Differential effects of cAMP and cGMP on in vitro epidermal cell growth.

Primary keratinocyte cultures free of dermal fibroblasts were used to investigate the effect of varying cyclic AMP (cAMP) concentrations on epidermal cell function. Addition of 10^?3, 10^?4 or 10^?5 M dibutyryl cAMP to plated cells (day 1) results by day 5 in a dose dependent increase of [³H]TdR incorporation into DNA as determined by increases in both the labeling index and incorporation of ³H label into an isolated DNA fraction. 8-Bromo cAMP, another cAMP analogue, likewise induced keratinocyte proliferation. The proliferative response was dose and time dependent, and 5- to 6-fold increases in ³H label incorporated into DNA were seen at day 6, 8 and up until day 15 of culture. Moreover, elevation of cellular cAMP by addition of cholera toxin, an irreversible stimulator of adenylate cyclase, also demonstrated a time dependent stimulation of [³H]TdR uptake into DNA and increased the labeling index. Specific histochemical staining for keratinaceous protein (Kreyberg technique) demonstrated that elevated cAMP levels also enhance the production of specialized (differentiated) epidermal cells. Determination of the level of cAMP and cyclic GMP (cGMP) by RIA of partially purified fractions of the cultures revealed that addition of 8-bromo cAMP or cholera toxin to the cultures increased the levels of cAMP but not of cGMP. Addition of 8-bromo cGMP to the keratinocytes on day 1 at concentrations of 10^?6, 10^?7 or 10^?8 M had no effect on culture proliferation on days 4, 6 and 8, although qualitative changes in the electron microscopic pattern of the culture stratification and specialization were observed. The results indicate (1) both large and moderate increases in cellular cAMP levels induce keratinocyte culture proliferation and specialization in the absence of fibroblasts or dermal influences, (2) the quantitative enhancement of keratinocyte growth and specialization occurs without apparent participation of cGMP, (3) cGMP may be a qualitative effector of epidermal cell differentiation.     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Modulation of proliferation in epidermal keratinocyte cultures by lowered oxygen tension

The modulation of proliferation and differentiation in primary epidermal keratinocyte cultures by lowered gas phase oxygen tensions was studied. Neonatal mouse epidermal keratinocyte cultures were grown in an Heraeus type B 5060 EK/O2 incubator in oxygen tensions between 5% and 15% (within the physiologic range); the oxygen tension of ambient air being 21%. Cell morphology was studied using histochemical stains and electron microscopy. Differentiation was assessed using autoradiography of SDS PAGE gels of six serially extracted cell protein fractions with [3H]leucine as a marker. Autoradiographs using [14C]glucosamine and 32Pi as markers were also assessed as a measure of other cell functions. Proliferation was studied using autoradiography of [3H]thymidine ([3H]TdR) pulse-labeled cultures and [3H]TdR incorporation into isolated DNA fractions. The results of these studies showed that lowering the oxygen tension in the gas phase reversibly inhibited cell proliferation. There was a direct arithmetic relationship between the proliferative rate of the cultures and the oxygen tension. No change in differentiation as defined by [3H]leucine indexing of protein synthesis was seen. Other markers of cell function, such as [14C]glucosamine glycosylation and [32P] phosphorylation of proteins were also unchanged. These results suggest that oxygen tension regulates only proliferation in epidermal keratinocytes. This epidermal response is well adapted to its role in the healing wound, and is an example of a tissue-specific modification of a regulatory function.     

