Epidermal Growth Factor In Culture Research Articles

Origins of Growth Factors: NGF and EGF

Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (1). My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (2). Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

Expression and regulation of gamma-glutamyl transpeptidase-related enzyme in tracheal cells.

Glutathione plays an essential role in protecting the pulmonary system from toxic insults. gamma-Glutamyl transpeptidase-related enzyme (GGT-rel) is a novel protein capable of cleaving the gamma-glutamyl peptide bond of glutathione and of converting leukotriene C4 to leukotriene D4. A rat homologue of GGT-rel was identified and was found to be highly expressed in cultures of differentiating rat tracheal epithelial (RTE) cells. The 2.6-kb cDNA predicts a 572-amino acid protein with 79% identity to human GGT-rel. GGT-rel was weakly expressed in normal trachea but was strongly induced by epidermal growth factor in cultures of RTE cells. GGT-rel was also highly expressed in lung tumors induced by inhalation of isobutyl nitrite. These results demonstrate that GGT-rel 1) is expressed in normal tracheal cells, 2) can be induced by epidermal growth factor, and 3) is elevated after chemical exposure. The induction of high levels of GGT-rel may play an important role in protecting the lung from oxidative stress or other toxic insults.

Effects of transforming growth factor-beta 1 on human arterial smooth muscle cells in vitro.

Control of the thickness of the arterial wall is critical, as excessive overgrowth of constituent smooth muscle cells (SMCs) may interfere with blood flow. Effects on SMCs in vitro of several growth factors that are present in blood and/or that are produced endogenously in the arterial wall under certain conditions suggest that influences of endocrine, paracrine, and autocrine nature from stimulating and inhibiting factors may control the smooth muscle tissue mass in the artery. This possibility was explored further by investigating the degree of myodifferentiation in terms of the presence of differentiation-specific filamentous alpha-smooth muscle actin and growth, as measured by the synthesis of DNA and cell number, of SMCs as influenced by their exposure to the mitogens, platelet-derived growth factor and epidermal growth factor, and the bifunctional growth factor, transforming growth factor-beta 1 (TGF-beta 1). Exposure to TGF-beta 1 markedly enhanced differentiation-specific filamentous alpha-smooth muscle actin. This effect did not require arrest of growth, which speaks against a direct causal relation between loss of myodifferentiation (modulation) and multiplication. When quiescent cultures were exposed to TGF-beta 1, alpha-smooth muscle actin was further increased, indicating a more specific differentiation-promoting effect by TGF-beta 1 than mere inhibition of growth. Exposure to TGF-beta 1 also increased spreading, which occurred in parallel with increased filamentous alpha-smooth muscle actin and appearance of stress fibers. Exposure to platelet-derived growth factor under serum-free conditions and to epidermal growth factor in cultures exposed to serum markedly decreased the number of alpha-actin-positive SMCs, indicating a dedifferentiating effect by these mitogens. Exposure of SMCs to TGF-beta 1 under serum-free conditions had pronounced effects on growth, with a concentration-dependent inhibition of platelet-derived growth factor-induced DNA synthesis and cell multiplication. The basal synthesis of DNA in the absence of added growth factors was also greatly inhibited. With serum-free cultures, some loss of cells occurred even with very low concentrations of TGF-beta 1 (5 pg/ml), against which platelet-derived growth factor or a dense cultural state had a protective effect. Enhancement of cell multiplication was not detected for cultivated human SMCs exposed to TGF-beta 1, irrespective of culture density, in contrast to that reported for dense cultures of rat SMCs. TGF-beta 1 is present in and may be released from platelets in situations that promote platelet adherence such as endothelial injury; TGF-beta 1 may also be released from activated macrophages and T lymphocytes either during an immune reaction or inflammation or from the endothelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization.

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.

The epidermal growth factor receptor tyrosine kinase in liver epithelial cells: The effect of ligand-dependent changes in cellular location

Abstract Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.

