Biochemical properties of the endothelium-derived growth factor: Comparision to other growth factors

Cultured bovine aortic endothelial cells (BAEC) can be maintained at saturation density for several weeks in the absence of serum. These cells retain viability and normal culture morphology, and continuously produce a growth factor for mesenchymally derived cells–the endothelium-derived growth factor (EDGF). The amount and specific activity of EDGF that is produced by BAEC under serum-free conditions remains constant for weeks. The levels of EDGF produced under these serum-free conditions is equivalent to levels produced in medium containing 5% plasma-derived serum. EDGF has been found to be trypsin sensitive, acetone and ammonium sulfate precipitable, and resistant to heat and sodium dodecyl sulfate treatment. Gel filtration on Sephacryl S-200 in the presence of formic acid (1%) yields two major peaks of activity corresponding to proteins of apparent molecular weights of approximately 24,000 and 14,000 daltons. This chromatographic step affords a ten-to 12-fold purification with a combined recovery of greater than 85%. Unlike brain or pituitary fibroblast growth factor, EDGF activity is destroyed by dithiothreitol or periodic acid. EDGF is not a somatomedin since it exhibits no detectable sulfation activity in a porcine cartilage assay. EDGF is not inhibited by antiserum to epidermal growth factor and is capable of stimulating DNA synthesis in a 3T3 variant cell line that is nonresponsive to and lacks receptors for epidermal growth factor. The majority of EDGF activity does not behave like the platelet-derived growth factor during ion exchange chromatography. Antisera prepared in rabbits and in mice to human platelet-derived growth factor has little effect on bivine or human EDGF activity. These biochemical and immunological properties of EDGF indicate that it is distinct from several other well-characterized polypeptide growth factors.     

Influence of chromatographic fractions of extracts derived from bovine neural retina on newt (Notophthalmus viridescens) lens regeneration in vitro

Removal of the ocular lens in adult newts (Notophthalmus viridescens) is followed by a series of cellular events leading to regeneration of a new lens by cell type conversion of pigmented iris epithelial cells at the dorsal pupillary margin (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). Following depigmentation and five to seven cell divisions, iris epithelial cells redifferentiate into lens fiber cells and synthesize crystallin proteins (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). This process is dependent upon neural retina in vivo (Stone, Anat. Rec. 131:151-172, 1958; Reyer, Dev. Biol. 14:214-225, 1966) and in vitro (Yamada et al., Differentiation 1:65-82, 1973). Acting on the hypothesis that the role of the neural retina is to promote passage of iris epithelial cells through the requisite number of cell cycles which will then allow them to redifferentiate as lens fiber cells (Yamada, in: Cell Biology of the Eye. Academic Press, New York, 1982), we undertook testing of the effects of eye-derived mitogenic substances, as well as other mitogens, on regeneration of lens from iris in organ culture. We have previously defined a critical period for the retinal influence in vivo and in vitro, and have shown that crude extracts of retina can enhance regeneration of lenses in culture (Connelly et al., J. Exp. Zool., 240:343-351, 1986). In this paper, we report on the lens regeneration enhancing activity (LRA) of more highly purified fractions of the retinal extracts. Heparin-sepharose chromatography of the crude retinal extract yields three fractions (Courty et al., Biochemie 67:265-269, 1985) called EDGF I, II, and III. EDGF I and II have affinity for heparin, while EDGF III does not. In our bioassay, LRA appears only in the EDGF III fraction. Dialysis of EDGF III against 0.1 N acetic acid yields a fraction which has affinity for cibacron blue sepharose (eluting at 2.15 M salt) and also has significant LRA. Because insulin at high doses has a marginal effect on lens regeneration in culture (Williams and McGlinn, Am. Zool. 19:923, 1979; Connelly, Differentiation 16:85-91, 1980), we tested IGF-I. Because of the putative neurotrophic effects of transferrin (Tf) (Mescher and Munaim, J. Exp. Zool., 230:485-490, 1986), we tested Tf for its ability to enhance regeneration of the lens in culture. IGF-I seems to have an enhancing effect on lens regeneration; Tf does not.     