Augmentation of keratinocyte differentiation by the epidermal mitogen, 8-bromo-cAMP

The effect of the epidermal mitogen, 8-bromo-cAMP, on keratinocyte differentiation was studied. A 3 X 10(-4) M dose of 8-bromo-cAMP was added to primary neonatal mouse epidermal keratinocyte cultures that slowly proliferate, stratify and differentiate over 2-3 weeks time. [3H]Thymidine autoradiography coupled with an NH4Cl plus reducing agent technic which separates basal and differentiating keratinocytes was used to determine the target cell for the 8-bromo-cAMP mitogenic effect. A histologic stain and a four buffer protein extraction protocol, in conjunction with PAGE and fluorographic technics, were used to assess the differentiation of the cultures. The data indicated that 8-bromo-cAMP primarily stimulated the proliferation of the basal cell monolayer. Simultaneous with the mitogenic effect was an increase in the production of keratohyalin granule, keratin and cell envelope proteins, which are specific markers of epidermal differentiation. The results indicate that keratinocytes stimulated by the epidermal mitogen 8-bromo-cAMP simultaneously express differentiation-related processes.     

Alterations of the incorporation of [3H]thymidine in cells in culture regulated by nucleoplasmic extract

Porcine skin nucleoplasmic extract (PSNE) was shown to alter the incorporation of [³H]thymidine into DNA of selected porcine, bovine, and human cell populations in culture. PSNE stimulated incorporation of [³H]thymidine into DNA of porcine and bovine dermal cells an average of 300 and 200% of control value, respectively. When porcine and bovine epidermal cells were exposed to PSNE the treatment inhibited [³H]thymidine incorporation into DNA by an average of 48 and 45%, respectively. Similar inhibitions were observed for porcine and bovine kidney, porcine lung, and human KB cells. Thus, the effect of PSNE on the incorporation of [³H]thymidine into DNA of various cultured cells was either stimulatory to dermal cells or inhibitory to a variety of other cell types, including skin epidermal cells. The stimulatory and inhibitory effects of PSNE were abolished by heating PSNE for 5 min in boiling water before its addition to cell cultures. This suggests that macromolecular structure is important in the action of PSNE. This project was supported by a grant from the Research Advisory Board, University of Nevada, Reno, NV.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Placental keratinocyte growth factor: partial purification and comparison with epidermal growth factor

A water-soluble extract of term human placenta, which was previously shown to promote proliferative growth of human keratinocytes in defined medium, enhanced both cellular attachment and proliferative growth. We have partially purified the activity which enhanced cell growth and examined its action in keratinocytes. Activity was precipitated from the crude extract by (NH4)2SO4 between 33 and 60% saturation and chromatographed by gel filtration. The activity did not bind to heparin-Sepharose at low ionic strength but was adsorbed to DEAE-cellulose from which it was eluted with NaCl and then passed over phenyl-HPLC to remove bovine serum albumin previously added to protect the activity. The active fraction was applied to gel exclusion HPLC in the presence of 0.02% octyl-beta-D-glucopyranoside, which yielded an apparent Mr 35,000 for the factor. Purification was approximately 200-fold with approximately 4% recovery. The factor appears to be a protein, since activity is destroyed by trypsin. Autoradiography of cultures treated with the placental factor or epidermal growth factor (EGF) revealed that approximately 50% of cells were labeled after treatment with either growth factor compared to 9% in control cultures after a [3H]thymidine pulse. Protein synthesis was increased by about 50% 42 h after treatment with either agent, consistent with a 50% increase in nuclear labeling. Cell number was increased fivefold after 6 days in the presence of the partially purified factor, whereas EGF increased cell number eightfold. Stimulation of [3H]thymidine incorporation by the partially purified factor, in contrast, was about twice that produced by EGF, indicating that thymidine incorporation is preferentially stimulated by the placental factor and does not correlate well with other parameters of proliferative growth. The placental keratinocyte growth factor is a unique factor with a novel effect on incorporation of thymidine into DNA.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc,Haliotis tuberculata

Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [³H]-leucine and [³H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [³H]-leucine or for [³H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.     