Activation of the Na+/H+ antiport is not required for epidermal growth factor-dependent gene expression, growth inhibition or proliferation in human breast cancer cells

Mitogen interaction with specific receptors in many cell types leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Since amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement for mitogenesis. However, concentrations of amiloride which inhibit the antiport also inhibit other cellular processes, including protein synthesis and phosphorylation. We have used an epidermal growth factor (EGF) receptor gene-amplified human breast cancer cell line, the growth of which is inhibited by high levels of EGF in culture (MDA-468) and a variant, the growth of which is stimulated by EGF (MDA-468-S4), along with two potent amiloride analogues to examine whether activation of the Na+/H+ antiport and cytoplasmic alkalinization is necessary for both EGF-dependent effects to occur. At concentrations of the amiloride analogues which block Na+/H+ exchange in both cell types by 76-98%, the EGF-dependent alterations in [3H]thymidine incorporation or induction in c-myc or c-fos gene transcription were unaltered. These results were confirmed by a lack of effect of the amiloride analogues on both the growth-stimulatory and growth-inhibitory effects on EGF in an anchorage-independent growth assay. Similarly, in pH-altered media that prevented normal cytoplasmic alkalinization, the response of both MDA-468 and MDA-468-S4 to EGF activation was unaltered. In addition, activation of the Na+/H+ antiport alone was not sufficient to induce c-myc and c-fos transcription in either cell type. Taken together, these data suggest that neither the Na+/H+ antiport nor cytoplasmic alkalinization are necessary or sufficient for either EGF-dependent growth stimulation or growth inhibition in MDA-468 human breast cancer cells.

In vivo selection of tumorigenic subline from non-tumorigenic human gastric carcinoma cells: in relation to proliferative properties in vivo and in nude mice.

Human gastric carcinoma cells from one of three long-term cultured cell lines (HPE-GAC-T) were injected into peritoneal cavities of BALB/c mice. The surviving celss in vivo were collected 3 days later. Following brief cultivation in vitro, those cells were reinjected into mice by the same route. This procedure was repeated 3 times. The cultured cancer cells recovered from the mice on the 3rd passage, at a 92.5% recovery rate, showed xenotransplantability in BALB/C nu/nu mice by subcutaneous injection. This subline (GAC-T.M-2) can be maintained in vitro but not in vivo while maintaining heterotransplantability. Three original cancer cell lines did not show tumorigenicity in nude mice. Animal passages by the same protocol failed to select tumorigenic sublines from the other cell lines (HPE-GAC-2 and -3). Factors affecting tumorigenic capacity of cancer cells in nude mice were studied in vivo and in vitro by comparing the properties of GAC-T.M-2 and parental cancer cells (GAC-T.O). Treatment of the hosts by injection of anti-asialoGM1 antibody or cyclophosphamide, adult thymectomy of BALB/c mice, and 400 rads whole body irradiation did not enhance the growth of either GAC-T.M-2 or -T.O cells. There was no detectable difference between in vitro growth properties of the original and variant cells at a rather high cell density. However, at a low cell density GAC-T.M-2 cells showed a higher cell growth rate and increased [3H] thymidine incorporation and possessed higher colony forming activity in the liquid medium than their parental cells. High dense expression of epidermal growth factor (EGF) receptors was evident equally in both GAC-T cells, however, GAC-T.M-2 cells were more sensitive to down-regulation by EGF in culture. Tumor cells of HPE-GAC-2 and -3 lines expressed minimum amount of EGF receptors on their cell surfaces and were refractory to additional EGF in culture. The results indicate that growth factors and their receptors are responsible for tumorigenicity in nude mice.

