Autocrine growth regulation of W12 and GCA cells in culture.
Two rat kidney cell lines transformed by two strains of ASV virus were investigated. It was demonstrated that these two lines (1) showed density-independent growth, (2) had a decreased requirement for serum in the culture medium, (3) had the ability to grow in a chemically defined medium (without serum), and the rate of this growth had increased with the increase in starting density of cells, and (4) had the ability of anchrage-independent growth, even without serum. These results confirmed autostimulation of growth of W12 and GCA cells. It was also shown that the crude conditioned media contained autocrine growth factors, which could be extracted with 1M acetic acid. The extracts (AEs) stimulated the growth of the parental cells and NRK-49F cells almost as well as 5% calf serum and the extraction resulted in several-fold purification of mitogenic substances. These substances were not only specific to parental lines, but also stimulated growth of other transformed lines and normal NRK-49F cells. Extracts from the conditioned media of W12 and GCA cells intensified the rate of anchorage-independent growth in the concentration-dependent manner. In AE-W12, two peaks of mitogenic activity were detected (F1, F2) and similarly in AE-GCA (F3, F4). Fractions F2 (approximately 8 kDa), F3 (approximately 25 kDa) and F4 (approximately 12 kDa) were thermostable but F1 (approximately 45 kDa) was thermolabile. All four fractions were sensitive to trypsin and DTT treatment, and were acid-stable. Using ELISA kit it was shown that W12 and GCA cells released TGFbeta1 and GCA cells released very small quantities of bFGF. These results confirmed the autocrine regulation of growth in both cell lines.
Szuster
Anna
A
Department of Molecular Genetics and Virology, Faculty of Biotechnology, Jagiellonian University, Kraków, Poland. szuster@awe.mol.uj.edu.pl
Kosz-Vnenchak
Magdalena
M
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000378:metabolism / Q000502:physiology*; D002448:Cell Adhesion; D002461:Cell Line, Transformed / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002472:Cell Transformation, Viral / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D006133:Growth Substances; D008934:Mitogens / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D013997:Time Factors; D015291:Transforming Growth Factors / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D006133:Growth Substances; D008934:Mitogens; D009856:Oncogene Proteins, Viral; D015291:Transforming Growth Factors