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.     

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.     

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 by EGF

Epidermal growth factor (EGF) has been noted to stimulate proliferation of a variety of normal and malignant cells including those of human breast epithelium. We report here that MDA-468, a human breast cancer cell line with a very high number of EGF receptors, is growth-inhibited at EGF concentrations that stimulate most other cells. The basis for the elevated receptor level is EGF receptor gene amplification and over-expression. An MDA-468 clone selected for resistance to EGF-induced growth inhibition shows a number of receptors within the normal range. The results are discussed in relation to a threshold model for EGF-induced growth inhibition.     

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.     

A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis.

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.     

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.     

Atypical receptor-mediated signal transduction events in the EGF-dependent growth-inhibited cell line, MDA-468.

It is now generally considered that early signalling from tyrosine kinases that induce mitogenesis is initiated through the formation of heteromeric complexes consisting of the autophosphorylated tyrosine kinase and a number of tyrosylphosphorylated proteins, including phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP). However, since much of this work has been performed on proliferative, chimeric cell lines expressing heterologous receptor molecules, we examined the nature of the epidermal growth factor receptor (EGFR) signalling complex formation in the human breast cancer cell line, MDA-468. This cell line has an amplified, native EGFR gene, correspondingly overexpresses the EGFR, and its growth in culture is inversely related to the EGF concentration. Our results indicate that in MDA-468 cells, both the EGFR and PLC-gamma are phosphorylated on tyrosine residues and can be co-immunoprecipitated. This occurs at both high and low EGF concentrations regardless of the proliferative endpoint. The molecular association is correlated with a significant increase in total inositol phosphates formed in response to the growth factor treatment. In contrast, however, there is no evidence that GAP is either phosphorylated on tyrosine residues or forms a complex with the activated EGFR in EGF-treated MDA-468 cells. These observations suggest that as a model for growth factor action, the formation of heteromeric protein signalling complexes may demonstrate considerable diversity depending upon both cell type and physiology.     

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.     

Two independent growth factor-generated signals regulate c-fos and c-myc mRNA levels in Swiss 3T3 cells

Polypeptide growth factors that stimulate cell proliferation bind to cell surface receptors and activate intracellular signal transduction pathways. One major signalling pathway, initiated by phosphatidylinositol (PI) turnover, involves activation of protein kinase C. Some polypeptide growth factors, including mitogens that activate protein kinase C, induce a rapid increase in expression of the proto-oncogenes, c-myc and c-fos. In order to characterize the signal transduction pathways responsible for proto-oncogene activation, we treated Swiss 3T3 cells with the tumor promoter phorbol dibutyrate to generate cells deficient in protein kinase C. These cells were then stimulated with platelet extract, bombesin, or epidermal growth factor (EGF) and the levels of c-myc and c-fos mRNA were determined. Platelet extract or bombesin, which stimulate PI turnover, were substantially weaker inducers of c-myc and c-fos mRNA levels in the protein kinase C-depleted cells, although some variability with platelet extract was noted. EGF, which does not stimulate PI turnover in several cell systems, was by contrast a potent inducer of both proto-oncogenes whether or not the cells were deficient in protein kinase C. Pretreatment of cells with phorbol dibutyrate caused little or no change in the basal levels of c-myc or c-fos mRNA, but led to a small but significant increase in basal levels of ornithine decarboxylase mRNA. These results demonstrate that EGF and growth factors that activate PI turnover induce expression of the c-myc and c-fos proto-oncogenes through different pathways.     

Comparative analysis of the involvement of p53, c-myc and c-fos in epidermal growth factor-mediated signal transduction

p53, a transformation-related protein located in the nucleus, shares several properties with the product of the nuclear proto-oncogene c-myc. The latter is transiently induced after different membrane-originating stimuli. A similar observation has been made with c-fos, a gene that also belongs to the 'nuclear' class of oncogenes. Here we show that p53, unlike the products of the c-myc and c-fos genes, is not induced by the signal generated by the interaction between epidermal growth factor (EGF) and its receptor. Hence, p53 does not appear to be involved in EGF signal transduction. In order to draw this conclusion we have used an EGF receptor gene-amplified human breast tumor cell line that is growth-inhibited by EGF, and exponentially growing normal human fibroblasts.     

Two independent mechanisms for escaping epidermal growth factor- mediated growth inhibition in epidermal growth factor receptor- hyperproducing human tumor cells

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.     

Suppression of the EGF-dependent induction of c-myc proto-oncogene expression by transforming growth factor beta in a human breast carcinoma cell line

Alterations in c-myc proto-oncogene expression after treatment of human mammary carcinoma MDA-468 cells with epidermal growth factor (EGF) and/or transforming growth factor beta (TGF beta) have been investigated. A stimulation of c-myc messenger RNA was detected within 60 min after treatment with EGF. This induction persisted for at least 24 hr, albeit to a lower extent. The early and late increase in c-myc mRNA levels induced by EGF were inhibited by the presence of TGF beta. TGF beta alone induced little change in c-myc mRNA levels. The effect of TGF beta represents a novel action of this hormone at the level of gene expression.     

Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells.

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.     

EGF-dependent cell cycle progression is controlled by density-dependent regulation of Akt activation

The normal human breast epithelial cell line, MCF10A, was used to investigate the mechanism by which high-density inhibits EGF-dependent cell cycle progression. EGF-dependent Akt activation was found to be transient in high-density cells and sustained in low-density cells. High-density cells also showed decreased EGF receptor (EGFR) autophosphorylation, decreased retinoblastoma protein phosphorylation, and increased p27 protein expression. Although EGFR activation was decreased in the high-density cells, the activation was sufficient to stimulate EGFR substrates comparable to low-density cells. EGF-dependent activation of the Erk1/2 pathway and the upstream activators of Akt (Gab1, erbB3, PI3 kinase, and PDK1) showed no density dependency. Antagonists of Akt activity provided further evidence that regulation of Akt activation is the critical signal transduction step controlling EGF-dependent cell cycle progression. Both adenovirus-mediated expression of dominant-negative Akt and inhibition of PI3 kinase-mediated Akt activation with LY294002 blocked cell cycle progression of low-density cells. In summary, we report the novel finding that high-density blocks EGF-dependent cell cycle progression by inhibiting EGF signaling at the level of EGF-dependent Akt activation rather than at the level of EGFR activation.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.