Search PubMed⌕ Search

Biomedical subjects

D D Cunningham

Publications and source records attributed to D D Cunningham.

At least 109 records · Page 6Linked to original sources
PubMed1978

Cell surface action of thrombin is sufficient to initiate division of chick cells.

Thrombin covalently linked to carboxylate-modified polystyrene beads initiated division of quiescent chick embryo (CE) cells either in medium containing low levels of serum or in serum-free medium. Release of thrombin was monitored by measuring acid-precipitable radioactivity released from 125I-thrombin beads into the medium during incubation with cells. Even if all of the acid-precipitable material released from the beads were active thrombin, it was not sufficient to account for any of the observed cell division, and was 10-30 fold less than the amount necessary to produce the increase in cell number caused by the thrombin beads. Two other kinds of experiments also showed that material released into the medium did not account for the observed initiation of cell division. First, medium taken from cultures incubated with thrombin beads did not initiate cell division when added to new quiescent cultures. Second, in coverslip experiments where populations of cells with an without thrombin feads shared the same medium, only bead-contacted cells divided. Several results suggested that the material which was released from the thrombin beads resulted from cell-associated proteolysis rather than from "leakage" of intact thrombin from the beads. For example, after incubating 125I-thrombin beads with or without CE cells, we were unable to detect any intact thrombin released into the medium. In addition, most of the material released from the beads was acid-soluble and was only released in the presence of CE cells. A few thrombin beads were endocytosed by CE cells, but they were surrounded by an intact plasma membrane. Thus they did not directly interact with the cytoplasm. The close association of many of the beads with the cell surface and the presence of a few beads in endocytic vesicles made it important to consider the possibility that thrombin might be released from the beads directly into the cells. This possibility was explored using ultrastructural (EM) autoradiography. With this technique (where one grain represented 700--900 thrombin molecules), we found that beads inside the cells had approximately the same number of grains as beads not in contact with cells. This suggested that little, if any, additional radioactive material had been released from the beads which were in contact with the cells. In addition, we were unable to detect any grains in the cytoplasm which could be attributed to released thrombin, even using an amount of 125I-thrombin beads which was 8 fold greater than the amount which produced maximal cell division. Taken together, these results provide direct evidence that thrombin action at the cell surface is sufficient to initiate division of CE cells.

Cell Count↗
PubMed1978

Dexamethasone modulates binding and action of epidermal growth factor in serum-free cell culture.

Experiments probing the mechanism by which glucocorticoids modulate cell proliferation were carried out on serum-free cell cultures of quiescent human diploid foreskin (HF) cells. Added alone, the synthetic glucocorticoid dexamethasone had no effect on cell number. However, dexamethasone enhanced the mitogenic response of HF cells to epidermal growth factor (EGF) by 50% at all EGF concentrations. The mitogenic action of EGF was maximally promoted by a dexamethasone concentration of 100 ng/ml (0.25 muM). Binding studies with (125)I-labeled EGF ((125)I-EGF) suggested that dexamethasone caused this "permissive" effect by modulating cell surface receptors for EGF. Paralleling their increased responsiveness to EGF growth stimulation, dexamethasone-treated cells exhibited a 50-100% increased ability to bind physiological concentrations of (125)I-EGF. A binding increase was apparent after a 4-hr dexamethasone treatment. The dexamethasone-treated cells maintained an increased ability to bind (125)I-EGF during the prolonged exposure to EGF that was required to stimulate cell division. Moreover, the increase in (125)I-EGF binding exhibited a dexamethasone dose-dependence similar to that for the enhancement of EGF mitogenesis, suggesting a relationship between the dexamethasone effects on binding and growth. An investigation of the binding increase showed that it was specific for glucocorticoids, and required protein synthesis. The enhancement of (125)I-EGF binding diminished with increasing concentrations of (125)I-EGF, indicating that dexamethasone caused a qualitative change in the EGF receptors (possibly a change in receptor affinity or cooperativity). The alteration in (125)I-EGF binding may occur as part of a far-reaching dexamethasone-mediated change in the cell surface, because dexamethasone treatment slightly increased the ability of HF cells to bind (125)I-insulin, and decreased by half their ability to bind (125)I-thrombin.

Cell Division↗
PubMed1977

Initiation of check cell division by trypsin action at the cell surface.

