Number Of Epidermal Growth Factor Receptors Research Articles

The epidermal growth factor receptor in the human endometrial adenocarcinoma cell line HEC-1-B

The binding characteristics and steroidal regulation of the EGF receptor were investigated in the human endometrial adenocarcinoma cell line HEC-1-B. The cell line was shown to possess a single, high affinity binding site for epidermal growth factor receptor (EGF) with a K d of 3.09±1.39 nM (mean±SD, n=6) and binding of 845±311 fmol/mg protein (mean±SD, n=6). The protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA) increased the K d of the EGF receptor in a dose dependent manner (PMA: 0, 1, 10, 100 nM; K d: 4.1, 5, 10, 50 nM, respectively). The effect of PMA (10 nM) was overcome by preincubating the cells with the protein kinase C inhibitor staurosporine (1 μM) prior to the addition of PMA. The effect of the ovarian steroids oestradiol and progresterone on EGF receptor accumulation was studied by pretreating the cells for 6 days with oestradiol or progesterone in phenol red free DMEM: F12, 1:1 supplemented with 5% charcoal stripped fetal calf serum. Both steroids were shown to increase EGF receptor number with a maximum 5- and 7-fold increase in the presence of 1 nM oestradior or 1μM progresterone, respectively. The study demonstrates the presence of a high affinity binding site for EGF in HEC-1-B cells which is regulated by oestradiol and progresterone.

Effect of tyrphostin on cell growth and tyrosine kinase activity of epidermal growth factor receptor in human gliomas

The effects of tyrphostin, a selective protein tyrosine kinase inhibitor, on epidermal growth factor (EGF)-stimulated cell growth and EGF-receptor tyrosine kinase activity were studied in four human glioma cell lines. Stimulation by EGF induced variable enhancements of cell growth as well as tyrosine phosphorylation of EGF receptor and intracellular target proteins in all glioma cell lines. The level of immunoreactive EGF receptor detected with antibodies against extra- and intracellular domains was moderate in all four glioma cell lines, but markedly decreased with the latter antibody in two glioma cell lines. This variation was associated with considerable reduction of the EGF-stimulated tyrosine autophosphorylation level. Tyrphostin inhibited dose-dependently the EGF-stimulated cell growth and tyrosine autophosphorylation in all glioma cell lines, and the optimum time for the maximum inhibitory effect on tyrosine autophosphorylation was 12 to 18 hours after treatment with tyrphostin. The antiproliferative activity of tyrphostin nearly correlated quantitatively with its potency as an inhibitor of the EGF-stimulated EGF receptor tyrosine kinase activity. Tyrphostin had no significant effect on the immunoreactive EGF receptor levels, on the affinity constants and numbers of EGF receptor, or on the down-regulation and specific internalization of EGF receptor in any glioma cell line, suggesting that the effects of tyrphostin are not likely to be the results of reduction in EGF receptor and EGF binding capacity. In addition, the serum-stimulated cell growth was also inhibited dose-dependently by higher concentrations of tyrphostin in all glioma cell lines. It might be suggested, therefore, that tyrphostin inhibits EGF-stimulated cell growth by a specific suppression of EGF receptor tyrosine kinase activity, and at higher concentrations there appears to be some degree of either nonspecific inhibition or inhibition of serum-stimulated protein tyrosine kinase activity to induce the cell growth inhibition of gliomas.

Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85–93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.

Response of atherosclerotic intimal smooth muscle cells to epidermal growth factor in vitro.

