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Biomedical subjects

G Carpenter

Publications and source records attributed to G Carpenter.

At least 127 records · Page 7Linked to original sources
PubMed1988

Presence of mannose phosphate on the epidermal growth factor receptor in A-431 cells.

In this report, we demonstrate a novel post-translational modification of the epidermal growth factor (EGF) receptor. This modification involves the presence of phosphate, previously thought to exist only on amino acid residues in the EGF receptor, on oligosaccharides of the receptor. We have utilized several independent approaches to determine that mannose phosphate is present on the EGF receptor in A-431 cells. Following metabolic labeling with 32P, immunoisolation of the EGF receptor, and digestion with Pronase radioactivity was determined to be present on high mannose type oligosaccharides by concanavalin A chromatography. Also, after acid hydrolysis of in vivo 32P-labeled EGF receptor, radioactivity was detected that co-migrated with mannose 6-phosphate on two-dimensional thin layer electrophoresis. This radiolabeled material co-eluted with a mannose 6-phosphate standard from a high pressure liquid chromatography anion exchange column. Last, an acid hydrolysate of [3H]mannose-labeled EGF receptor contained two radiolabeled fractions, as analyzed by thin layer electrophoresis, and the radioactivity in one of these fractions was substantially reduced by alkaline phosphatase treatment prior to electrophoresis. These experiments indicate that the mature EGF receptor in A-431 cells contains mannose phosphate. This is a novel modification for membrane receptors and has only been reported previously for lysosomal enzymes and a few secreted proteins.

Animals↗
PubMed1988

Regulation of epidermal growth factor-stimulated formation of inositol phosphates in A-431 cells by calcium and protein kinase C.

Epidermal growth factor (EGF) treatment of A-431 cells induces a biphasic increase in the levels of inositol phosphates. The growth factor produces an initial, rapid increase in the level of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) due to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (Wahl, M., Sweatt, J. D., and Carpenter, G. (1987) Biochem. Biophys. Res. Commun. 142, 688-695). The level of inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) also rises rapidly in response to treatment with EGF. The initial formation (less than 1 min) of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 does not require Ca2+ present in the culture medium. However, the addition of Ca2+ to the medium at levels of 100 microM or greater potentiates the growth factor-stimulated increases in the levels of all inositol phosphates at later times after EGF addition (1-60 min). The data suggest that EGF-receptor complexes initially stimulate the enzyme phospholipase C in a manner that is independent of an influx of extracellular Ca2+. The presence of Ca2+ in the medium allows prolonged growth factor activation of phospholipase C. Treatment of A-431 cells with Ca2+ ionophores (A23187 and ionomycin) did not mimic the activity of EGF in producing a rapid increase in the formation of the Dowex column fraction containing Ins-1,4,5-P3, Ins-1,3,4,5-P4, and inositol 1,3,4-trisphosphate (InsP3). However, the initial EGF-stimulated formation of inositol phosphates was substantially diminished in cells loaded with the Ca2+ chelator Quin 2/AM. EGF receptor occupancy studies indicated that maximal stimulation of InsP3 accumulation by EGF requires nearly full (75%) occupancy of available EGF binding sites, while half-maximal stimulation requires 25% occupancy. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an exogenous activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), causes a dramatic, but transient, inhibition of the EGF-stimulated formation of inositol phosphates. Tamoxifen and sphingosine, reported pharmacologic inhibitors of protein kinase C activity, potentiate the capacity of EGF to induce formation of inositol phosphates. Neither TPA nor tamoxifen significantly affects the 125I-EGF binding capacity of A-431 cells; however, TPA appeared to enhance internalization of the ligand. Ligand occupation of the EGF receptor on the A-431 cell appears to initiate a complex signaling mechanism involving production of intracellular messengers for Ca2+ mobilization and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta.

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.

Animals↗
PubMed1988

Similarities in glycosylation and transport between the secreted and plasma membrane forms of the epidermal growth factor receptor in A-431 cells.

We have studied the synthesis and oligosaccharide processing of the 110,000 dalton form of the epidermal growth factor (EGF) receptor that is secreted into the medium of A-431 cells. Its 90,000 dalton precursor is soluble within the lumen of intracellular membrane vesicles shortly after synthesis, indicating that it lacks a membrane anchor. Analysis of labeled glycopeptides reveals that the glycosylation of the 110,000 dalton, secreted receptor is very similar to that of the 170,000 dalton, plasma membrane receptor. Based on Concanavalin A-Sepharose elution profiles of its glycopeptides, the secreted receptor has both complex and high-mannose N-linked oligosaccharides. Also, like the plasma membrane receptor, the secreted receptor contains N-acetylgalactosamine residues in its complex chains. Not only are major features of oligosaccharide processing of the soluble and membrane-bound forms of the receptor similar, but the kinetics of transport to the cell exterior is the same for each. These data indicate that the glycosylation pattern and kinetics of cellular transport of the EGF receptor are determined by factors other than the sequence of its cytoplasmic and transmembrane domains.

