescence of alpha-PKC. In normal cels, alpha-PKC is tightly associated with the cytoskeleton and appears to be organized into focal contacts because it colocalizes with talin. In contrast, in SV40-REF52 cells, alpha-PKC is not tightly associated with the cytoskeleton and does not colocalize with talin. The difference in subcellular localizations correlates with a loss of two alpha-PKC-binding proteins in the transformed cells. These results indicate that inappropriate subcellular location of alpha-PKC may contribute to maintenance of the transformed phenotype.

Topics: Animals; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Transformation, Viral; Cells, Cultured; Cytoskeleton; Down-Regulation; Enzyme Activation; Fibroblasts; Fluorescent Antibody Technique; Isoenzymes; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Rats; Receptors, Drug; Simian virus 40; Vasopressins

Four subclones of the originally cloned Harvey murine sarcoma virus-transformed Madin Darby canine kidney (MDCK) cells have been isolated. These subclones fall into two general classes. Two subclones have a fibroblastic morphology, have lost the growth requirement for prostaglandin E1 (PGE1), do not respond to glucagon or vasopressin, and, in general, appear transformed. Two other subclones have epithelioid morphologies, are growth-stimulated by PGE1, respond to vasopressin with an increase in intracellular cAMP. We propose that these cells represent revertants to a more non-transformed phenotype. Unlike normal cells, however, these revertants grow under anchorage-independent conditions, express detectable but reduced amounts of the transforming gene product, p21, and grow in nude mice. The appearance of such revertants may be one cause of the observed heterogeneity of tumor cells.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Viral; Culture Media; Cyclic AMP; Dinoprostone; Dogs; Glucagon; Growth Substances; Harvey murine sarcoma virus; Mutation; Oncogene Proteins, Viral; Prostaglandins E; Sarcoma, Experimental; Vasopressins