Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells.

Stromal-epithelial interactions play a profound role in regulating normal and tumor development in the mammary gland. The molecular details of these events, however, are incompletely understood. A novel serum-free transwell coculture system was developed to study the natural paracrine interactions between mammary epithelial cells (MEC) and mammary fibroblasts (MFC) isolated from normal rats during puberty. The MEC were cultured within a reconstituted basement membrane (RBM) in transwell inserts with or without MFC in the lower well. The presence of MFC stimulated epithelial cell growth, induced alveolar morphogenesis, and enhanced casein accumulation, a marker of the functional differentiation of MEC, but did not induce ductal morphogenesis. Potent mitogenic, morphogenic, and lactogenic effects were observed when the MFC were cultured either on plastic or within a layer of RBM. Although most MFC maintained on plastic died after 1 wk in serum-free medium, fibroblast survival was enhanced significantly when the MFC were cultured within the RBM. Taken together, this in vitro model effectively reconstitutes a physiologically relevant three-dimensional microenvironment for MEC and MFC, and seems ideal for studying the locally derived factors that regulate the developmental fate of the epithelial and fibroblast compartments of the mammary gland.

Studies with a new antifolate 2,4-diamino-5-adamantyl-6-methylpyrimidine (DAMP): tissue distribution and disposition of 2,4-diamino-5-adamantyl-6-methylpyrimidine and its metabolite.

The pharmacokinetics of a potentially useful antitumor agent, 2,4-diamino-5-adamantyl-6-methylpyrimidine (DAMP) has been studied in rat using 14C-labeled drug. It is reported that DAMP is metabolized rapidly by liver microsomes to an unidentified compound. The metabolite seems to be excreted by liver more rapidly than the unchanged drug, and within 24 hours all radioactivity is eliminated, 80% through kidney and 20% through the bile. Both DAMP, and its metabolite accumulate readily in pancreas and kidney. In contrast, in the brain mostly unchanged DAMP could be detected. The accumulation of the drug in tissues other than liver is much greater after i.v. than after i.p. administration.

Synthesis and antifolate activity of new diaminopyrimidines and diaminopurines in enzymatic and cellular systems.

The following new 2,4-diamino-6-methylpyrimidines, 5-cyclohexylmethyl, 5-cyclohexylethyl,and 5-(2-naphthyl), as well as 2,6-diaminopurines, 8-adamantyl and8-adamantylmethyl, were synthesized as potential antifolates. Tese, as well as three known compounds, 2,4-diamino-5-cyclohexyl-6-methylpyrimidine, 2,4-diamino-5-(1-naphthyl) -6- methylpyrimidine, and 2,6-diaminopurine, were compared with respect to the inhibition of growth of mammalian cells in culture (TA 3) and with respect to the inhibition of partially purified dihydrofolate reductase. All of the pyrimidines except for the 5-(1maphthyl) derivative were competitive inhivitors of dihydrofolate reductase, with K values ranging from 0.07 to 0.04 pM. They were 2-5 times better as inhibitors of the isolated dihydrofolate reductase than of the cell growth. 2,4-Diamino-5-(1-naphthyl)-6-methylpyrimidine was a noncomptive inhivitor of the enzyme with a Kvalue of 56 pM. This compound was more potent in inhibiting cell growth than the isolated enzyme. indicating that its biological activity was not related to the inhibition of dihydrofolate reductse. All of the purine derivatives were poor growth inhibitors and although some of them inhibited isolated dihydrofolate reductase, their mode of action in cellular system did not seem to concern folate metabolism, as judged by the inability of hypoxanthine, and glycine to provide protection. The implication of these findings as to the structural requirements for inhibition of dihydrofolate reductase is discussed. The implication of these findings as to the structural requirements for inhibition of dihydrofolate reductase is discussed. The pitfalls of the determinition of ID-50 values instead of a complete kinetic analysis in structure-activity studies are emphasized.