Mouse submandibular salivary epithelial cell growth and differentiation in long-term culture: Influence of the extracellular matrix

Summary The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and its receptor and signal transduction pathway. This work was supported by Public Health Service grant DE07766 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel,collagen, fibrin,and plasma clot

Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of³H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis. This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes of Health, Bethesda, MD.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Fibronectin,not laminin,mediates heparin-dependent heparin-binding growth factor type I binding to substrata and stimulation of endothelial cell growth

Summary Fibronectin and heparin-binding growth factors (HBGF) are essential for growth of cultured endothelial cells. The stimulation of endothelial cell growth by HBGF type one (HBGF-1) in particular requires heparin or a similar glycosaminoglycan. The requirement for fibronectin and heparin for HBGF-1-stimulated endothelial cell growth may be related. HBGF-1 absorbed to the natural subcellular matrix of endothelial cells supports cell growth. [¹²⁵I]HBGF-1 specifically associates with a sequentially reconstituted matrix of collagen-fibronectin-heparin, and HBGF-1 absorbed to the reconstituted matrix supports growth of the endothelial cells. A reconstituted matrix of collagen-laminin-heparin neither supported binding of [¹²⁵I]HBGF-1 nor HBGF-1-stimulated endothelial cell growth. Association kinetics of [¹²⁵I]HBGF-1 to heparinlike sites and membrane receptor sites on endothelial cell monolayers suggest that fibronectin-heparinlike binding sites in the subcellular matrix may be an obligatory reservoir of active HBGF-1 that binds to specific cell membrane receptors. This work was carried out in the laboratory of Dr. W. L. McKeehan and supported in part by grants CA37589, DK35310 and DK38639 from the Public Health Service, Department of Health and Human Services, Washington, DC.     

高寒草甸生态系统牲畜种群结构和动态

本文用Leslie矩阵模型研究了高寒草甸生态系统牲畜种群结构及动态。模型考虑了更加精确的年龄组转移关系,出栏率是种群波动的主要因子。目前,牲畜种群结构不合理,种群数量不能保持平衡。     

A comparison of fertilization envelope development in three species of Strongylocentrotus (S. franciscanus, S. droebachiensis, and S. purpuratus)

The ultrastructure of fertilization envelope (FE) development and the polypeptide spectra of Strongylocentrotus franciscanus and S. droebachiensis envelopes were compared to S. purpuratus. In S. franciscanus, the FE reached its maximum thickness of 67 nm by 3 minutes postinsemination (PI), and final structuralization was observed by 40 minutes PI. The fully formed FE did not have microvillar impressions (casts) and was symmetrical, with outer double laminar elements surrounding an amorphous central region. Isolated S. franciscanus FEs were soluble in reducing and denaturing solvents and the same set of 33 polypeptides ranging from 18.5 to 260 kD was detected in FEs isolated from 10 to 180 minutes PI. The S. droebachiensis FE retained microvillar casts, assumed its definitive form by 3 minutes PI, and was 70 nm thick between microvillar impressions. Isolated S. droebachiensis FEs were partially soluble in reducing and denaturing solvents, and the polypeptide spectra of FEs isolated between 10 and 60 minutes PI were identical and showed 14 polypeptides from 18.5 to 265 kD. Antisera against extracted FEs and the FE extract from S. purpuratus were immunologically cross-reactive (using an enzyme-linked immunosorbent assay) with S. franciscanus and S. droebachiensis FE preparations; immunoblots identified 13 and 5 cross-reactive polypeptides, respectively. Most of the cross-reactive polypeptides were of slightly different molecular weight. Based on comparative ultrastructural, solubility, and electrophoretic data, we suggest that S. droebachiensis FE development is most like that observed in S. purpuratus.     

Altered matrix metalloproteinase expression associated with oncogene-mediated cellular transformation and metastasis formation

Matrix metalloproteinase expression was examined in a series of mammalian cell lines of varying degrees of malignant progression. The expression of MMP-2 and MMP-9 was found to correlate with ras-mediated cellular transformation and as a function of malignant potential. Altered MMP-2 and MMP-9 expression was found to correlate also in other oncogene transformed cell lines and the level of expression of both MMP-2 and MMP-9 correlated with metastatic potential. Increased expression of both MMP-2 and MMP-9 was also found in cells which constitutively over-express MAP kinase kinase suggesting that one of the consequences of the persistent activation of the MAP kinase pathway is elevated expression of MMP-2 and MMP-9. Additionally, this study demonstrated a correlation between the expression of MMP-3 (stromelysin-1) and the level of ras expressed in cells and with the cells' ability to form tumors and with malignant potential. The existence of a novel 80 kDa caseinase activity which correlates with ras expression and the ability of the cell to form tumors was also demonstrated. The growth status of transformed cells was also found to be important in determining the expression of MMP-2 mRNA but not MMP-9 mRNA expression, and this expression was cell-type specific. This study also demonstrates that oncogenes can interact to influence and to determine the nature of the matrix metalloproteinases expressed and that this interaction results in a tumorigenic phenotype and, most importantly, contributes to the metastatic phenotype. Alterations in the expression and the regulation of MMPs, particularly MMP-2 and MMP-9, constitute an integral part of the altered growth regulatory program found within transformed cells and in particular, in transformed cells capable of malignant progression.