In culture, cAMP is known to be mitogenic for mammary cells and several other epithelia, but evidence for a similar role in vivo has been only correlative. We have used plastic implants to cause slow release of cholera toxin and other cAMP-active agents to local areas of mammary glands in ovariectomized mice. Elevated levels of intracellular cAMP around the implants promoted vigorous growth and normal ductal morphogenesis, while distant sites were unaffected. Local effects of cAMP included restoration of normal ductal caliber, formation of new end buds, and reinitiation of DNA synthesis in both epithelium and surrounding stroma. Thus, cAMP is both a mitogenic and a morphogenetic factor in this tissue.Numerous in vitro studies have shown cAMP to be growth promoting for mouse, rat, and human mammary epithelia (1-4) and for a variety of other epithelial cell types (5-8). In situ, cAMP levels increase as the mammary gland grows during pregnancy (9, 10), and they are elevated in several breast carcinomas (11,12). To obtain more direct evidence as to whether cAMP may function as an intracellular mediator of hormone or growth factor action in the mammary gland, we investigated the effects of raising cAMP levels in small regions of the mouse mammary gland by using slow-release plastic implants.End buds, the primary sites of mammary epithelial proliferation in subadult virgin mice, are complex highly mitotic structures comprised of the stem cell progenitors of myoepithelial and ductal cells (13). As the growth points for the ductal tree, end buds are the focus of mammary growth regulatory influences during this stage in development of the gland (14). We here present evidence that, in situ, local elevation of cAMP stimulated formation of new end buds in regressed ducts, apparently replacing the normal hormonal requirements for this type of development. CT Dosage. Commercial CT contains 4.8% by weight of toxin protein, the bulk of the preparation being NaCl and Tris HCl (55% and 37.6%, respectively). The most commonly used implant was prepared with 1 mg of the commercial preparation dissolved in 0.25 ml of Elvax. The dried Elvax pellet weighed -40 mg and contained 48 ,ug of toxin protein or 1.2 ,ug in an implant-sized piece weighing 1 mg. The useful dose range for this implant type is 0.1-1.2 ,ug of toxin.
MATERIALS AND METHODSRelease of CT from Elvax. The release kinetics of CT was studied in vitro. Pellets prepared with either 1 mg or 10 mg of the toxin were cut into implant-sized pieces, weighed, and sealed in nylon bags (Nitex), which were then placed in 1.5 ml of normal saline at 37°C on a rotator (1 rpm). At 24-hr intervals, the bags were removed and the saline was assayed for protein against a CT standard using the Bradford method (16). Bags were then rinsed and replaced in fresh saline. An adequate amount of Elvax with toxin was available for three trials, one of which is shown here (Fig. 5); the release profile was the same for all three trials. Surgical Implantation. The abdominal skin of Nembutalanesthet...