Insulin-like growth factors, IGF-1, IGF-2 and somatomedin C trigger cell proliferation in mammalian epithelial cells cultured in a serum-free medium

A single insulin-like growth factor which constitutes part of a defined serum-free medium is sufficient to stimulate DNA synthesis and mitosis in mammalian lens epithelial cells. Rabbit lenses were cultured in KEI-4, a medium which mimics rabbit aqueous humor, or in KEI-4 containing insulin growth factor I (IGF I), insulin growth factor II (IGF II) or somatomedin C. The magnitude of DNA synthesis and mitosis was evaluated on whole mount preparations of the epithelium at various times of culture. IGF I and II, the most highly purified of the insulin-like growth factors, and somatomedin C were equipotent lens mitogens, were active at the ng level, were more mitogenic toward lens epithelial cells than insulin, and initiated cell proliferation throughout the normally amitotic central region of the lens epithelium. The time course of the mitotic response elicited by the insulin-like growth factors was identical to that noted in lenses cultured in medium supplemented with serum or insulin. The present results, coupled with those of other investigators, suggest that insulin-like factors may regulate cell division in the mammalian lens in vivo.     

Human and bovine vascular endothelial cells. Comparative effect on cell growth and longevity of an eye-derived growth factor (EDGF) and of extracellular matrix

Human and bovine vascular endothelial cells from the umbilical vein and the aorta, respectively, were cultured in the presence of EDGF (a growth factor prepared from bovine retina) on plastic or on extracellular matrix (ECM). Both EDGF and ECM are required to allow the maximal proliferation of human cells and their organization in a typical monolayer. Conversely, bovine aortic endothelial cells grow perfectly in the absence of both factors in 6% fetal calf serum. However, a requirement for EDGF can also be demonstrated in low serum conditions, or in cells at high passage number. ECM had no growth promoting activity by itself. Thrombin acts similarly to EDGF on bovine serum-starved cells. EDGF prolongs the in vitro lifespan of both types of cells. Cells at all stages still synthesize factor VIII antigen as revealed by immunofluorescence. Thus EDGF, like other growth factors from brain, FGF or ECGF, may have an important role in angiogenesis, a critical problem in pathological retinas.     

Interaction of bovine epithelial lens (BEL) cells with extracellular matrix (ECM) and eye-derived growth factor (EDGF): I. Effects on short-term adhesiveness and on long-term organization of the culture

A growth factor (EDGF) derived from the retina controls the proliferation and shape of adult bovine epithelial lens (BEL) cells in vitro as well as extracellular matrix (ECM) assembly. In order to analyse this mechanism and the specificity of the interactions between BEL cells and the extracellular matrix we have investigated the adhesion and growth of BEL cells on various substrata (fibronectin, laminin, ECM). BEL cells treated with EDGF adhered more slowly to plastic Petri dishes than untreated cells, in part due to EDGF inhibition of fibronectin deposition. The untreated BEL cells spread less well on ECM or laminin than on fibronectin-coated plastic. The preferential adhesiveness of BEL cells on fibronectin vs laminin was confirmed by attachment experiments performed on replicas of SDS-PAGE of these proteins. However, in long-term cultures, 8 days after seeding, BEL cells were very differently arranged on plastic or on ECM. ECM by itself did not increase the proliferation rate but helped to restore an organized cell monolayer. BEL cells stimulated to grow on ECM by treatment with EDGF exhibited at least transiently contact inhibition producing a perfectly organized epithelium similar to the one observed in vivo. These results suggest specific interactions between ECM or ECM components with BEL cell that restrain excessive cell spreading and restore an original polarized phenotype of the cells seen in vivo.     

