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Biomedical subjects

D Gospodarowicz

Publications and source records attributed to D Gospodarowicz.

At least 145 records · Page 8Linked to original sources
PubMed1981

Up-regulation in vascular endothelial cells of binding sites of high density lipoprotein induced by 25-hydroxycholesterol.

Exposure of bovine vascular endothelial cell cultures to 25-hydroxycholesterol (50--100 microgram/ml) result in a 5--10-fold increase in cell surface binding sites of high density lipoprotein (HDL). This increase in HDL-binding sites was dependent on time and temperature. After a 48-h exposure to the oxygenated sterol, a maximal increase in HDL binding could be observed, and newly binding sites disappeared rapidly once 25-hydroxycholesterol was removed from the medium. No increase in HDL-binding sites was observed when cells were maintained at 4 degrees C. In contrast, cultures maintained at 37 degrees C did show an increase in HDL-binding sites when exposed to 25-hydroxycholesterol. Since simultaneous exposure of the cells to 25-hydroxycholesterol and cycloheximide resulted in an inhibition of HDL binding to the cells, it is suggested that de novo synthesis of HDL-binding sites is induced by 25-hydroxycholesterol. When the abilities of HDL2 and HDL3 to bind to newly synthesized HDL-binding sites were compared, HDL3 was found to bind more efficiently than HDL2. It is therefore unlikely that apoprotein E plays a major role in the binding of HDL to newly synthesized HDL-binding sites. When the properties of newly synthesized HDL-binding sites were analyzed, they were found to have a high affinity for HDL, since half-maximal binding was reached at a concentration as low as 5 microgram HDL protein/ml, and were saturable. Such HDL-binding sites had a relaxed specificity, since they were capable of binding low density liprotein (LDL). However, when LDL bound to newly synthesized HDL binding sites, it was no longer internalized, as reflected by a 90% reduction of LDL degradation, and instead of being cytotoxic it became mitogenic.

Animals↗
PubMed1981

Factors controlling proliferation and progesterone production by bovine granulosa cells in serum-free medium.

Bovine granulosa cells seeded in the presence of serum on extracellular matrix-coated dishes proliferate actively when exposed to serum-free medium supplemented with insulin (2 microgram/ml), fibroblast growth factor (FGF, 100 ng/ml), and high density lipoprotein (HDL, 30 microgram protein/ml). The final density of the cultures is 80-120% that of cultures grown in the presence of medium supplemented with optimal concentration (10%) of calf serum. Insulin has the greatest effect on cell proliferation when added alone to serum-free medium, since it induced an increase in cell number that was 35-60% that observed with optimal serum concentration. Somatomedin C can replace insulin when added alone. FGF, epidermal growth factor, or HDL had no significant effect on cell proliferation by themselves. When these factors were added together with insulin, they acted synergistically in stimulating cell proliferation. When cultures were seeded in the total absence of serum, the addition of transferrin (10 microgram/ml) to serum-free medium was required in order for insulin and FGF to be mitogenic. Cultures maintained on extracellular matrix and exposed to serum-free medium alone have a lifespan in culture of 4 generations. Addition of insulin, FGF, and HDL increases the lifespan of the cultures to 12 generations. Bovine granulosa cells, which proliferate in a defined medium, respond to dibutyryl cAMP by releasing progesterone into the medium. Addition of FSH to the defined medium resulted in a 30% decrease in cell proliferation and in a 2.1-fold increase in the amount of progesterone released into the medium in response to dibutyryl cAMP. This release of progesterone reached a level similar to that observed with cultures grown in medium supplemented with optimal concentration of serum and exposed or not to FSH during their growth phase and at confluence. These results demonstrate that bovine granulosa cells can actively proliferate in a serum-free medium and maintain their differentiated function, as indicated by their ability to produce progesterone.

Animals↗
PubMed1981

Corneal endothelial replacement. I. In vitro formation of an endothelial monolayer.