Comparative evaluation of the antiproliferative effect of cyclosporin A and γ-interferon on normal and HPV-transformed keratinocytes by cell counting, MTT assay and tritiated thymidine incorporation

We compared three techniques, the MTT tetrazolium assay, cell counting, and tritiated thymidine ([3H]TdR) incorporation assay to measure the antiproliferative effect of cyclosporin A (CsA) and interferon-γ (IFN-γ) on normal human skin keratinocyte cultures (NHK) used at the second passage and human papillomavirus type 16- and 18-transformed cell lines (EK16 and EK18) exposed continuously to the drugs for 3 days. The three techniques showed that under CsA (0.5 and 8 μ/ml) and IFN-γ (5 and 160 U/ml) treatments the cells remained viable and that the growth of keratinocytes was inhibited. For IFN-γ, the MTT colorimetric assay consistently underestimated its growth inhibitory activity as compared to cell counting or [3H]TdR incorporation, whatever the cells used. For high doses of CsA, MTT and cell counting gave similar percentages of inhibitory activity whatever the cells; MTT underestimated this activity as compared to [3H]TdR incorporation only in NHK and EK18 cells, whereas similar results were obtained with EK16 cells. In conclusion, this investigation shows that MTT sensitivity differed with the drug and also according to the keratinocyte cultures. The MTT test is clearly not appropriate for study of IFN-γ treatment whatever the keratinocytes used. Such discrepancies indicate that the MTT test should be done with care on cultures to measure the effects of drugs on cell growth; the growth inhibition should be carefully considered and it would be best if two different methods were used.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Enhancement of intracellular glutathione promotes lymphocyte activation by mitogen

We have examined the effect of chemically modulating intracellular glutathione (GSH) levels on murine lymphocyte activation. Lymphocyte activation was determined by the induction of polyamine synthesis (ornithine decarboxylase (ODC) induction) and DNA synthesis ([3H]thymidine([3H]Tdr) incorporation). Intracellular GSH levels were enhanced using L-2-oxothiazolidine-4-carboxylate (OTC), which delivers cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO), which inhibits gamma-glutamylcysteine synthetase. In addition, the thiol 2-mercaptoethanol (2-ME) was tested for its ability to augment intracellular GSH levels. Our results indicate that both OTC and 2-ME enhance GSH concentrations and [3H]Tdr incorporation in resting and mitogen (concanavalin A)-stimulated cells. The induction of ODC by concanavalin A (Con A) was augmented by the addition of OTC or 2-ME. The GSH concentration of Con A-stimulated cells was reduced when compared to resting cells; however, it was markedly enhanced by OTC or 2-ME. The stimulatory effects of 2-ME on GSH concentrations, [3H]Tdr incorporation, and ODC induction in both resting and Con A-stimulated cells were much more potent than those of OTC. In contrast, BSO suppressed intracellular GSH and [3H]Tdr incorporation in resting and Con A-stimulated cells. BSO also inhibited the promotion of intracellular GSH concentrations and [3H]Tdr uptake by OTC or 2-ME. However, BSO did not affect the induction of ODC by Con A or its enhancement by OTC or 2-ME. We conclude that enhancement of intracellular GSH concentration results in an increased lymphocyte response to mitogen stimulation.     

Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.     

Human chorionic gonadotropin promotes thyroid growth via thyrotropin receptors in FRTL-5 cells

To ascertain the presence of thyroid growth-promoting activity (TGA) in the sera of pregnant women, we measured TGA in the sera of pregnant women by means of a bioassay based on [3H]-thymidine [( 3H]Tdr) incorporation in cultured rat FRTL-5 thyroid cells. Furthermore, to elucidate the mechanisms of human chorionic gonadotropin (hCG) in promoting the thyroid growth, we evaluated the effects of blocking type TSH receptor antibody (blocking IgGs) from patients with primary hypothyroidism on the activity of hCG. After the PEG-pretreated serum or the serum plus blocking IgGs was incubated for 72 h at 37 degrees C with FRTL-5 cells and [3H] Tdr, [3H] Tdr incorporated in the cells was counted. Although 9 normal pregnant women had normal TGA, two patients with hydatidiform mole, whose hCG levels were 966,500 and 497,100 IU/L, had positive TGA, but the activity showed normal when analyzed with the addition of a blocking IgG. hCG also showed a dose-dependent increase in [3H]Tdr incorporation, and it was inhibited by the addition of blocking IgGs. Furthermore, the inhibition of hCG-induced [3H]Tdr incorporation by 16 blocking IgGs correlated with their TBII and the inhibition activity of hCG-induced cAMP accumulation. Analysis by the Lineweaver-Burk plots of dose response curves of TSH- and hCG-induced [3H]Tdr incorporation showed the same inhibition pattern as with the addition of the same blocking IgGs. In conclusion, 1) hCG-related TGA exists in the sera of some patients with hydatidiform mole; and 2) hCG and the sera of some patients with hydatidiform mole promote thyroid growth, at least in a part, via TSH-receptors in FRTL-5 cells.     