Growth and characterisation of primary bovine colon epithelial cells in vitro

Epithelial crypts from the bovine colon were obtained by using a combined mechanical and enzymatic isolation method, followed by differential D-sorbitol gradient centrifugation. By using this isolation technique, a pure fraction of epithelial crypts with minimal mesenchymal contamination was obtained. The crypts were seeded in collagen-coated plastic flasks. The attached epithelial cells proliferated and formed a confluent monolayer after 6 days in culture. Under low-serum culture conditions (1% fetal calf serum), the cells had a population doubling time of 21-22 hours. During the culture period, the colonocytes were characterised morphologically and enzymatically. The morphology of the cultured cells was confirmed by scanning electron microscopy and transmission electron microscopy. The presence of microvilli, tight junctions and desmosomes demonstrated the ability of the cultured cells to restore an epithelial-like cell monolayer. The epithelial origin of the cells was demonstrated by labelling the cells with antibodies against epithelial-specific cytokeratins 7 and 13. The functional integrity of the cells was evaluated by measuring various marker enzymes (gamma-glutamyltranspeptidase, acid phosphatase, alkaline phosphatase, NADH-dehydrogenase) and membrane-associated Na+-K+-ATPase activity. Membrane integrity was determined by measuring the leakage of lactate dehydrogenase into the culture medium. This new culture system for bovine colon epithelial cells could be used as an in vitro model of the colon epithelium in physiological and toxicological studies.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

Isolation, culture and characteristics of epididymal epithelial cells from adult cats

A tissue-culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the epididymal duct in the cat. A widely used culture protocol for rat epididymal epithelium was used as a starting point and subsequently modified. The cellular population of the cat's epididymal epithelium was isolated by successive collagenase and trypsin digestion. A high yield of isolated cells obtained with good viability, were cultured in DMEM/F12 medium supplemented with foetal bovine serum, in absence or in presence of additional dihydrotestosterone (1 nM). The plated primary cultures reached confluence within 5-8 days, producing a monolayer of cohesive cells. Samples taken after 6 days in culture were processed for transmission and scanning electron microscopies. Immunocytochemical staining was used to estimate the purity of the epithelial cell population in the monolayers. The cell cultures displayed several functional traits of in vivo epithelia, including [35S] hypotaurine and [35S] taurine production. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually mature cats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell functions, metabolic activities and their regulation in cats.     

Influence of microenvironment on mammary epithelial cell survival in primary culture.

Mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix form multicellular structures termed mammospheres, in which cells and matrix become arranged around a central luminal space. In the presence of lactogenic hormones, cells within mammospheres become polarized, form tight intercellular junctions, and secrete milk proteins vectorially into the luminal space. This study examined the mechanism of lumen formation. Histological examination of developing mammospheres showed that cavitation was associated spatially and temporally with the appearance of fragmented nuclear material in apoptotic bodies, and with the presence of cells positively labeled by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labeling (TUNEL). Analysis of [(32)P]-deoxynucleotide end-labeled genomic DNA by electrophoresis and autoradiography showed DNA laddering indicative of apoptosis. A transient increase in laddering coincided with both lumen formation and the presence of TUNEL-positive cells. Lumen formation, DNA laddering, and detection of TUNEL-positive cells were all accelerated when matrix composition was altered. They were also impaired coordinately when caspase inhibitor was present during the first two days of culture. Therefore, lumen formation in mammosphere cultures is due to selective apoptosis of centrally located cells. Mammosphere cavitation was accompanied by redistribution of matrix constituents to the mammosphere periphery. Western blotting and Western ligand blotting of culture medium showed that lumen formation was also associated with a transient increase in insulin-like growth factor binding protein-5 (IGFBP5), a factor implicated in mammary apoptosis in vivo. We propose that epithelial cell survival during mammosphere development is induced selectively through stabilization by basement membrane constituents, which may act directly on the epithelial cell or confer protection against autocrine apoptotic factors.     

