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C M Stoscheck

Publications and source records attributed to C M Stoscheck.

29 records · Page 2Linked to original sources
PubMed1984

Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts.

The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to the nonradioactive medium, the half-life of prelabeled receptor was decreased to 1.2 h in human fibroblasts. These data demonstrate by direct analysis of receptor protein that during "down regulation" the EGF receptor is rapidly degraded. Enhanced receptor degradation was observed 5-10 min after the addition of EGF. The EGF-induced degradation of the receptor was blocked by methylamine, chloroquine, iodoacetate, or incubation at 25 degrees C. We have also shown that EGF-induced down regulation in human fibroblasts results in a decrease in the total amount of EGF receptor protein present. The amount of EGF receptor protein has been quantitated by radiolabeling cellular protein and immunoprecipitation of the receptor. The EGF receptor constitutes approximately 0.0035% of the cellular protein in human fibroblasts.

Cells, Cultured↗
PubMed1984

Visualization of epidermal growth factor receptors in human epidermis.

The localization of epidermal growth factor (EGF) receptors in normal human epidermis was examined with two independent experimental methods. The distribution of EGF receptor sites was studied using light microscopic autoradiography with [125I]EGF and direct immunocytochemical techniques with EGF receptor antibodies and protein A-colloidal gold complexes. Direct visualization by autoradiography indicated that the concentration of EGF receptors was greatest in the lower epidermal layers. Ultrastructural morphometric analysis of protein A-gold complexes showed that EGF receptors were primarily associated with the plasma membranes although intranuclear and cytoplasmic localization was also evident. This postembedment immunolocalization method also confirmed the relative differences in the number of EGF receptors found in individual epidermal layers (basalis greater than spinosum greater than granulosum greater than corneum layers). This inverse relationship between numbers of EGF receptors and the degree of epidermal differentiation and/or keratinization may suggest a physiologic role for EGF in these processes in human epidermis.

Autoradiography↗
PubMed1984

Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages.

To localize epidermal growth factor (EGF) receptors in normal human epidermis and other skin structures, two different light microscopic methods were used. EGF binding [( 125I]EGF/R) to the extracellular portion of the EGF receptor was studied by incubating intact skin samples with [125I]EGF, sectioning the tissues, and performing autoradiography. Immunoreactive EGF receptor molecules (IR-EGF/R) were localized with a mono-specific anti-EGF receptor antibody using a 2-step indirect immunocytochemical method (horseradish peroxidase) and detergent permeabilized tissues. This latter method measured the total pool of EGF receptors: occupied and/or internalized forms, precursor forms, and partially degraded forms of the EGF receptor that retain immunoreactivity. Both the [125I]EGF/R and IR-EGF/R localization studies indicated that EGF receptors were present in basal epidermal keratinocytes, sebocytes, outer root sheath cells in hair follicles, smooth muscle cells of arrector pili muscles, and dermal arteries. The highest levels of [125I]EGF/R and IR-EGF/R were found in the dermal ducts of eccrine sweat glands. The distribution of both [125I]EGF/R and IR-EGF/R was not consistent with the concept that EGF exclusively is involved in cellular division and proliferation in normal human epidermis and its appendages, i.e., EGF receptors were also found in tissues that do not undergo rapid proliferation. The present study indicates that EGF may have a more complex regulatory role in the skin than was previously thought.

Autoradiography↗
PubMed1983

Characteristics of antibodies to the epidermal growth factor receptor-kinase.

Polyclonal antibodies to different antigenic forms of the epidermal growth factor (EGF) receptor-kinase from human A-431 cells have been produced, and their properties have been characterized and compared. Biochemically active receptor-kinase purified by affinity chromatography was employed as one type of antigen. Denatured receptor-kinase prepared by sodium dodecyl sulfate-gel electrophoresis of the affinity-purified receptor was used as the second type of antigen. Animals immunized with either type of antigen produced antibody capable of immunoprecipitating the receptor-kinase molecule. Antibodies produced in response to the biochemically active antigenic form of the receptor-kinase are capable of blocking 125I-EGF binding to the receptor and inhibited EGF-stimulated biological responses. These antisera are not species specific in their ability to inhibit growth-factor binding to the EGF receptor of various mammalian cells. However, these rabbit antisera were unable to inhibit 125I-EGF binding to rabbit cells. Although antisera produced in response to the denatured receptor-kinase molecule are not able to block 125I-EGF binding or EGF-stimulated biological responses, they are particularly efficient for the immunoprecipitation of solubilized 125I-EGF:receptor complexes. None of the antisera contain antibodies capable of interfering with basal receptor-kinase phosphorylation activity. Although each of the antisera immunoprecipitated this kinase activity, none of the antisera contained antibody which served as a phosphorylation substrate for the EGF receptor-kinase in contrast to the immunoglobulins present antisera to the src gene product of the Rous sarcoma virus.

Animals↗
PubMed1983

Antibodies to the epidermal growth factor receptor block the biological activities of sarcoma growth factor.

The role of the epidermal growth factor (EGF) receptor system in mediating the biological activities of sarcoma growth factor (SGF) has been assessed by using specific anti-EGF receptor antibodies. There are two classes of anti-EGF receptor antibodies, those that block binding of 125I-labeled EGF (125I-EGF) and those that do not block binding but do interact with a portion of the EGF receptor on the surface of intact cells. Antisera of both types have been assayed for their capacity to affect the biological activities of SGF. The antisera that block 125I-EGF binding to its receptor block the induction of DNA synthesis in human fibroblasts by either EGF or SGF but not by other polypeptide mitogens. Titration of the anti-EGF receptor antiserum indicates the presence of one population of antibody that blocks the site of both EGF and SGF action. Antisera to the EGF receptor that block 125I-EGF binding also inhibited the SGF-dependent anchorage-independent growth of normal cells in soft agar. The antisera to the EGF receptor that does not block 125I-EGF binding or EGF activity did not inhibit any of the biological activities of SGF. The results suggest that occupation of the EGF receptor is required for both the mitogenic and colony-forming activity of SGF.

Animals↗
PubMed1982

Effects of inhibitors of ornithine and S-adenosylmethionine decarboxylases on L6 myoblast proliferation.

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.

Adenosylmethionine Decarboxylase↗
PubMedDate not supplied

Epidermal growth factor binding and receptor distribution in term human placenta.

Human placenta has a large number of epidermal growth factor (EGF) receptors when measured either by [125I]iodoEGF binding or by protein yield after purification. To localize EGF receptors in situ in normal human term placenta, two different light microscopic methods were used. To detect unoccupied, accessible EGF binding sites on the extracellular surface of placental cells in intact blocks of tissue, samples were incubated with [125I]iodoEGF, sectioned and autoradiography performed. To detect the total pool of intracellular and extracellular EGF receptors, placental tissue was sectioned, treated with detergent, and then anti-EGF receptor antibody was localized by immunohistoperoxidase techniques. Both [125I]iodoEGF and anti-EGF receptor antibody methods showed that EGF receptors were primarily present on syncytiotrophoblast cells of placental villi. Smooth muscle cells of placental blood vessels also contained EGF receptors. Neither connective tissue cells within the core of terminal chorionic villi nor endothelium of fetal blood vessels had detectable [125I]iodoEGF binding or immunoreactive EGF receptors. Since the quantity of placental smooth muscle cells is only a small fraction compared to trophoblast cells, we conclude that syncytiotrophoblast cells are primarily responsible for the high levels of EGF receptors found in extracts prepared from human term placenta.

Autoradiography↗