Increased proliferation of intimal smooth muscle cells (SMCs) plays an important role in the early phase of atherogenesis. To investigate growth mechanisms of these cells, we used intimal SMCs from rabbits fed an atherogenic diet and examined the sequential events that may facilitate induction of intimal SMC proliferation as well as the possible effects of growth-promoting factors secreted by these cells. In serum-free medium, epidermal growth factor (EGF) stimulated [3H]thymidine uptake by quiescent intimal SMCs at a rate six times higher than quiescent medial SMCs. There was no significant difference between the two cell types in terms of the number of specific EGF receptor per cell, the dissociation constant of EGF, and the time course of EGF binding and internalization. Furthermore, in both types of cells, c-fos, c-jun, and c-myc mRNAs were induced after 1, 1, and 4 hours of EGF treatment, respectively, whereas they required 8 hours of contact with EGF to induce proliferation. Growth response of medical SMCs to EGF was greatly enhanced when rabbit serum, deficient in lipoproteins and free of platelet-derived growth factor, was added to the medium. Moreover, EGF induced a twofold to fourfold increase in DNA synthesis in medial SMCs cocultured with intimal SMCs compared with medial SMCs incubated alone. Likewise, DNA synthesis of medial SMCs grown in medium conditioned by intimal SMCs was six times higher than that observed in medium conditioned by medical SMCs. Adding EGF to the medium conditioned by intimal SMCs increased their DNA synthesis even further.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone increases the expression of receptors for epidermal growth factor in UMR 106-01 cells

PTH administration in vivo increases osteoblast number and activity, resulting in increased bone formation, and also increases osteoclast-mediated bone resorption. Studies in vitro, however, have shown that the actions of PTH on osteoblast-like cells are inhibitory and catabolic, as shown by decreases in growth rate and collagen synthesis and increases in collagenase production. The present studies were designed to investigate possible mechanisms for these observations by examining the effects of PTH on the response of osteoblast-like cells to the osteoblast growth factor, epidermal growth factor (EGF). Confluent cultures of UMR 106-01 cells were treated with rat PTH-(1-34) for periods up to 72 h, and EGF receptors were measured with [125I]EGF. PTH, in a dose- and time-dependent manner, increased the number of EGF receptors 2-fold. The half-maximal effect of PTH occurred at a concentration of 1 nM, the same PTH concentration that resulted in half-maximal increases in cAMP generation. The increase in EGF binding was associated with an enhanced biological effect, as shown by augmentation of EGF-stimulated diglyceride production. The effect of PTH could be reproduced by the addition of 8-bromo-cAMP, but not by the phorbol ester phorbol myristate acetate. In the presence of cyclohexamide, the effect of PTH on EGF binding was abolished, suggesting that new protein synthesis was required to increase the number of EGF receptors. Northern blots of total RNA, using a cDNA probe encoding the extracellular domain of the rat EGF receptor, revealed that PTH treatment resulted in a 2- to 3-fold increase in the level of EGF receptor mRNA. These data suggest that the proliferative effects of PTH on the osteoblast may be mediated indirectly by a PTH-induced increase in the number of EGF receptors.

Parathyroid hormone increases the expression of receptors for epidermal growth factor in UMR 106-01 cells.

PTH administration in vivo increases osteoblast number and activity, resulting in increased bone formation, and also increases osteoclast-mediated bone resorption. Studies in vitro, however, have shown that the actions of PTH on osteoblast-like cells are inhibitory and catabolic, as shown by decreases in growth rate and collagen synthesis and increases in collagenase production. The present studies were designed to investigate possible mechanisms for these observations by examining the effects of PTH on the response of osteoblast-like cells to the osteoblast growth factor, epidermal growth factor (EGF). Confluent cultures of UMR 106-01 cells were treated with rat PTH-(1-34) for periods up to 72 h, and EGF receptors were measured with [125I]EGF. PTH, in a dose- and time-dependent manner, increased the number of EGF receptors 2-fold. The half-maximal effect of PTH occurred at a concentration of 1 nM, the same PTH concentration that resulted in half-maximal increases in cAMP generation. The increase in EGF binding was associated with an enhanced biological effect, as shown by augmentation of EGF-stimulated diglyceride production. The effect of PTH could be reproduced by the addition of 8-bromo-cAMP, but not by the phorbol ester phorbol myristate acetate. In the presence of cyclohexamide, the effect of PTH on EGF binding was abolished, suggesting that new protein synthesis was required to increase the number of EGF receptors. Northern blots of total RNA, using a cDNA probe encoding the extracellular domain of the rat EGF receptor, revealed that PTH treatment resulted in a 2- to 3-fold increase in the level of EGF receptor mRNA. These data suggest that the proliferative effects of PTH on the osteoblast may be mediated indirectly by a PTH-induced increase in the number of EGF receptors.

