© 1991 Oxford University Press
research-article |
Cyclic AMP response in SV40 immortalized human bladder cells
Geriatric Research, Education, and Clinical Center, VA Medical Center, and Departments of Biochemistiy and Internal Medicine, St Louis University School of Medicine St Louis, MO 63125 USA
1Department of Human Oncology, University of Wisconsin Clinical Cancer Center Madison, WI 53792, USA
2To whom reprint requests should be sent
Forskolin-mediated increase in cyclic AMP and subsequent activation of protein kinase A were evaluated in SV4O-immor-talized human urothelial cells. This cell line is being used to evaluate the multistep carcinogenic process. Forskolin elicited a time- and dose-dependent increase in cyclic AMP. Increases in intracellular cyclic AMP preceded media increases in cyclic nucleotide. Large increases in intracellular cyclic AMP occurred within 5 min of forskolin addition. The lowest effective concentration of forskolin was between 0.1 and 1.0 µM. Cyclic AMP increases as large as 20- to 100-fold were observed in cells and media following forskolin addition. A 60 min preincubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) did not reduce the magnitude of the forskolin increase in cyclic AMP. TPA has been shown to affect cyclic AMP metabolism in many types of cells including primary and secondary cultures of urothelial cells. In the latter, preincubation with TPA reduces the magnitude of the forskolin increase. A 4.2-fold increase in protein kinase A activity was observed within 0.5 min of forskolin addition, while only small increases in cyclic AMP (1.6-fold) were detected within 1 min. Much more cyclic AMP is synthesized than is needed to maximally activate protein kinase A. While results demonstrate a forskolin-responsive cyclic AMP system, this system does not appear to be regulated by TPA. Lack of regulation of this second messenger system by TPA may be part of the immortalization process.