Trypsin immobilised on polystyrene beads causes initiation of cell division which cannot be accounted for by trypsin released into the medium or into the cells. Also, initiation by soluble trypsin is inhibited by immobilised soybean trypsin inhibitor. These results demonstrate that trypsin can initiate proliferation at the cell surface.

Animals↗
PubMed1977

Serum-stimulated phosphate uptake and initiation of fibroblast proliferation.

Previous studies have shown that initiation of proliferation of density-inhibited fibroblasts by fresh serum is accompanied by a rapid increase in phosphate uptake. This increase might be a key event in the initiation of DNA synthesis. The present studies examined this possibility. Mouse 3T3, secondary chick embryo, or human diploid foreskin cultures were grown to quiescence in medium containing varying levels of serum. When proliferation of the cultures was initiated by addition of fresh serum, the changes in phosphate uptake were inversely related to the final increases in cell number. Additional experiments showed that the change in phosphate uptake following serum addition was determined by the level of phosphate uptake prior to serum addition. Addition of dexamethasone to quiescent 3T3 cultures caused them to proliferate but did not increase phosphate uptake. Similarly, trypsin or insulin stimulated proliferation of quiescent secondary chick embryo cultures, but caused little or no change in phosphate uptake. Quiescent 3T3 cultures switched to medium containing fresh serum and reduced levels of phosphate showed a decrease in both phosphate uptake and intracellular phosphate pool size. Cell proliferation in these cultures, however, was stimulated to the same degree as cultures switched to medium containing fresh serum and the normal amount of phosphate. In addition, quiescent secondary chick embryo cultures switched to medium containing fresh serum and no phosphate showed a decrease in the intracellular phosphate pool size. Thymidine incorporation and final cell number in these cultures, however, was stimulated to the same or higher degree than in cultures switched to medium containing fresh serum and the normal amount of phosphate. These results demonstrate that the rapid increase in phosphate uptake following addition of fresh serum to quiescent fibroblasts is not a necessary event for the initiation of proliferation.

Animals↗
PubMed1975

Growth limitation of 3T3 mouse fibroblasts by available growth surface area and medium components.

Studies with untransformed fibroblasts demonstrate that growth of these cells in culture can be limited by the availability of both growth surface and medium components. Experiments using cells grown on coverslips, in which the only variable was available growth surface, indicate that when the medium to cell ratio is high, surface area is the principal factor limiting growth. At low medium to cell ratios, however, growth of cells is predominantly limited by medium components. The final number of cells per culture is almost directly proportional to available surface area when the culture medium is changed daily.

Cell Count↗
PubMed1973

Maximal concanavalin A-specific agglutinability without loss of density-dependent growth control.

Density-inhibited 3T3 mouse fibroblasts were treated for 10 min with nine concentrations of Pronase ranging from 0.25 to 40 mug/ml. Concanavalin A-specific agglutinability increased from control values of about 25% to plateau values of about 80%. None of these Pronase concentrations brought about an increase in cell number within 84 hr. Pronase-treated cells remained responsive to growth stimulation by serum and cortisol. Therefore, the protease-mediated surface change measured by increased concanavalin A-specific agglutinability is not an event sufficient by itself to bring about cell division.

Agglutination↗
PubMed1969

Transport changes rapidly initiated by serum addition to "contact inhibited" 3T3 cells.

Two- to fourfold increases in uridine and phosphate uptake were brought about within 10 to 15 minutes after adding fresh serum to confluent 3T3 cells. The stimulation of transport was specific, since no serum effect was observed for 3-O-methyl-D-glucose or amino acids. The early increase in RNA labeling, previously identified by (14)C-uridine incorporation, could be accounted for by this transport effect. Increased labeling with (32)P-phosphate of at least five phospholipids occurred soon after adding serum. This increase, however, could not be accounted for by increased transport. Both of these results suggest that specific early membrane changes are involved in "contact inhibition."The following results are consistent with this suggestion. Uptake of uridine and phosphate by nonconfluent 3T3 cells was higher than by confluent cells and was only slightly increased by fresh serum. In contrast, uptake of these substrates by Polyoma virus-transformed 3T3 cells, which are not subject to "contact inhibition," was not significantly decreased after the cells became confluent, and serum addition had no effect on transport by either nonconfluent or confluent Polyoma virus-transformed 3T3 cells. Fractionation of serum on Sephadex G-200 demonstrated that the factor which stimulated phosphate transport was different from the previously identified factor which stimulates DNA synthesis by confluent 3T3 cells.

Animals↗