Epidermal growth factor secretion by submandibular glands is not perturbed in the early phase of streptozotocin-induced diabetes in mice

In rodents, diabetes mellitus is accompanied by a decreased concentration of epidermal growth factor (EGF) in plasma and urine and by a reduced number of EGF receptors on the surface of target cells. A combination of these two abnormalities may reduce the effects of EGF and could be implicated in some complications of diabetes. The aim of the present work was to investigate the possible implication of the major source of EGF in the organism, the submandibular glands, upon the reduced EGF concentration in blood and urine. Firstly, we measured the content of mice submandibular gland EGF by radioimmunoassay and by morphometric determinations of the relative volume occupied by granular convoluted tubules in the gland and by the EGF-containing granules in the cells at the light and electron microscopical levels respectively. We found no differences in the EGF content of submandibular glands of streptozotocin-treated diabetic animals compared to control ones. Secondly, we estimated stimulus-secretion coupling by EGF-secreting cells, present in an enriched preparation of granular convoluted tubules (GCT) perifused in thermoregulated chambers. Phenylephrine was the only agent tested that was capable of stimulating EGF secretion. The stimulation was dose-dependent and similar in streptozotocin-diabetic and control mice. Thus, the EGF deficiency observed in diabetic mice is related neither to a defect of EGF content in the submandibular glands nor to an abnormal EGF secretion by the glands.

Internalization and excretion of epidermal growth factor-dextran-associated radioactivity in cultured human squamous-carcinoma cells.

Certain tumor cells, such as squamous carcinomas and gliomas, can have an increased number of epidermal-growth-factor (EGF) receptors. The EGF receptors can in these cases be targets for toxic conjugates with specific binding. EGF-based toxic conjugates are potential targeting agents. We have analyzed the internalization and excretion of 125I administered in the form of 125I-EGF-dextran in squamous-carcinoma A431 cells. 125I-EGF without dextran was used for comparison. A431 cells have large numbers of EGF receptors and are capable both of recycling and of degradation of internalized receptor-ligand complexes. The binding of 125I-EGF-dextran and 125I-EGF was receptor-specific, since both ligands competed with non-radioactive EGF for binding. The amount of internalized 125I as a function of time increased continuously within 24 hr following administration of radioactivity as 125I-EGF-dextran. The time pattern was quite different when 125I-EGF without dextran was applied. In the latter case, the amount of internalized radioactivity decreased already after a few hours, probably depending on degradation of EGF. Pre-incubation of the cells with 125I-EGF-dextran or 125I-EGF and analysis of retained and released 125I activity at different times after washing showed that the 125I activity was retained for longer periods of time when EGF-dextran was used instead of EGF. About 30% of the internalized 125I activity was retained after 24 hr when EGF-dextran was used, compared with excretion of nearly all the radioactivity within 5 hr when EGF was used. In some experiments a high concentration of non-radioactive EGF, 5 micrograms/ml, was given to the cells after pre-incubation with 125I-EGF-dextran. This changed the retention and excretion patterns, so that a larger amount of 125I was excreted in the macromolecular fraction and a smaller amount of 125I activity was retained in the cells. Gel chromatography of the 125I activity released into the culture medium showed that the variations in molecular weight were larger after administration of a high concentration of non-radioactive EGF, most likely due to partial degradation of EGF-dextran. The results regarding excretion are in conformity with a model of competition between recycling of EGF-dextran-EGF-receptor complexes and "trapping" of EGF-dextran in the lysosomes followed by slow degradation. For targeting purposes, it is worth noting that the radioactivity administered in the form of 125I-EGF-dextran had a longer retention time than when 125I-EGF without dextran was used, and that the retention and excretion rates could be modified by post-treatment with the receptor ligand itself.

Epidermal growth factor in acute renal failure.

