Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells

The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and -independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and -independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibodies were acting to block access of an endogenously produced ligand to the EGF receptor. In the presence of antibody and increasing concentrations of EGF, MDA-468 cell growth was first stimulated then inhibited as the EGF concentration increased, thus, uncovering the growth stimulatory potential of low concentrations of EGF in these cells. Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities. The growth of MDA-231 and Hs578T cells, which contain activated ras oncogenes, was not inhibited by the antibodies and the growth of these cell lines was not stimulated by EGF. Of the cell lines studied only MDA-468 cells appear to possess an autocrine growth stimulatory capacity.     

Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants.

We have recently reported (J. Filmus, M. N. Pollak, R. Cailleau, and R. N. Buick, Biochem. Biophys. Res. Commun. 128:898-905, 1985) that MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited in vitro pharmacological doses of EGF. We have derived several MDA-468 clonal variants which are resistant to EGF-induced growth inhibition. These clones had a number of EGF receptors, similar to normal human fibroblasts, and had lost the EGF receptor gene amplification. Karyotype analysis showed that MDA-468 cells had an abnormally banded region (ABR) in chromosome 7p which was not present in the variants. It was shown by in situ hybridization that the amplified EGF receptor sequences were located in that chromosome, 7pABR. Five of the six variants studied were able to generate tumors in nude mice, but their growth rate was significantly lower than that of tumors derived from the parental cell line. The variant that was unable to produce tumors was found to be uniquely dependent on EGF for growth in soft agar.     

EGF-dependent growth inhibition in MDA-468 human breast cancer cells is characterized by late G1 arrest and altered gene expression.

The MDA-468 human breast cancer cell line displays the unusual phenomenon of growth inhibition in response to pharmacological concentrations of EGF. This study was initiated with the objective of elucidating the cellular mechanisms involved in EGF-induced growth inhibition. Following EGF treatment the percentage of MDA-468 cells in G1 phase increased, together with a concomitant depletion in S and G2/M phase populations, as revealed by flow cytometry of DNA content. The apparent G1 block in the cell cycle was confirmed by treating the cells with vinblastine. DNA synthesis was reduced to about 35% of that measured in control, untreated cells after 48 h of EGF treatment, as measured by the incorporation of [3H]thymidine. DNA synthesis returned to normal following the removal of EGF from the growth-arrested cells. In order to locate the EGF-induced event responsible for the G1 arrest more precisely, we examined the expression of certain cell cycle-dependent genes by Northern blot analysis. EGF treatment did not alter either the induction of the early G1 marker, c-myc, or the expression of the late G1 markers, proliferating cell nuclear antigen, and thymidine kinase. However, EGF-treated cells revealed down regulation of p53 and histone 3.2 expression, which are expressed at the G1/S boundary and in S phase, respectively. These results indicate that EGF-induced growth inhibition in MDA-468 human breast cancer cells is characterized by a reversible cell cycle block at the G1/S boundary.     

EGF effects on p53 in MDA-468 human breast cancer cells: implications for G1 arrest

EGF, in pharmacological concentrations, inhibits cell proliferation of the MDA-468 human breast cancer cell line. Previously, we have demonstrated that this was characterized by a reversible cell cycle arrest at the G₁-S boundary, concomitant with downregulation of mRNA levels for p53 (a point mutant, p53^273.His). Since p53^273.His is regarded as a gain-of-function mutant and acts to enhance cell proliferation, we hypothesized that the G₁ arrest induced by EGF might be mediated by p53^273.His. In this study, we report an EGF-dependent altered conformation as indicated by immunofluorescence, while no significant immediate effects of EGF-treatment on p53^273.His protein levels and synthesis were observed. These experiments demonstrated a decreased PAb 240 (mutant-specific) reactivity of nuclear p53^273.His in EGF-treated cells, while that of PAb 1620 (wild-type specific) was enhanced. Staining with PAb 1801 (pan specific), on the other hand, showed little change upon EGF treatment. Further studies indicated a decreased phosphorylation of nuclear p53²⁷³. His in EGF-treated cells. These EGF-dependent events were detected early enough to be attributed as causative of cell cycle arrest. We suggest that EGF-mediated, phosphorylation-dependent conformational change in nuclear p53^273.His, and in turn altered p53 function, may be responsible for EGF-dependent growth inhibition MDA-468 cells.     