Characterization of cells isolated and cultured from human bone

Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJb culture medium supplemented with penicillin (100 units/ml), streptomycin (100 micrograms/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (less than 1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJb supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [3H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [3H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 micrograms/ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.     

Proliferation, differentiation and maturation of a mouse epidermal keratinocyte cell line

The spontaneous development of a cell line of neonatal mouse C3H/He epidermal cells is described. The culture has been serially passaged at 29 °C over 18 months in the absence of any dermal support. The cell morphology of the 18th passage is reported. During early growth phase, the morphology of the cell layers was similar to that observed in the basal and differentiating strata of the epidermis: numerous tonofilament bundles and desmosome-filament complexes were observed. During late growth phase, maturation and vertical stratification occurred: demonstrated by the tonofilament accumulation, cell organelle degradation, nuclear pyknosis, presence of keratohyalin granules and horny cell layers with thickened membranes. Hemidesmosome-like structures were shown. No basal lamina or membrane coating granules were detectable. The 18th passage cultured cells did not induce tumors in nude mice. This keratinocyte cell line is not permanent, however: a malignant transformation occurred after 25 subcultures which resulted in an undifferentiated cell population.     

HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE : Growth and DNA Synthesis

Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [³H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [³H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [³H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration.     

Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: Protein secretion,steroid metabolism,and responsiveness to growth factors

A bovine granulosa cell line (BGC-1) has been obtained by spontaneous immortalization of primary cultures. BGC-1 cells have retained some characteristics of primary cultures, such as the hormonal regulation of fibronectin biosynthesis. In the present study we have compaed BGC-1 cells and primary cultures of bovine granulosa cells in terms of protein secretion, steroid metabolism, and mitogenic responses to growth factors. The pattern of protein secretion in BGC-1 cells was qualitatively similar to that of primary cultures. The main differences were a higher proportion of fibronectin and the relative amounts of several other unidentified proteins. Progesterone levels in BGC-1 cultures were undetectable. When BGC-1 cells and primary cultures were incubated with [³H]-pregnenolone, the former showed a lower conversion rate to progesterone. In contrast, the conversion rate of [³H]-progesterone to 5α-reduced metabolites was markedly increased in BGC-1 cells. We also examined the effects of epidermal growth factor (EGF), insulin like growth factor-1 (IGF-I), and transforming growth factor-b? (TGF-b?) on DNA synthesis under serum-free conditions. Both primary cultures and BGC-1 cells exhibited a stimulatory response to EGF and IGF-I on [³H]-thymidine incorporation. Neither BGC-1 cells nor primary cultures showed a significant response to TGF-b? when added alone. However, in the presence of a combination of EGF and IGF-I, TGF-b? displayed an inhibitory effect on primary cultures while it stimulated DNA synthesis in BGC-1 cells even further. The addition of conditioned medium from BGC-1 cells (BGC-1-CM) stimulated DNA synthesis on primary cultures to a greater extent than the addition of conditioned medium from primary cultures. These results suggest that BGC-1 cells may be a useful model to study the regulation of granulosa cell function during the period previous to the preovulatory stage of follicular development. The differential responses of the immortalized cells to growth regulators may offer some clues on the mechanisms that control cell proliferation in normal tissues. © 1995 Wiley-Liss, Inc.