Epidermal growth factor (EGF) is produced in large amounts in the kidney in the form of a membrane-bound high molecular weight precursor. This precursor is inserted in the apical plasma membrane of the EGF-producing cells, which are localized in the thick ascending limb and distal convoluted tubule in mouse and rat kidney, and probably also in human kidney. High levels of EGF are excreted in urine, although renal tissue contains little mature EGF. It modulates renal cell proliferation and differentiation in vitro, but the role of the distal tubular EGF and/or its precursor in vivo is unknown. The expression of EGF in the kidney and its liberation into the urine are quickly abolished during several types of drug- or ischemia-induced acute renal failure and also in ureteral obstruction. Moreover, its expression is restored only after morphological and functional recovery of the kidney. This absence of EGF in conditions in which its mitogenic properties would be most appropriate suggests that the EGF of renal origin is not acting as a mitogen during kidney regeneration. Nevertheless, since the number of EGF receptors, which are localized at the basolateral cell surface in most nephron segments, is increased in regenerating renal epithelia, EGF of systemic origin or other members of the EGF family of growth factors, released from infiltrated inflammatory cells at the sites of injury, could enhance cellular proliferation by interacting with the EGF receptor. Administration of EGF indeed has a mildly beneficial effect on recovery from acute renal injury.

Secretion of transforming growth factor alpha and expression of its receptor in human mammary cell lines.

The secretion of transforming growth factor alpha (TGF alpha) and the expression of cell-surface receptors for epidermal growth factor (EGF) were measured in a series of human mammary cell lines. The amount of TGF alpha secreted by the cells did not correlate with the phenotype of the cells (epithelial or myoepithelial), the mechanism of immortalization of the cells (SV40 or spontaneous) or the source of the cells (normal mammary gland, benign hyperplastic lesion, malignant tumour). The level of expression of cell-surface receptors for EGF was markedly increased as a consequence of SV40-immortalization of mammary cells, but otherwise did not correlate with the phenotype of the cells or the source of the cells. Much of the increase was accounted for by the appearance of a large number of low-affinity receptors for EGF in the SV40-immortalized cells. It is suggested that one of the mechanisms whereby SV40-immortalization suppresses the senescence of primary cultures of human mammary epithelial cells involves increasing the level of expression of receptors for EGF. In contrast the level of secretion of TGF alpha by cells in culture is probably a consequence of the mechanisms of adaptation of each cell line to culture conditions, and does not reflect the level of secretion of TGF alpha by cells in vivo.

Epidermal growth factor and transforming growth factor alpha characteristics of human oral carcinoma cell lines.

This study examined the expression of epidermal growth factor (EGF) cell-surface receptors, the response to exogenous ligand and the autocrine production of transforming growth factor alpha (TGF-alpha) in normal and carcinoma-derived human oral keratinocytes. One of eight malignant cell lines overexpressed EGF receptors, while the remainder expressed receptor numbers similar to normal cells. Exogenous EGF stimulated incorporation of tritiated thymidine in a dose-dependent manner. In keratinocytes expressing normal numbers of EGF receptors, the cellular response to exogenous EGF correlated positively with total EGF receptor number. SCC-derived keratinocytes produced more TGF-alpha than normal cells. There was no statistical correlation between the autocrine production of TGF-alpha, EGF cell-surface receptor expression and cellular response to exogenous EGF. While the growth-stimulatory effects of exogenous TGF-alpha were inhibited by the addition of a neutralising antibody, the presence of this antibody in conditioned medium failed to produce a similar decrease in growth. The results indicate that overexpression of EGF receptors is not an invariable characteristic of human oral squamous carcinoma-derived cell lines. Further, the contribution of TGF-alpha to the growth of normal and carcinoma-derived human oral keratinocytes in vitro may be less significant than previously documented.

Cell cycle arrest induced by epidermal growth factor on human squamous cell carcinoma cell lines

Although human squamous cell carcinoma (SCC) cell lines frequently contain an elevated number of epidermal growth factor (EGF) receptor accompanied with amplification of EGF receptor/c-erbB gene, it is well known that EGF inhibits the growth of these cells in culture at doses that stimulate the growth of epidermal keratinocytes and dermal fibroblasts. To study this growth inhibitory effect of EGF on the SCC cell lines, 3H-thymidine incorporation into DNA and cell cycle distribution were analyzed. In HSC-1 and NA cells, which contain the highest number of EGF receptor among these SCC cell lines, the inhibition of 3H-thymidine incorporation was apparent 2 to 4 hours after treatment with 100 ng/ml of EGF and reached more than 95% inhibition after 24 hours. Two-color cell cycle analysis using fluorescein isothiocyanate (FITC)-conjugated anti-Bromodeoxyuridine (BrdU) antibody and propidium iodide revealed that this inhibitory effect was due to cell cycle arrest not only in G1 but also in G2 phase. This effect was well correlated to the sensitivity to the growth inhibitory effect of EGF among the 4 SCC cell lines. These observations suggest that the SCC cells contain altered machineries which regulate the normal cell growth in both G1 and G2 phases, and the EGF affects these machineries via overexpressed its receptor.

