Site selective cyclic amp analogues are antagonistic to estrogen stimulation of growth and proto‐oncogene expression in human breast‐cancer cells

Abstract

Studies from our laboratory suggested that regulation of the growth of hormone-dependent mammary tumors may depend on the antagonistic action between estrogen and cyclic AMP (CAMP). Estrogen stimulates whereas cAMP arrests the growth of mammary carcinomas induced by 7,12-dimethylbenz(a)anthracine (DMBA) in the rat (Cho-Chung and Gullino, 1974). During growth arrest of the tumors after either hormone removal (ovariectomy) or treatment of the hosts with N6, d'-dibutyryl-CAMP (DBcAMP), estrogen binding decreases whereas cAMP binding and CAMP-dependent protein kinase activity increase in the cytosol and nuclei of the tumor cells (Bodwin et al., 1978). It was further demonstrated that the growth of DMBA-induced mammary tumors is associated with an enhanced expression of a cellular oncogene, c-rasH, The p21-transforming protein of the ras gene product (Shih et al., 1979) was a predominant in vitro translation product of mRNAs of the growing tumors, and a sharp reduction of the translated p2 1 protein preceded regression of these tumors after either ovariectomy or DBcAMP treatment (Huang and Cho-Chung, 1984).Recent studies on cAMP binding kinetics, using purified preparations of CAMP-dependent protein kinases, identified cAMP analogues that are potent activators of protein kinase and selectively bind to either one of the 2 different cAMP binding sites (Rannels and Corbin, 1980) of protein kinase. Generally, analogues modified at the C-8 position of adenine ring are Site 1-selective and those modified at the C-6 position are Site 2-selective (Rannels and Corbin, 1980). Furthermore, the Site 1-and Site 2-selective analogues in combination demonstrate synergism of binding to and activation of protein kinase (Robinson-Steiner and Corbin, 1983; Qgreid et al., 1985). the effect of site-selective cAMP analogues on the growth and cellular proto-oncogene expression that can be stimulated by estrogen in human MCF-7 breast-cancer cells. MATERIAL AND METHODS MaterialCAMP, DBcAMP, and 8-bromo-CAMP were from Boehringer Mannheim (Indianapolis, IN). N6-monobutyryl CAMP, N6-benzoyl CAMP, and 17P-esrradiol were obtained from Sigma (St. Louis, MO). All other cAMP analogues were provided by Dr. R.K. Robins (Revankar and Robins, 1982). Tamoxifen (ICI-46474) was obtained from Stuart (Wilmington, DE). Improved minimal essential medium (IMEM) was obtained from NIH Media and Glassware Section. Dulbecco's phosphate-buffered saline (PBS), fetal bovine serum (FBS), trypsin-EDTA solution, penicillin-streptomycin solution, Lglutamine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer 1 M stock solution, PH 7.3, were obtained from GIBCO (Grand Island, NY). The MCF-7 breastcancer cell line was provided by Dr. M. Lippman. Cell cultureMCF-7 cells were grown in IMEM with no phenol red (Berthois et al., 1986), containing 6.5 % of charcoal-stripped serum, HEPES 20 mM, penicillin-streptomycin, and extra glutamine. Cells were grown at 37°C in humidified incubators in an atmosphere of 5 % C02.For cell growth experiments, 1-2 X lo5 cells/...