Adenosine stimulation of DNA synthesis in mammary epithelial cells.

Adenosine increased the DNA synthesis rate and the percentage of S-phase cells 2-3-fold in mouse mammary epithelial cells (NMuMG), with an optimum concentration of 10-100 microM. This effect was not mimicked by adenosine metabolites adenine, hypoxanthine, or inosine. N-ethylcarboxamidoadenosine (NECA, a relatively nonselective adenosine receptor agonist) and 2-p-(2-carboxyethyl) phenethylamino-5'-N ethylcarbox-amidoadenosine (CGS-21680, an A2 selective agonist) also increased DNA synthesis by mammary epithelial cells. However, N6-cyclohexyladenosine (CHA, an agonist for A1 type receptors) decreased DNA synthesis. The A1 selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no effect on basal or adenosine-induced DNA synthesis, whereas the A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) decreased adenosine-induced DNA synthesis. Similar effects were observed in another nontumorigenic mouse mammary epithelial line, HC11, as well as the nontumorigenic human lines MCF-10A and 184.A1. Binding studies indicated that NMuMG cells contained approximately 3200 A1 receptors and about 5300 A2 receptors per cell. Both CGS-21680 and CHA increased GTPase activity in isolated cell membranes, whereas only CGS-21680 increased activity of adenylyl cyclase. Adenosine and CGS-21680 increased expression of a cyclic AMP responsive reporter gene. In addition, the protein kinase A inhibitor H-89 blocked the ability of adenosine and CGS-21680 to induce DNA synthesis, but did not affect EGF-induced DNA synthesis. These results indicate that adenosine appears to be a possible growth promoting agent in mammary tissue, and this effect may be mediated by extracellular receptors of the A2 type.

Effects of adenosine and epidermal growth factor on the growth of mouse mammary epithelial cells.

The objective of this study was to determine if adenosine alters growth of mammary epithelium. Mouse mammary epithelial cells (NMuMG) were cultured in DMEM supplemented with 10% fetal calf serum. After serum starvation for 24 h, EGF (0-100 ng/ml) and/or adenosine (0-100 microM) was added. Adenosine at concentrations of 1, 10 or 100 microM increased DNA synthesis significantly, when compared to control. Addition of epidermal growth factor (EGF) (10 ng/ml) into 1 or 10 microM adenosine showed the interaction in DNA synthesis between EGF and adenosine. A similar result was observed when 100 microM adenosine added to various concentrations of EGF (0-100 ng/ml). In the second mammary gland (thoracic) organ culture studies, mammary development scores were increased by adenosine (100 microM), EGF (100 ng/ml) and adenosine plus EGF. These results indicate that the purine nucleoside adenosine stimulates mammary epithelial cell growth and interacts with EGF in DNA synthesis of mouse mammary epithelial cells.

Photoreactive (caged) cyclic AMP analogs induce DNA synthesis in mammary epithelial cells.

Dimethoxy-2-nitrobenzyl adenosine-3',5' cyclic monophosphate (DMNB) is a metabolically active, photolabile cyclic AMP analog that yields free cyclic AMP upon UV hydrolysis. The analog is useful in that it permits short term, transient elevations of intracellular cyclic AMP. Addition of DMNB (1-10 microM) to mouse mammary epithelial cells, followed by UV irradiation of cells, caused a significant increase in DNA synthesis over that observed with controls, UV irradiation alone or DMNB alone. In subsequent studies, DMNB exhibited a modest, but statistically significant, interaction with epidermal growth factor in promoting DNA synthesis. Effects of DMNB were observed if DNA synthesis was measured as either 3H-thymidine incorporation into DNA or as percent S-phase cells. These results indicate that previously observed effects of agents such as cholera toxin and phosphodiesterase resistant cyclic AMP analogs on mammary epithelial proliferation can be mimicked, at least in part, by a short term pulse of cyclic AMP.

Influence of epidermal growth factor on growth of bovine mammary tissue in athymic nude mice.

Bovine mammary tissue obtained from midpregnant Holstein heifers by surgical biopsy was transplanted subcutaneously to ovariectomized athymic nude mice (n = 5 heifers). After 3 weeks recovery, mice were either sham operated or sialoadenectomized (submandibular salivary glands removed). After an additional week, mice were injected with saline or 17 beta-estradiol + progesterone (1 microgram + 1 mg/day) for 2 days. In addition, half of the sialoadenectomized mice were injected with epidermal growth factor (5 micrograms/day). Grafted tissue was removed and rate of deoxyribonucleic acid (DNA) synthesis estimated by incorporation of 3H thymidine. Estradiol + progesterone increased the incorporation of 3H thymidine from 77 +/- 20 dpm/micrograms DNA to 472 +/- 53 dpm/micrograms DNA. In sialoadenectomized mice, DNA synthesis was increased from 88 +/- 16 dpm/micrograms DNA (saline treated) to 360 +/- 29 dpm/micrograms DNA (estradiol + progesterone treated). In sialoadenectomized mice treated with epidermal growth factor, DNA synthesis in estradiol + progesterone treated mice was 529 +/- 36 dpm/micrograms DNA, compared to 112 +/- 30 dpm/micrograms DNA in sialoadenectomized mice treated with epidermal growth factor. These data indicate that sialoadenectomy of athymic nude mice decreased the ability of transplanted bovine mammary tissue to increase DNA synthesis in response to estradiol and progesterone. This inhibition was removed by epidermal growth factor treatment. These data suggest a physiological role of epidermal growth factor in regulating development and hormone responsiveness of bovine mammary tissue.