Effect of epidermal growth factor and transforming growth factor β1 on growth and invasive potentials of newly established rat bladder carcinoma cell lines

We established 5 rat bladder cell lines (MYU3L, MYU4, MYU6s, MYKU1L and MYP3). EGF stimulated DNA synthesis of all the cells in monolayer culture, regardless of the number of EGF receptors. In soft agar, only MYU3L formed colonies, and EGF enhanced their growth. However, EGF did not induce the other cells to grow in soft agar. In contrast, TGF-beta 1 inhibited the growth of the cells, but a tumorigenic cell and the cells which were established from large in vivo tumors were more resistant than the others to TGF-beta 1. We tested the effect of growth factors on the invasive potential of MYP3 cells (non-tumorigenic), MYU3L cells (tumorigenic/highly invasive but not metastatic) from newly established cell lines, and another metastatic cell line, LMC19. MYP3 expressed only a trace amount of 92-kDa gelatinase (MMP-9), whereas MYU3L expressed interstitial collagenase (MMP-1) and MMP-9, and LMC19 expressed 72-kDa gelatinase (MMP-2) and MMP-9. The release of MMP-2 in LMC19 was stimulated by TGF-beta 1, but EGF had no effect on the release of any MMPs in either type of cells. These observations suggest that EGF acted as a mitogen on all the cells tested, but did not enhance the malignant phenotype. Further, the loss of responsiveness to the suppressive effect of TGF-beta 1 may be an important step toward a malignant phenotype. Some of malignant tumors may utilize TGF-beta 1 for enhancing their invasive and metastatic potential.

Transient effect of epidermal growth factor on the motility of an immortalized mammary epithelial cell line.

The effects of growth factors on epithelial cell motility and dispersion have been examined on an immortalized human mammary epithelial cell line, the 184A1 nontumorigenic cell line. Among all the molecules tested, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) were demonstrated to stimulate an increase in mammary epithelial cell motility and wound closure that was associated with a morphological transformation of the cells and was accompanied by modifications in cell-cell and cell-substrate adhesion systems. The EGF-induced increase in cell motility and monolayer wound closure occurred over a 24 hour period and was not dependent on an increase in cell number. The effect of EGF was abolished by inhibiting alpha 2 integrins with specific antibodies, indicating that part of the mechanism for the increase in cell motility and accelerated wound closure depends on alpha 2 integrin functional expression. After 72 hours of exposure to EGF, the EGF-induced alterations in cell morphology, motility and cell adhesion systems underwent a spontaneous reversion that was correlated with a 10-fold reduction in the number of EGF receptors. The ability to regulate the scattering response induced by growth factors might be an important feature distinguishing normal epithelial cells from their tumoral counterparts.

Effect of Donor Age on Epidermal Growth Factor Processing in Man

Epidermal growth factor (EGF) is a well-characterized mitogen whose effectiveness decreases with age both in vivo and in vitro. Previous studies utilizing the Hayflick aging model of early versus late-passage fibroblasts failed to demonstrate changes that might account for the unresponsiveness. In contrast, we now report striking differences in EGF receptor (EGFR) number, affinity, and rate of EGF/EGFR internalization in early-passage dermal fibroblasts derived from newborn versus young adult versus old adult donors. These data demonstrate critical differences between the two models of cellular aging, provide an explanation for the age-associated loss of EGF responsiveness, and may explain in part the tendency toward impaired wound healing in the elderly.

Epidermal growth factor stimulates substrate-selective protein-tyrosine-phosphatase activity.