Animals↗
PubMed1988

Prostatropin and acidic FGF also support proliferation of an EGF-dependent keratinocyte cell line.

Despite the relevance of epithelial cells to the biology of cancer, considerably less is known about the capacity of specific growth factors to control the proliferation of these cells compared to information available on fibroblastic cells. Epidermal growth factor is the most widely recognized mitogen for epithelial cells. We demonstrate that prostatropin and acidic fibroblast growth factor, structurally similar molecules, are potent growth factors for the mouse keratinocyte cell line Balb/MK. This indicates that these growth factors in addition to epidermal growth factor may be of physiological relevance to epidermal cell proliferation.

Animals↗
PubMed1988

The effect of short term treatment with growth hormone and ethinyl estradiol on lower leg growth rate in girls with Turner's syndrome.

An important consequence of Turner's syndrome is short stature. We previously reported that the optimal doses of ethinyl estradiol (EE2) and GH for the stimulation of short term growth in such patients were 100 ng/kg.day and 0.15 U/kg (administered sc three times weekly), respectively. The aim of this study was to determine whether the combination of low dose estrogen and GH would stimulate short term growth more than either agent administered alone. Thirty-nine girls with Turner's syndrome (aged 5-15 yr) underwent one to three 6-month cycles, each consisting of a 2-month baseline period, a 2-month treatment period, and a subsequent 2-month washout period. During the first 2 yr of the study, the girls were assigned to receive the three treatments in random order. The treatments were EE2 (100 ng/kg.day, orally), GH (0.15 U/kg, sc, three times weekly), or the combination of EE2 and GH. Subsequently, some of the girls were treated with reduced doses of EE2 (50 ng/kg.day) and GH [0.09 U/kg, three times weekly (tid)] according to the same protocol. Lower leg length was measured every 2 months throughout the study. EE2 increased lower leg growth rate significantly at the dose of 100 ng/kg.day, but not at the dose of 50 ng/kg.day. Similarly, the higher dose of GH (0.15 U/kg, tiw) increased lower leg growth rate significantly, whereas the lower dose (0.09 U/kg, tiw) did not. However, combined treatment with the lower doses of EE2 (50 ng/kg.day) and GH (0.09 U/kg, tiw) stimulated lower leg growth rate significantly and to a similar degree as the higher dose of GH (0.15 U/kg, tiw). This higher dose of GH appeared to cause a maximal increase in lower leg growth rate, which was not further increased by combined administration with the higher dose (100 ng/kg.day) of estrogen. Thus, addition of low dose EE2 to an optimal dose of GH did not cause any apparent increase in short term lower leg growth rate in girls with Turner's syndrome. Whether the long term outcome of GH treatment would be altered by concurrent administration of low dose estrogen will require long term clinical trials.

Adolescent↗
PubMed1988

Accumulation of epidermal growth factor receptors in retinoic acid-treated fetal rat lung cells is due to enhanced receptor synthesis.

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung (FRL) cells is increased approximately 2 to 3-fold within 18 h of retinoic acid addition. Analysis of 125I-EGF binding assays at 0 C reveals approximately 25,000 receptors per cell, while analysis of growth factor binding to retinoic acid-treated cells demonstrates an increase in receptor levels to approximately 70,000 receptors per cell with no detectable changes in receptor affinities. We show by immunoprecipitation of 35S-methionine labeled EGF receptors that retinoic acid addition produces an increase in the accumulation of EGF receptor protein. Using brief pulses of 35S-methionine, an increase in EGF receptor synthesis can be identified within 3 h after retinoic acid addition. These results are the first to demonstrate that a retinoic acid-induced increase in 125I-EGF binding capacity is due to increased EGF receptor protein synthesis. Also, we find that a transient decrease in the rate of EGF receptor turnover occurs when retinoic acid is initially added to FRL cells. On the basis of our data, we conclude that the retinoic acid-induced accumulation of EGF receptors in FRL cells is primarily due to increased receptor synthesis. The effect of retinoic acid on EGF receptor turnover may be a secondary factor, influencing the rate at which receptors accumulate.