Expression and effects of epidermal growth factor on human periodontal ligament cells

Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1β were found to be upregulated in a perilesional pattern. Interleukin-1β induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue.     

Reduced growth rate accompanied by aberrant epidermal growth factor signaling in drug resistant human breast cancer cells

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.     

A novel calcium signalling response in the breast cancer cell line MDA-468

In the human breast carcinoma cell line MDA-468 addition of epidermal growth factor (EGF) is growth inhibitory. Calcium signalling was investigated in this cell line using the calcium sensitive fluorescent probe Indo-1. Addition of EGF to MDA-468 cells resulted in a novel biphasic calcium response. In the first phase of the response EGF raised calcium to levels significantly above basal. This was followed by a prolonged fall in calcium to levels significantly lower than original basal levels. The G-protein activator aluminum fluoride (AlF), stimulated a rise in calcium which was not proceeded by a fall below basal levels. Conversely addition of PMA, an activator of protein kinase C (PKC), induced a fall in calcium from basal without a prior increase. Down regulation of PKC eliminated the response to PMA, however the biphasic nature of the EGF response was maintained. Pretreatment of the cells with pertussis toxin did not alter the response to EGF nor to AlF. We conclude that in the MDA-468 cell in which EGF is growth inhibitory: 1) EGF results in a biphasic calcium response which ultimately leads to reduction below baseline levels, 2) a rise in calcium itself is not sufficient to account for the subsequent fall below basal levels, 3) G-proteins may be involved in the initial phase of the EGF response, 4) activation of PKC can also reduce intracellular calcium, however the response to EGF is not dependent on this pathway.     

Colocalization of somatostatin receptors and epidermal growth factor receptors in breast cancer cells

Background  

Somatostatin receptor (SSTR) expression is positively correlated with tumor size and inversely correlated with epidermal growth factor receptor (ErbB) levels and tumor differentiation. In the present study, we compared SSTR1-5 and ErbB1-4 mRNA and protein expression in two breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ERα-).     

Inhibition of epidermal growth factor receptor by ferulic acid and 4-vinylguaiacol in human breast cancer cells

Objectives

To examine the potential of ferulic acid and 4-vinylguaiacol for inhibiting epidermal growth factor receptor (EGFR) in human breast cancer cells in vitro.

Results

Ferulic acid and 4-vinylguaiacol limit the EGF (epidermal growth factor)-induced breast cancer proliferation and new DNA synthesis. Western blot analysis revealed both ferulic acid and 4-vinylguaiacol exhibit sustained inhibition of EGFR activation through down-regulation of Tyr 1068 autophosphorylation. Molecular docking analysis shows ferulic acid forming hydrogen bond interaction with Lys 745 and Met 793 whereas, 4-vinylguaiacol forms two hydrogen bonds with Phe 856 and exhibits stronger hydrophobic interactions with multiple amino acid residues at the EGFR kinase domain.

Conclusions

Ferulic acid and 4-vinylguaiacol could serve as a potential structure for the development of new small molecule therapeutics against EGFR.

     

Receptors for epidermal growth factor in breast cancer

Epidermal growth factor receptor (EGFr) and cytosolic (cER) and nuclear (nER) estradiol receptors were quantified in 220 primary breast cancers. The EGFr was significantly more frequent (X2 = 5.9; P less than 0.025) and its concentration was significantly higher (P less than 0.001) among ER- tumors than in ER+ tumors. There was a significantly greater proportion (X2 = 6.4; P less than 0.05) of node involvement in EGFr+/ER+ tumors than in EFGr/ER+. Increases in the proportion of EGFr+ in ER- tumors are parallel to Scarff-Bloom scores (X2 = 6.1; P less than 0.05) and there is a significant trend towards increased EGFr concentrations with histologic dedifferentiation. In ER+ tumors the median concentrations of EGFr in the different age groups show linear correlation and follow a parallel profile with the medians of nER. These findings support the hypothesis that considers EGFr as a bad prognosis factor and suggest that EGFr expression and concentration in ER+ tumors might be considered an estrogenic action mediated through the binding of ER to their nuclear acceptors.     