Endothelial cell differentiation of human breast tumour stem/progenitor cells

Breast tumour stem cells have been reported to differentiate in the epithelial lineage but a cross-lineage potential has not been investigated. We aimed to evaluate whether breast tumour stem cells were able to differentiate also into the endothelial lineage. We isolated and cloned a population of breast tumour stem cells, cultured as mammospheres that expressed the stem markers nestin and Oct-4 and not epithelial and endothelial differentiation markers, and formed serially transplantable tumours in SCID mice. When cultured in the presence of serum, mammosphere-derived clones differentiated in the epithelial lineage. When cultured in the presence of VEGF, the same clones were also able to differentiate in the endothelial lineage acquiring endothelial markers and properties, such as the ability to organize in Matrigel into capillary-like structures. In the transplanted tumours, originated from mammospheres, we demonstrate that some of the intratumour vessels were of human origin, suggesting an in vivo endothelial differentiation of mammosphere-derived cells. Finally, endothelial cell clones originated from mammospheres were able, when implanted in Matrigel in SCID mice, to form after 7 days a human vessel network and, after 3–4 weeks, an epithelial tumour suggesting that in the endothelial-differentiated cells a tumourigenic stem cell population is maintained. In conclusion, the results of the present study demonstrate that stem cells of breast cancer have the ability to differentiate not only in epithelial but also in endothelial lineage, further supporting the hypothesis that the tumour-initiating population possesses stem cell characteristics relevant for tumour growth and vascularization.     

Cellular changes in cultured lenses.

Observations were made on the frog lens epithelium after culture of the entire lens or of capsular explants. General deviations from normal lens structure as well as specific changes in two media were studied. DNA synthesis and mitosis were induced in the central epithelial cells. Disruption of the orderly, single, epithelial layer that is characteristic of normal lenses was accompanied by the appearance of multilayered plaques of epithelial cells and invasion of vacuolated regions of the lens fibers by epithelial cells. Cells that are fibroblast-like in appearance were observed in regions of the capsule depleted of cells and at the free edges of epithelial sheets in cell culture. Epithelial cells were surrounded by capsule-like material even situated in the lens interior. Nuclie derived from central epithelial cells of lenses cultured in L-15 medium and medium 199 had served as donors in previous nuclear transfer experiments in this laboratory. In our current observation of L-15-cultured lenses, cells were sparsely distributed on the capsule and nuclei were abnormally shaped; in 199-cultured lenses, cells were more densely distributed and nuclei resembled those of normal lenses. Medium 199 without serum could better maintain normal lens structure than L-15 medium without serum. In addition, the percentage of epithelial explants demonstrating cellular outgrowth was greater in medium 199. The differences in cellular behavior were shown not to be the result of different sugars, pH, or the presence of CO2. The nuclear transfer results may reflect the structural changes in the epithelium after lens culture in the two media.     

Serial culturing of human bronchial epithelial cells derived from biopsies

Summary In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.     

A granulosa cell differentiation-stage-specific cytotoxic activity in bovine and rat sera

Heat-inactivated serum is cytotoxic to granulosa cells from preantral follicles but not to cells from preovulatory follicles. A dominant feature of the granulosa cells of preovulatory follicles is the presence of luteinizing hormone (LH) receptors on the surface of the cells. In the present study, we have examined the relationship between the process of LH receptor induction and the acquisition of serum tolerance in granulosa cells in vitro. Granulosa cells from the ovaries of immature rats primed with diethylstilbestrol (DES) were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium containing 30 ng of ovine follicle-stimulating hormone (oFSH; NIH-15). At either 0, 24, or 48 h of culture, heat-inactivated fetal bovine serum (FBS) was added (10% by volume) to separate groups of culture tubes. All cells were cultured for a total of 72 h, at which time the cultures were assessed for LH receptor (specific 125I-human chorionic gonadotropin [hCG] binding) and DNA content. LH receptors were induced in all FSH-containing serum-free cultures by 48 h. Receptors were not induced, however, when serum was added after either 0 or 24 h of culture. Furthermore, serum addition at these times resulted in a cell loss (assessed by DNA) of 40-60%. Serum addition at 48 h to FSH-containing cultures resulted in an inability to detect LH receptors at 72 h and with no significant effect on the culture DNA content. Addition of a protein extract of FBS at the initiation of cell culture prevented FSH-stimulated LH receptor induction and was cytotoxic. A lipid extract of FSH did not interfere with receptor induction and was not cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of medium and substratum conditions on the rates of DNA synthesis in primary cultures of bile ductular epithelial cells