This study investigates the regulation of protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) activity by epidermal growth factor (EGF). Cytosol from EGF-treated A-431 human epidermoid carcinoma cells was used as a source of PTPase activity, and tyrosine-phosphorylated ErbB2, EGF receptor, phospholipase C-gamma 1, and the Ras GTPase-activating protein were used as substrates to monitor PTPase activity. EGF stimulated PTPase activity that was selective toward these substrates, as it dephosphorylated ErbB2 and the EGF receptor, but not phospholipase C-gamma 1 and the Ras GTPase-activating protein. EGF stimulated PTPase activity in several cell lines, regardless of EGF receptor number, and the activity was localized in the cytosol. The dephosphorylation activity in vitro was dependent on the presence of reducing agents and was inhibited by orthovanadate. Agonists such as phorbol 12-myristate 13-acetate, isoproterenol, or ATP were unable to stimulate PTPase activity. Physiological relevance is indicated by experiments showing that EGF treatment of a human mammary cancer cell line rapidly induced the dephosphorylation of ErbB2.

The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.

Prolactin inhibits epidermal growth factor (EGF)-stimulated signaling events in mouse mammary epithelial cells by altering EGF receptor function.

We have previously shown that lactogenic hormones stimulate epidermal growth factor (EGF) mRNA accumulation in mouse mammary glands in vivo and in mouse mammary epithelial cells (NMuMG line). However, our in vitro studies indicate that the lactogenic hormone prolactin (PRL) completely inhibits EGF-stimulated DNA synthesis. PRL does not alter cholera toxin or insulin-like growth factor-1-stimulated cell growth, thus the inhibition appears to be specific for EGF. Our current studies are designed to evaluate the effects of PRL on EGF-stimulated signaling events in the NMuMG cell line. Cells treated with PRL for 30 min demonstrated a loss of high affinity EGF-binding ability. After long-term PRL treatment (18 h) there was a decrease in EGF receptor (R) number, as determined by [125I]EGF binding. PRL treatment (8 h) also decreased EGF-R mRNA levels. An EGF-stimulated increase in EGF-R mRNA observed 2-4 h after treatment was decreased when PRL was added to the cultures. Furthermore, levels of EGF-stimulated tyrosine phosphorylation of the EGF-R (170 kDa) and phospholipase C gamma (145 kDa) are dramatically decreased in cells treated with PRL. Also of great interest was a decrease in EGF-stimulated c-myc mRNA in PRL-treated cells. We conclude that PRL is acting to down-regulate the EGF-R, thus limiting EGF-stimulated cell signaling in mammary tissue.

CHARACTERIZATION OF CAL39, A NEW HUMAN CELL-LINE DERIVED FROM A VULVAR SQUAMOUS-CELL CARCINOMA

We have obtained a permanent cell line from a squamous cell carcinoma of the vulva. These cells, named CAL39, exhibited morphological, ultrastructural, and immunochemical characteristics of epithelial cells. They were tumorigenic in nude mice. Our observations indicate that the behavior of the cell line in the nude mouse and in culture is similar to that of the original cancer from which it was derived. We have compared these cells with A431 which are currently the best studied model cells of the same tumor origin. Like A431, CAL39 had an elevated number of high affinity EGF receptors, and showed an amplification of DNA sequences at 11q13. We have compared the cytogenetic features of the amplification units in the two cell lines. Unexpectedly, an HSR was present, in both cases, on a marker chromosome which was a derivative of chromosome 11. This result addresses the issue of the in situ amplification of chromosomal DNA.

Estrogen enhances epidermal growth factor-induced DNA synthesis in mammary epithelial cells.

Estradiol (E2) priming (1 nM for 48 h) of normal murine mammary gland epithelial cells significantly increased the response of those cells to epidermal growth factor (EGF)-induced DNA synthesis. The synergism between E2 and EGF was evident in two aspects: After serum-free synchronization for 24 h, more cells entered the S-phase of the cell cycle after E2 priming and when treated with 0.17 nM EGF (13%) than did control cells (1.3%) or cells treated with EGF (4%) or E2 (3.5%) alone; further, the dose of EGF required to elicit maximal response was reduced an order of magnitude in estrogen-primed cells (0.17 nM) compared to controls (1.7 mM). Estrogen alone, however, did not increase DNA synthesis in these cells. Ligand binding studies indicate that these effects of estrogen on proliferating mammary epithelial cells may be explained, at least in part, by a 3.7-fold increase in the number of high affinity EGF-receptors observed in estrogen primed cells (7,300 receptors per cell) compared to estrogen deprived cells (1,960 receptors/cell).