Cultured bovine corneal endothelial cells were seeded onto a corneal button denuded of its own endothelium. After an incubation time of 30 min to 1 hr, these cultured cells were able to repopulate and reconstitute a new endothelium over Descemet's membrane. Three basic cellular processes were involved in the formation of the new endothelial layer. (1) Adhesion of the cells to the tissue substrate occurred rapidly because Descemet's membrane is the natural substrate for these cells. (2) Expansion of the cells formed a continuous monolayer and, at the concentration used in our experiments, was achieved with a minimum of cell thinning. (3) Cohesion was developed within 1 hr after seeding when cells contacted each other by means of long thin cytoplasmic processes (filopodia). The special junctional complexes associated wih the fluid barrier found in the corneal endothelium were only partially formed 72 hr after plating the cultured cells. Finally, the durability of the reconstituted endothelium was examined by using the corneal buttons at keratoplasty. It appears that corneal buttons incubated for 1 to 2 hr may be used at keratoplasty.

Animals↗
PubMed1980

Nuclear accumulation of epidermal growth factor in cultured rat pituitary cells.

Epidermal growth factor (EGF) is a mitogen for epidermal cells in vivo and for a wide variety of cells in culture. Recently, we and others have reported that EGF can also regulate the cellular levels of various hormones and fibronectin at concentrations which only minimally influence cell division. In addition, EGF treatment of GH3 cells affects chromatin structure such that isolated nuclei from treated cells have an increased capacity to bind bacterial RNA polymerase in initiation site complexes. Thus, the data suggest that various nuclear functions are modulated by EGF in GH3 cells despite its failure to affect DNA synthesis or cell proliferation. Recently, Yanker and Shooter have reported on the nuclear accumulation of nerve growth factor (NGF) in PC12 cells in which NGF does not promote cell division but does influence RNA and protein synthesis while inducing overt differentiation (neurite outgrowth). The similarities between the two systems and the various theories regarding the mechanism by which mitogens exert their growth-promoting and other effects led us to investigate whether an interaction between EGF and the cell nucleus can be demonstrated after surface binding and internalization of EGF in GH3 cells. We report here that when its lysosomal degradation is inhibited by chloroquine, EGF accumulates in the nucleus.

Animals↗
PubMed1980

Morphological appearance, growth behavior and migratory activity of human tumor cells maintained on extracellular matrix versus plastic.

Growth of human tumor cells (hepatocarcinoma, Ewing's sarcoma) on an extracellular matrix (ECM) produced by bovine corneal endothelial cells is associated with the adoption of a morphological appearance and growth properties that are not expressed when the cells are maintained on plastic. Within minutes after seeding cell aggregates onto an ECM, the aggregates attached firmly. Active cell migration leading to the formation of flattened and nonoverlapping cell clusters was subsequently observed. In contrast, no firm attachment, migratory activity or disorganization of cell aggregates was observed when the same cells were maintained on plastic. Cells seeded on ECM, instead of growing as floating or loosely attached aggregates, formed a cell monolayer composed of firmly attached, highly flattened and closely apposed epithelioid-like cells. Cell overlapping and subsequent detachment were observed only late at confluence. Cells maintained on ECM had a higher growth rate as well as a lower serum requirement than those maintained on plastic. These results demonstrate that the phenotypic expression as well as the proliferation of tumor cells can be modulated by their adhesive interaction with the extracellular matrix. Both tumor cells and normal cells of epithelial origin are more likely to resemble their in vivo counterparts when maintained on extracellular matrix than on plastic, and when so maintained can therefore provide a better model for oncogenic studies.

Cell Adhesion↗
PubMed1980

Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response.

Cultured rat pituitary tumor cells, GH3/D6, which synthesize both growth hormone and prolactin, have cell-surface epidermal growth factor (EGF) receptor sites (34,000 per cell) that bind 125I-labeled EGF with a high affinity (Kd approximately 1 nM). Prolonged treatment of the cells with EGF did not stimulate cell division but did inhibit thyroid hormone-stimulated cell growth. In addition, EGF altered the morphology of the cells from a rounded to an elongated conformation. EGF also induced a perturbation of chromatin structure in GH3 cell nuclei that was detected by an increase (40%) in the number of rifampicin-resistant initiation sites for bacterial RNA polymerase. This was accompanied by an increased synthesis of prolactin and an inhibition of synthesis of growth hormone. In the presence of EGF, the synthesis of growth hormone was no longer inducible by thyroid hormone, but it remained responsive to glucocorticoids. The results demonstrate that EGF can elicit major effects on the cellular phenotype and expression of specific genes in the absence of a proliferative response. This suggests that EGF can also regulate differentiated cellular functions.

