Using a monoclonal antibody to the human epidermal growth factor (EGF) receptor (EGF-R1), we have followed the metabolism of the receptor and the pathway of its internalization in KB cells after the addition of EGF. Measurement of surface binding of '251-labeled EGF showed that about 80% of EGF binding activity disappeared from the plasma membrane after a 10-min exposure to EGF at 37TC. Immunoprecipitation Other receptors, such as the EGF receptor (7) and the insulin receptor (8), are subject to down-regulation. The kinetics of down-regulation vary depending on the cell type studied. It is believed that down-regulation is due to internalization of the receptor and its subsequent intracellular degradation (7, 9-11). The availability of monoclonal antibodies to the protein portion of the human EGF receptor (12-14) has made it possible to study the fate of the receptor in cells treated with EGF and undergoing receptor internalization. Waterfield et al. (14) have described an antibody designated EGF-R1 that binds to the EGF receptor in human cells and have shown that, when antibody EGF-R1 is bound to the receptor of living cells, it is internalized upon EGF addition. Using antibody EGF-R1 as an immunocytochemical probe to detect the location of the EGF receptor after fixation, we show here that the EGF receptor is efficiently internalized when EGF is added to KB cells. The results demonstrate that EGF causes the receptor to move first to coated pits, next into receptosomes, and finally into lysosomes, where it is degraded with a half-life of approximately 40 min. EXPERIMENTAL PROCEDURESBinding of 125I-Labeled EGF ('2-IkEGF). KB cells, propagated as described (15,16), were planted in 35-mm plastic tissue culture dishes at 5 x i05 cells per dish (80% confluent). To measure the effect of EGF on receptor number, unlabeled EGF (Bethesda Research Laboratories) was added to the medium to a final concentration of 1.8 ng/ml (0.3 nM), and the cells were incubated at 370C for various times. At 0, 10, 30, and 60 min, the cells were placed on ice and washed with pH 4.0 buffer (0.2 M acetic acid/0.5 M NaCl) for 6 min to remove surface-bound EGF, followed by two rinses with medium and one with medium containing 10% fetal calf serum (ice cold). Finally, 2 ml of ice-cold fresh medium was added. To measure the binding of labeled EGF to the remaining cell-surface receptors, 1251I-EGF (2.6 x 105 cpm/ng) at a final concentration of 0.5 nM was added. Nonspecific binding was determined by coincubation with 100 nM unlabeled EGF. After 90 min on ice, the cells were washed four times with ice-cold medium, solubilized in 1 M NaOH, and placed in a mini-y-counter to measure their content of 125i.
One objective of periodontal therapy after resolution of the infectious process is to facilitate a new connective tissue attachment to the root dentin surface. Here we report that a selective advantage for attachment and growth can be conferred on human gingival fibroblasts by biochemical manipulation of the dentin surface. Tetracycline HCL treatment of the dentin surface increases binding of fibronectin. The adsorbed fibronectin stimulates fibroblast attachment and growth, while suppressing epithelial cell attachment and growth. This biochemical manipulation may provide a useful approach for the treatment of periodontally involved teeth.
The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10 -9 M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10 -a to 10 -l° M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, Iit, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.
Selective use of tetracyciine HCl was studied to evaluate a potential treatment methodology. Tetracycline HCl adsorbed to dentin surfaces, binding 4.7 /ig/mm-after a 5 min exposure to a 50 mg/ml tetracycline HCl solution. Desorption in a discontinuous flow assay maintained biologically active concentrations of tetracycline HCl in the fluid phase for at least 48 h. The tetracycline HCl bound, and subsequently released from dentin retained antimicrobial activity with an ID50 of 3.7 ,«g/ml. Tetracycline HCl conditioning removed an amorphous surface layer, thereby exposing dentin with open tubules, as determined by scanning electron microscopy. These data suggest that tetracycline HCl-treated root surfaces may act as a depot for release of active antibiotic, as well as serve as an improved substrate for connective tissue components vital to healing at the interface between hard and soft tissues.
Acidic fibroblast growth factor (aFGF), a polypeptide with a mol. wt of approximately 16,000, is a potent mitogen for a variety of cells and shares 55% amino acid sequence identity with basic FGF. The recent isolation of three new oncogenes which share 35‐45% amino acid sequence similarity with the FGFs suggests that the coding sequences for the FGFs themselves may be oncogenic under certain circumstances. To test this hypothesis, we cotransfected 3T3 NR6 cells with factors expressing the aFGF coding sequence and the bacterial neomycin gene. The aFGF produced by cotransfected cells was found only in the cellular homogenate and not in medium conditioned by the cells. Cells expressing aFGF grew to 10 times the density of control cells at saturation and were multilayered and disorganized, similar to transformed cells. The cotransfected cells do not grow in soft agar, but show enhanced soft agar growth relative to controls in the presence of added aFGF and heparin. The aFGF‐producing cells formed small, non‐progressive tumors when injected subcutaneously into nude mice. Our data suggest that expression of aFGF in NR6 cells results in enhanced growth, and that several traits characteristic of the transformed phenotype are partially expressed.
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