Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell–cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

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Optimization of host cell lines both for transient and stable protein production is typically hampered by the inherent heterogeneity of cells within a population. This heterogeneity is caused not only by "hard fact" gene mutations, but also by subtle differences in the cellular network of regulation, which may include epigenetic variations. Taking advantage of this heterogeneity, we sorted for naturally occurring variants of CHO-K1 and CHO-S host cells that possess an improved cellular machinery for transient antibody production. The long-term goal of this study was both to identify host cells that yield recombinant cell lines with on average higher productivity, but also to study the molecular differences that characterize such cells, independent of the site of gene integration or gene amplification. To identify such cells we optimized the procedure for transient transfection by electroporation to a degree that gave uniform transfer of plasmid DNA into nearly 100% of the cells and resulted in reproducible average productivities, with a standard deviation of 16% between independent experiments. Using this optimized protocol, the 1% of cells with the highest specific productivity was sorted and subcloned with a cold capture secretion assay. Upon re-transfection, the resulting subclones showed the same specific productivity as their respective parental cell line. To enrich for cells with potentially stable improved properties, the 1% highest producers were sorted three times, 2 days after transient transfection each, and the enriched population was again sorted into microtiter plates for subcloning. For each of the two parental cell lines tested, three subclones were obtained that had a threefold higher specific productivity after transient transfection. This property was stable for approximately 3 months, indicating that the changes in productivity were regulatory and not mutational.

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In this study the luteotropic activity of mouse placental lactogen I (mPL-I) at midpregnancy was assessed using in vivo and in vitro methodologies. Ovaries from 10-day pregnant mice were enzymatically dispersed and plated on fibronectin-coated wells in serum-free medium. The percentage of ovarian cells that stained for the presence of 3 beta-hydroxysteroid dehydrogenase activity was 24.4 +/- 2.7% at the time of plating and remained constant (26.1 +/- 5.0%) after a 20-h attachment period. Two types of 3 beta-hydroxysteroid dehydrogenase-staining cells, with distinct differences in size and morphology, were present in the culture. Large luteal cells (26-45 microns) were characterized by a small round nucleus and spherical shape with abundant cytoplasm. In contrast, small luteal cells (< 20 microns) were stellate, with little cytoplasm and a large oval nucleus. Basal progesterone secretion was maintained without a change in cellular DNA content and cell number for 168 h of culture. Treatment of ovarian cells with mPL-I (0.05-10 micrograms/ml) caused a dose-dependent increase in the progesterone concentration in the medium. The magnitude and time course of mPL-I-stimulated progesterone accumulation in culture were dependent on the time after plating that mPL-I treatment was initiated. The effects of mPL-II and mouse PRL (mPRL) on progesterone production were similar to those of mPL-I. The ability of sera from 10-, 14-, and 17-day pregnant mice to maintain progesterone production in bromocryptine-treated hysterectomized mice was also examined. Mice were hysterectomized on day 9 of pregnancy, and serum progesterone, mPL-I, mPL-II, and mPRL concentrations were measured 72 h later. Twice daily injections of 0.5 ml day 10 pregnancy serum maintained the circulating progesterone concentration at values not different from those present at the time of hysterectomy. In contrast, serum progesterone concentrations were not maintained in mice treated with serum of 14- or 17-day pregnant mice or with saline. Depletion of mPL-I from day 10 pregnancy serum by affinity chromatography on an anti-mPL-I column removed all luteotropic activity, as determined by the inability of this modified serum to maintain the serum progesterone concentration in bromocryptine-treated hysterectomized mice. A similar pool of day 10 pregnancy serum chromatographed on a nonspecific IgG control column did maintain progesterone production, but at somewhat lower concentrations than those present at the time of surgery. These studies offer direct evidence that mPL-I and mPL-II are luteotropic and support progesterone production at midpregnancy in the mouse.

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