Induction of Differentiated Trophoblast Function by Epidermal Growth Factor: Relation of Immunohistochemically Detected Cellular Epidermal Growth Factor Receptor Levels*

The effects of epidermal growth factor (EGF) on the production and secretion of hCG and human placental lactogen (hPL) by cultured placental tissues were investigated in relation to immunohistochemical measurements of cellular EGF receptor levels in the placenta. Explants of trophoblastic tissues obtained from normal early and term placentas were cultured in the presence or absence of EGF (100 ng/mL) with or without processing inhibitors (bacitracin, 1 mg/mL; colchicine, 100 microM; chloroquine, 100 microM) for 5 days, with EGF present for the first 2 days. Addition of EGF to the medium increased the release of hCG, hCG alpha, and hPL by the cultured early placental tissues. This EGF-stimulated hCG, hCG alpha, and hPL release was markedly inhibited by concomitant treatment with processing inhibitors. The time course of EGF effects indicated that the EGF-stimulated increase in hCG alpha secretion required a lag period of approximately 1 day, whereas significant increases in hCG and hPL secretion became apparent only after 3 days of EGF treatment. By contrast, in term placental tissues EGF stimulated only hCG alpha and hPL release, with a lag period of approximately 3 days. A possible direct action of EGF on the cultured placental tissues was reinforced by the immunohistochemical demonstration of EGF receptors in the placenta. When determined using the avidin/biotin immunoperoxidase method with monoclonal antibody to the mouse EGF receptor, EGF receptors were found predominantly on the syncytiotrophoblasts. Immunohistochemical measurements of cellular EGF receptor levels in the syncytiotrophoblasts revealed remarkably higher levels in early placenta compared to those in midterm and term placentas. Since EGF is likely to interact with its receptor, the lesser biological effects of EGF in cultures of term placental tissues may be due to the lower cellular EGF receptor levels in term placenta. These results demonstrate that EGF, via its receptors on the syncytiotrophoblasts, stimulates the release of both hCG and hPL in normal early placenta. They also suggest that EGF may play a significant role in the induction and regulation of the differentiated function of trophoblasts.

Nuclear accumulation of epidermal growth factor in cultured bovine corneal endothelial and granulosa cells.

Intracellular accumulation of intact 125I-labeled epidermal growth factor (125I-EGF) in corneal or granulosa cell cultures exposed to either chloroquine or leupeptin can be 10-fold higher than that observed in cultures not exposed to the lysosomal inhibitors. This has made it possible to study the translocation and accumulation of 125I-EGF in a nuclear fraction of both cell types. The accumulation of 125I-EGF was found to be dependent on the inhibitor's concentrations. Chloroquine at a concentration of 5 X 10(-5) M yielded a maximal nuclear accumulation which amounted to 18% of the total 125I-EGF present within the bovine corneal endothelial cells exposed to chloroquine. Nuclear accumulation could be detected in that cell type as early as 4 h after cultures were exposed to both chloroquine and 125I-EGF and was maximal by 24 h. Saturation of nuclear accumulation of 125I-EGF in corneal endothelial and granulosa cell cultures was observed at 20 and 10 ng/ml of 125I-EGF, respectively. After 24 h, 1.06 X 10(4) and 1.4 X 10(4) 125I-EGF molecules were found associated with granulosa or corneal endothelial nuclei, respectively. In corneal endothelial cell cultures exposed to chloroquine, a small fraction (0.6%) of 125I-EGF associated with the cell was found to be irreversibly found to a polypeptide with a molecular weight of 185,000, and 50% of these irreversible 125I-EGF . receptor complexes were found to be associated with the nuclei.

Vitamin A potentiates the mitogenic effect of epidermal growth factor in cultures of normal adult human skin fibroblasts.

The polypeptide hormone, epidermal growth factor (EGF) and Vitamin A (retinoic acid) were added to log phase cultures of adult human skin fibroblasts and the effect on cell growth observed. At the end of seven days EGF alone stimulated cell growth 68%, while trans-retinoic acid alone had no significant effect. However, when EGF was added in combination with trans-retinoic acid, cell growth was increased 159 to 214%. Cis-retinoic acid gave similar results. These results indicate that, under certain conditions, a presumed inhibitor of fibroblast growth (retinoic acid) can potentiate the mitogenic action of a hormone, namely, epidermal growth factor.