Imaging epidermal growth factor receptor phosphorylation in human colorectal cancer cells and human tissues

In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.     

Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.     

Uptake and metabolism of epidermal growth factor in the perfused human placenta

Uptake of 125I-labelled epidermal growth factor into trophoblast, and its subsequent fate, was studied in an isolated dually-perfused lobule of term human placenta. 125I-EGF added into the maternal circulation was rapidly taken up into the placental tissue where a portion was degraded and most of the breakdown products released back into the maternal circuit. At the end of the 2 h perfusion, radioactivity in the tissue accounted for 52% of the initial dose. 12.9% of the radioactivity remaining in the maternal circuit at the end of the perfusion, amounting to only 5.2% of the initial activity, could be identified as intact EGF by immunoaffinity chromatography. About 45 min after the start of the perfusion there was a sustained rise in the 125I activity in the fetal circulation accounting for 4.6% of the initial activity, and a small proportion of this (0.22% of the dose) could be immunologically characterised as EGF. In the presence of the acidotrophic agent chloroquine, there was a similar rapid clearance from the maternal circulation, which was not associated with breakdown. The tissue retention was slightly enhanced and there was very little transfer of activity into the fetal circulation.     

Effects of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on metabolism of the epidermal growth factor receptor in normal human fibroblasts.

The biosynthesis, phosphorylation, and degradation of the epidermal growth factor (EGF) receptor were examined in normal human fibroblasts. The receptor was initially synthesized as an Mr = 160,000 immature form which matured to an Mr = 170,000 form in a monensin-sensitive manner. Tunicamycin treatment led to the accumulation of an Mr = 130,000 protein. The receptor was phosphorylated on serine and threonine residues in normally growing and quiescent cells, and treatment with EGF or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a two- to threefold increase in receptor-bound phosphate. EGF increased the amount of phosphoserine and phosphothreonine and caused the appearance of a minor amount of phosphotyrosine. TPA increased the levels of phosphoserine and phosphothreonine exclusively. Prior treatment with TPA inhibited the EGF-dependent appearance of phosphotyrosine in the receptor. Analysis of tryptic phosphopeptides revealed that six of the seven major peptides were common to the receptor from cells treated with EGF or TPA. EGF strongly stimulated [3H]thymidine incorporation in confluent cells, increased final saturation density three to fourfold, and increased whole-cell levels of phosphotyrosine about threefold. Treatment of cells with TPA before addition of EGF inhibited all three of these EGF-dependent responses. EGF also decreased the receptor half-life from 15 h to 1 h, but this was not inhibited by TPA. TPA alone had no detectable effect on the receptor half-life.     

Partial purification and characterization of epidermal growth factor in human breast milk

Human epidermal growth factor (hEGF), a potent growth stimulator of many tissues in culture, has been isolated from human urine and subsequently identified in many human biological fluids including breast milk. In this study, partial purification and characterization of hEGF-like substance(s) in human milk were performed using homologous hEGF radioimmunoassay (RIA) and radioreceptor assay (RRA). hEGF-like material(s) was extracted from pooled human milk by ethanol precipitation, followed by adsorption to cation- and anion-exchange resin. DEAE-Sephadex G-25 ion-exchange chromatography of human milk extracts revealed three major components with hEGF activity (peak I, II, III) eluted with a linear gradient by ammonium acetate. The competitive binding curves for these components were parallel to those for standard hEGF in both RIA and RRA. The apparent molecular weight of peak I was approximately 6,500 and that of peak II and III was approximately 7,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The pI value for peak I was approximately 4.5 and that for peaks II and III was approximately 5.0 by isoelectric focusing. These data are comparable to the size and charge heterogeneity of hEGF in human urine extracts. In conclusion, the major components of hEGF in human milk appear to be physicochemically, immunologically and biologically (receptor binding activity) indistinguishable from hEGF of urinary origin.     

Characterization of insulin-like growth factor binding proteins from human breast cancer cells

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)