Summary Select medium and substratum conditions were investigated for their effects on semiconservative DNA synthesis in essentially pure primary cultures of bile ductular epithelial cells that were initially isolated from cholestatic rat livers at 6 to 10 wk after bile duct ligation. DNA synthesis in these cultured cells was serum-dependent, being at its highest level when the concentration of fetal bovine serum present in the medium was maintained at 10%. This serum-dependent DNA synthesis was completely inhibited when 10 mM hydroxyurea was also included in the medium, and bile ductular cells cultured in the continued presence of 1.0% fetal bovine serum showed only marginal DNA synthesis during 8 to 10 d of primary culture when compared with no-serum controls. Maximum rates of serum-dependent DNA synthesis were obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with either fibronectin from bovine plasma or type I rat-tail collagen. Cells cultured on plastic coated with basement membrane Matrigel exhibited the lowest levels of DNA synthesis, whereas those on plastic alone had intermediate amounts. Furthermore, the addition of epidermal growth factor (50 ng·ml⁻¹·d⁻¹) to medium supplemented with 1.0% fetal bovine serum greatly enhanced the rate of DNA synthesis in bile ductular cells after 6 d in primary culture on type I collagen-coated plastic over that measured in solvent control cultures. These findings indicate that our bile ductular epithelial cell culture model is potentially useful in the study of biliary cell growth regulation and carcinogenesis. This investigation was supported by USPHS grant RO1 CA 39225 to A. E. Sirica by the National Cancer Institute, Department of Health and Human Services, Bethesda, MD. During the period of this study, G. A. Mathis was a recipient of a Fellowship from the Fund for Academic Career Development of the State of Zurich, Switzerland.     

Growth characteristics, morphology, and phospholipid composition of human type II pulmonary alveolar cells grown in a collagen-free microenvironment

Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro.     

Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia-prostaglandin E2

Cell strains and cell lines rat mammary (Rama) 350-353 have been isolated from the slowly adherent stromal fraction of enzymatically digested rat mammary glands. Primary cultures of this fraction yield fat cels on extended culture. Their proportion can be increased with horse serum or growth hormone in the medium, and this increase is associated with a 100-fold or more increase in the release of radioimmunoassayable prostaglandins of the E type (PGE). The stromal cell strains and lines that are capable of yielding fat cells also secrete elevated levels (greater than 100 ng/mg/24 hr) of PGE; the fast-sticking epithelial fraction in primary cultures and the epithelial cell lines derived from it secrete 10-100 times less. Chromatography and radioisotopic labeling of the culture media from Rama 352 cells identify the PG as PGE2. PGE2 with insulin and hydrocortisone maximally stimulates [3H]DNA synthesis of epithelial cell lines and primary cultures from normal and tumorous glands by 2-4-fold at concentrations (10-20 ng/ml) well below those released by the preadipocytic stromal cells (20-100 ng/ml). Medium exposed to most cultured cells stimulates [3H]DNA synthesis of one epithelial cell line, Rama 25, by 2-4-fold. Prevention of the synthesis of PGE2 in Rama 352 cultures with indomethacin or flurbiprofen abolishes the mitogenic activity present in the culture medium, and the PG receptor antagonist polyphloretin phosphate inhibits completely the mitogenic activity for Rama 25 cells. Myoepithelial-like cell lines normally secrete moderate levels of PGE (10-100 ng/mg/24 hr) but the mitogenic activity for Rama 25 cells released from one such line, Rama 29, is not abolished by preventing the synthesis of PG's nor by PG-receptor antagonists.     