Animals↗
PubMed1980

Role of the degradation process in the mitogenic effect of epidermal growth factor.

The protease inhibitor leupeptin inhibits the degradation process of (125)I-labeled epidermal growth factor ((125)I-EGF) by cultured bovine granulosa cells. At 80 mug/ml, leupeptin inhibited the appearance of degradation products of (125)I-EGF in the medium by 95% during 1 hr of incubation and by 90% during 24 hr of incubation when the cells were exposed to 5 ng of (125)I-EGF per ml. In contrast, cultures exposed to either saturating (10 ng/ml) or nonsaturating (0.1 ng/ml) concentrations of EGF in the presence of leupeptin (80 mug/ml) exhibited an increase in DNA synthesis that was 70-80% that of cultures exposed to EGF alone. Cultures responded to either EGF or fibroblast growth factor with a logarithmic increase in cell number and, over a period of 8 days, the number of cells increased 10- to 18-fold. Addition of leupeptin did not diminish the growth rate of the cultures. In the presence of leupeptin, (125)I-EGF accumulated within the granulosa cells and was in a form that was precipitable with antiserum against EGF and that comigrated on isoelectric focusing with native (125)I-EGF. That a full mitogenic response can be obtained despite a 90-95% inhibition of EGF degradation at either saturating or nonsaturating concentrations of the mitogen suggests that a proteolytic degradation of a given mitogen may not be involved in the induction of a proliferative response.

Animals↗
PubMed1980

Do plasma and serum have different abilities to promote cell growth?

The abilities of plasma and serum to support the growth of vascular smooth muscle cells maintained on uncoated tissue culture dishes or dishes coated with an extracellular matrix (ECM) have been compared. Vascular smooth muscle cells maintained on plastic dishes and exposed to plasma proliferate poorly; when exposed to serum they proliferate actively. Addition of fibroblast growth factor (FGF) incrases the growth rate of the cultures in both cases. In contrast, when vascular smooth muscle cells are maintained on an ECM, they proliferate equally well exposed to either plasma or serum. Because the cultures had an average doubling time (15 hr) that was already at a minimum, FGF no longer had an effect on vascular smooth muscle cell proliferation. These results raise the possibility that the lack of response of vascular smooth muscle cells, as well as that of other cell types in vitro, to plasma factors is not an intrinsic property of the cells but is rather due to the substrate upon which the cells rest. Because cells maintained on an ECM respond to plasma factors, it is likely that the close contact of the cells with the ECM restores their sensitivity to physiological factors present in plasma.

Animals↗
PubMed1980

Permissive effect of the extracellular matrix on cell proliferation in vitro.

Corneal endothelial cells maintained in tissue culture retain the ability to synthesize and secrete an extracellular matrix (ECM) along their basal cell surface. Treatment of confluent cultures with 0.5% Triton X-100 results in the removal of the cell monolayer, thereby exposing the ECM, which adheres strongly to the tissue culture dish. Dishes coated with ECM were used to study the permissive effect of such a substrate on cell proliferation. The proliferation of bovine granulosa and adrenal cortex cells maintained on plastic tissue culture dishes was compared to that on dishes coated with ECM. Neither cell type, even when exposed to optimal serum concentration, replicated when seeded at low cell density on plastic. In contrast, when seeded on ECM they proliferated actively. None of the cultures maintained on ECM required fibroblast growth factor in order to reach confluence, although when maintained on plastic they were totally dependent on fibroblast growth factor for proliferation. Because cells maintained on plastic do not respond to factors present in serum or plasma, although they do so respond when maintained on ECM, it is likely that the close contact of the cells with the ECM restores their sensitivity to agents present in serum and plasma.

Adrenal Cortex↗