Animals↗
PubMed1987

Transforming growth factor alpha and beta expression in human colon cancer lines: implications for an autocrine model.

Three human colon cancer lines (SW480, SW620, WIDR) secrete different levels of transforming growth factor beta (TGF beta)-like and transforming growth factor alpha (TGF alpha)/epidermal growth factor (EGF)-like molecules into serum-free conditioned media as measured by competing activity in TGF beta and EGF radioreceptor assays. SW480 cells, the highest producers of TGF beta-like activity, lack detectable TGF beta receptors while SW620 cells, the highest producers of TGF alpha/EGF-like activity, lack EGF receptors. This study investigated the production of these growth factors at the mRNA level and examined the mechanism of loss of detectable receptors. Using complementary DNA probes for TGF beta and TGF alpha, it was demonstrated that mRNA levels correlated with the amounts of TGF beta and TGF alpha produced; TGF beta gene expression was highest in SW480 cells and TGF alpha gene expression was highest in SW620 cells. Acid washing of the SW480 cells prior to performing the TGF beta binding assay resulted in the unmasking of substantial levels of TGF beta receptors. Neither acid washing nor preincubation with suramin uncovered EGF receptors in SW620 cells. Also, and in contrast to the other two lines, EGF receptor expression could not be detected in SW620 cells by Northern gel analysis of receptor messenger RNA or by immunological analysis of receptor protein. Thus two distinct mechanisms (occupation of TGF beta receptor in SW480 cells, or absence of EGF receptor in SW620 cells) explain the lack of detectable TGF beta and EGF receptors in the binding assays. The autocrine hypothesis remains viable for TGF beta in SW480 cells but not for TGF alpha in SW620 cells; this would not discount a paracrine role in this latter case.

Cell Line↗
PubMed1987

Epidermal growth factor (EGF) stimulates inositol trisphosphate formation in cells which overexpress the EGF receptor.

EGF is a low molecular weight polypeptide hormone which acts as a regulator of cell growth and differentiation. The A-431 cell line has been used frequently to examine receptor-mediated biochemical effects of EGF, since this cell line has an increased (20-50 fold) level of EGF receptors. We have utilized A-431 cells to examine the influence of EGF on formation of an intracellular second messenger, inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3), and other inositol phosphates. The results show that EGF induces rapid formation of Ins-1,4,5-P3 as well as Ins-1,3,4-P3 and Ins-1,3,4,5-P4. There is a concurrent decrease in the level of the lipid precursor for Ins-1,4,5-P3, phosphatidylinositol 4,5-biphosphate (PIP2). Furthermore, we have examined five other cell lines that overexpress the EGF receptor and find that EGF treatment induces formation of inositol polyphosphates in those cell lines also.

Calcium↗
PubMed1987

Role of growth factors and their receptors in the control of normal cell proliferation and cancer.

Polypeptide growth factors modulate proliferation of nontransformed cells in vivo and in vitro, while cancer appears to reflect an alteration of growth-regulatory mechanisms found in normal cells. Some provocative clues for understanding the cellular biochemical events involved in growth regulation have come from the study of transforming retroviral oncogenes. Some of these oncogenes encode proteins similar to those implicated in growth factor-mediated growth control. Of particular interest is the study of growth factor receptors present on the cell surface, which are cellular homologs of members of the largest class of oncogenes, the tyrosine kinases. It is likely that the study of the interplay of growth factors, and the molecular basis of pleiotropic effects elicited by growth factors, will help to explain how growth factor-signaling pathways affect gene expression and cell division in normal and transformed states.

Animals↗
PubMed1986

Polysomnographic findings in recently drug-free and clinically remitted depressed patients.

Thirteen patients were examined by sleep polysomnograph (PSG) and the dexamethasone suppression test when clinically depressed and later when clinically remitted for six months and no longer receiving antidepressant medication for two to five weeks. None of the PSG variables (rapid eye movement [REM] latency, total sleep time, stage 1 through 4 times, REM time, and REM densities in periods 1 through 3) was significantly changed between symptomatic depression and symptom remission. While symptomatic, 11 of 13 patients exhibited a reduced REM latency (65 minutes or less). After clinical remission, eight of the 11 continued to exhibit reduced REM latencies, whereas the dexamethasone suppression test tended to show nonsuppression only during clinical depression. These data represent either longer-term (ie, slow to normalize) biologic consequences of a depressive episode or biologic antecedents of clinical depression that may herald a return of the depression in individuals vulnerable to recurrence. Whether PSG abnormalities identify clinically remitted patients who are prone to develop another depressive episode requires longitudinal follow-up studies.

Adult↗