Initiation of DNA synthesis in mammary epithelium and mammary tumors by lithium ions

The growth promoting effects of lithium and insulin on cultures of mammary gland epithelium and CZF mouse mammary tumor cells were investigated. Lithium chloride exerts a 450-fold increase in the rate of DNA synthesis in mammary epithelium from mid-pregnant mice in organ culture or monolayer culture. There is an increase in both the percentage of cells initiating DNA synthesis and the net accumulation of DNA. The most effective lithium concentration is 10 mM, and the maximally effective rate of stimulation is reached 48 hours after addition. The magnitude of response to lithium varies with the physiological state of the mammary epithelial cell donor: epithelium from non-pregnant or lactating mice is less responsive than that from mid-pregnant mice. In combination, insulin and lithium produce either a synergistic or an additive effect on the growth of epithelium dependent upon the physiological state of the donor animal. Lithium also promotes the growth of mammary tumor cells in the absence of serum or other mitogens. The action of lithium on DNA synthesis appears to be a direct effect on the epithelial cells.     

Cellular changes in cultured lenses

Summary Observations were made on the frog lens epithelium after culture of the entire lens or of capsular explants. General deviations from normal lens structure as well as specific changes in two media were studied. DNA synthesis and mitosis were induced in the central epithelial cells. Disruption of the orderly, single, epithelial layer that is characteristic of normal lenses was accompanied by the appearance of multilayered plaques of epithelial cells and invasion of vacuolated regions of the lens fibers by epithelial cells. Cells that are fibroblast-like in appearance were observed in regions of the capsule depleted of cells and at the free edges of epithelial sheets in cell culture. Epithelial cells were surrounded by capsule-like material even when situated in the lens interior. Nuclei derived from central epithelial cells of lenses cultured in L-15 medium and medium 199 had served as donors in previous nuclear transfer experiments in this laboratory. In our current observation of L-15-cultured lenses, cells were sparsely distributed on the capsule and nuclei were abnormally shaped; in 199-cultured lenses, cells were more densely distributed and nuclei resembled those of normal lenses. Medium 199 without serum could better maintain normal lens structure than L-15 medium without serum. In addition, the percentage of epithelial explants demonstrating cellular outgrowth was greater in medium 199. The differences in cellular behavior were shown not to be the result of different sugars, pH, or the presence of CO₂. The nuclear transfer results may reflect the structural changes in the epithelium after lens culture in the two media. This work was supported by grants 2RO1 EY 00555-06 and 5SO1 RR 05510-10 from the National Institutes of Health.     

Stimulation in cell culture of mesenchymal cells of newt limb blastemas by EDGF I or II (basic or acidic FGF)

After amputation of a newt limb, a blastema forms on the amputation plane and later differentiates to regenerate all the missing parts of the limb. Proliferation of blastema cells is under the control of severed nerves which deliver a 'neurotrophic factor' (NTF) of unknown nature. In order to characterize this factor we use a primary culture of blastema mesenchymal cells; changes in mitotic index after 48-h colchicine treatment indicate mitogenic activity of potential growth substances. These cells, which are stimulated by nerve extracts (mitotic index X 6), were tested with two purified growth factors extracted from bovine retina or brain (EDGF I = basic FGF and EDGF II = acidic FGF). We show that these two growth factors stimulate proliferation of blastema cell cultures in a dose-dependent manner. Maximal stimulation was obtained at 3 pM for EDGF I (mitotic index X 5.7) or 300 pM for EDGF II (mitotic index X 4.9). So it appears that these two growth factors have a mitogenic activity on blastema mesenchymal cells similar to that obtained with nerve extracts. The fact that two different growth factors can stimulate these cells raises the question of whether both are present in NTF and/or whether there are receptors to both EDGF I and EDGF II on mesenchymal cell membranes.