Antisense inhibition of amphiregulin expression reduces EGFR phosphorylation in transformed human breast epithelial cells.

The activation of epidermal growth factor receptor (EGFR) by its ligands constitutes an important step in the metastatic process but the clinical response to its inhibition in breast cancer patients has so far been very low. In this work, we investigated the role of the EGFR ligand amphiregulin (AR) in modulating EGFR activation. For this, transformed epithelial mammary tumor cells NS2T2A1 were used in which AR or EGFR expression was down-regulated by antisense cDNA technique. This down-regulation was associated with a significant inhibition of matrix metalloproteinase-9 production as well as cell proliferation, but this inhibition was only minimally reversed by exogenously added AR or EGF. EGFR protein levels were not affected but EGFR-tyrosine phosphorylation in response to EGF was markedly reduced. Thus, the inhibition of AR expression, which impairs EGFR response to its exogenously available ligands, may represent an alternative anti-EGFR therapeutic strategy in breast cancer.

Determination of TGFbeta1 protein level in human primary breast cancers and its relationship with survival.

Transforming growth factor-beta (TGFbeta)1 is thought to be implicated in breast cancer progression. However, data about the influence of TGFbeta1 on breast cancer development are conflicting. To clarify the clinical relevance of TGFbeta1, TGFbeta1 protein level has been measured by enzyme-immunoassay in 193 breast tumour samples. We found that 94.3% of patients expressed TGFbeta1 with a range of 0-684 pg mg(-1) protein. In the overall population, an increase of tumoral TGFbeta1 was observed in premenopausal patients when compared to postmenopausal subgroup (P=0.0006). When patients were subdivided according to nodal status, TGFbeta1 was correlated to type-1 plasminogen activator inhibitor in the node-negative subgroup (P=0.040). Multivariate analysis revealed that, after lymph node status (P=0.0002) and urokinase-type plasminogen activator (P=0.004), TGFbeta1 was an independent prognostic marker for DFS (P=0.005) in the overall population. In the node-negative population, TGFbeta1 was the prominent prognostic factor (P=0.010). In the same population, Kaplan-Meier curves demonstrated that high TGFbeta1 level was correlated with a shorter disease-free survival (P=0.020). These data suggest that the measurement of tumoral TGFbeta1 protein level, especially for node-negative patients, might help to identify a high-risk population early in tumour progression.

SR31747A is a sigma receptor ligand exhibiting antitumoural activity both in vitro and in vivo.

SR31747A is a recently described sigma receptor ligand that binds SR31747A-binding protein 1 (SR-BP) and emopamil-binding protein (EBP) (also called the sigma 1 receptor and the human sterol isomerase (HSI), respectively), and has immunoregulatory and antiproliferative activities. To further investigate its antitumour activity and focusing on cancers, which are sensitive to the molecule, we measured the proliferation of different human epithelial breast or prostate cancer cell lines following in vitro and in vivo SR31747A treatment. Firstly, in vitro, we found that nanomolar concentrations of SR31747A dramatically inhibited cell proliferation in both hormono-responsive and -unresponsive cancer cell lines. Secondly, tumour development was significantly decreased in mice treated with SR31747A. In an attempt to decipher the SR31747A mode of action, we found that the two binding sites may not fully account for this activity. Indeed, while competitive experiments indicated that EBP prevails in mediating SR31747A antiproliferative activity, an analysis of the expression of both receptors indicated that the cellular sensitivity to SR31747A is not correlated with either EBP or SR-BP expression. These data suggest that additional binding sites may exist. Preliminary binding studies demonstrated that SR31747A also binds to sigma 2, a protein that has not yet been cloned, but which is considered as a potential marker of the proliferative status of tumour cells. Altogether, our data demonstrate the antitumoural activity of SR31747A both in vitro and in vivo in two different cancer models, broaden the spectrum of its binding proteins and enhance the potential for further therapeutic development of the molecule.

Differential regulation of cell proliferation and protease secretion by epidermal growth factor and amphiregulin in tumoral versus normal breast epithelial cells.

Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dose-dependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10(-8)M EGF stimulated AR expression in HMECs to 140-300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5-10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system.

Morphological evidence for a subpopulation selection effect by estrogen and antiestrogen treatments in the heterogeneous MCF-7 cell line.

Recently, we developed a method to quantitatively study tumour cell heterogeneity in terms of both nuclear size and estrogen receptor (ER) content by image cytometry. The method, previously used to analyse the proliferation of the breast cancer cell line MCF-7, was applied here to analyse the growth of this cell line under estradiol (E2), hydroxytamoxifen (OH-TAM), and both E2 and OH-TAM treatments. The method extracts characteristic parameters of single nuclei and features that measure the global and local organisation of the cells in their growing phase. Modifications of the heterogeneity of the cell line are emphasised through phenotypic changes and modifications of the spatial organisation of the cells. The hormone (E2) generates a very fast growth of cells with small nuclei that became ER negative in the long term. The antihormone (OH-TAM) produces a gradual selection of ER negative or poorly positive cells with large nuclei. These modifications are reversed when E2 and OH-TAM are simultaneously used. Moreover, estradiol induces a permissive context of proliferation, whereas hydroxytamoxifen acts only on some subpopulations. The combination of cell count, cytomorphology, and cell organisation revealed the magnitude of the potential of structuration of hormones or antihormones on in vitro growing cells.

Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line.

The epidermal growth factor (EFG) family of receptors and their respective ligands play a major role in breast cancer progression and are the targets of new therapeutic approaches. Following immortalization with SV40 T antigen of normal human breast epithelial cells, a transformed variant cell line (NS2T2A1) was selected for its increased tumorigenicity in nude mice. This cell line was shown to have a higher expression of EGF receptors (EGFR) and amphiregulin (AR) when compared to their normal counterparts or less aggressive transformed cells. Dual staining of EGFR and AR was observed in 50-60% of NS2T2A1 cells, while 30-40% cells expressed AR only. To explore the potential tumorigenic role of AR, a 1.1 kb AR cDNA in an antisense orientation was transfected in NS2T2A1 cells. Three clones, selected by hygromycin B, expressed AR antisense RNA (AR AS1, AR AS2 and AR AS3 cell lines) in which AR protein expression was reduced (ranging from about 50 to < 5%). The anchorage-independent growth of AR AS cell lines was reduced to levels ranging from 32.4-6.8% relative to the control cell line transfected with the vector alone. The clones expressing AR antisense RNA showed a reversion of the malignant phenotype when injected in nude mice, since a significant reduction of tumor intake was observed coincident with a significant tumor mass reduction (> 96%). Moreover, intra-tumoral vascularization decreased significantly in tumors derived from AR AS cells (26.7, 70.7 and 50.4% of control). These in vitro and in vivo data reveal the oncogenic nature of AR in transformed breast epithelial cells and imply a role for AR in tumor angiogenesis.

Differential regulation of normal and tumoral breast epithelial cell growth by fibroblasts and 1,25-dihydroxyvitamin D3.

Mesenchymal-epithelial interactions are of paramount importance during normal and tumoral breast developments. We have investigated the paracrine growth regulation of normal and tumoral breast epithelial cells by fibroblasts derived from normal or pathological breast tissues. In some cases, breast cancer MCF-7 cells or normal epithelial cells in primary culture were cocultured with fibroblasts in a Transwell system allowing diffusible factor exchanges. Alternatively, conditioned medium produced by fibroblast cultures was added to epithelial cell cultures. Fibroblasts were shown to stimulate the proliferation of normal and carcinoma cells through paracrine mechanisms. However, the paracrine exchanges appeared to be different in normal versus tumoral breast epithelial cell growth regulation. Moreover, vitamin D-related compounds that have been proposed as anti-tumoral drugs were studied for their ability to affect normal and tumoral mammary epithelial cell proliferation and to interfere with the growth-regulatory activity of fibroblasts. Whereas vitamin D compounds inhibited MCF-7 cell growth, they led to a marked stimulation of the proliferation of normal mammary epithelial cells. Moreover, it was shown that the vitamin D analog EB 1089 can block the mitogenic effect of fibroblast-conditioned medium on tumoral but not normal breast epithelial cells. The differential effects of vitamin D compounds on cell proliferation provide further data in favor of the different behaviours of normal and tumoral mammary epithelial cells. The potential therapeutic use of vitamin D derivatives in the treatment of breast cancer is supported by these results but their growth-stimulatory properties on normal epithelial cells cannot be overlooked.

Role of epidermal-growth-factor receptor in tumor progression in transformed human mammary epithelial cells.

The presence of epidermal-growth-factor receptors (EGFR) and of its ligands (TGFalpha and amphiregulin) in breast-cancer tissues suggests that they play a paracrine/autocrine role in tumor growth or progression. This hypothesis was tested on 3 cell lines, S2T2, NS2T2A and NS2T2A1. These epithelial cells are derived from a normal human breast-epithelial-cell culture transformed by SV40-T Ag, are of the same clonal origin, have respectively increasing levels of EGFR, TGFalpha, amphiregulin and of thymidine-kinase activity associated with increasing tumorigenic potential in nude mice (tumor intake and tumor volume). The monoclonal antibody MAb 425, which blocks ligands interaction with EGFR, reduced by more than 90% anchorage-independent growth of the most tumorigenic cells, NS2T2A1. Another anti-EGFR MAb, 528, reduced to 25% of controls the mean tumor mass after NS2T2A1 grafting in mice. Anti-sense RNA expression of EGFR in these cells confirmed the importance of this receptor in tumor progression, since it reduced significantly the tumor volume and tumor weight of NS2T2A1 cells to 16% of those in mock-transfected control cells.

Differential dose-dependent effects of epidermal growth factor on gene expression in A431 cells: evidence for a signal transduction pathway that can bypass Raf-1 activation.

Epidermal growth factor (EGF), which plays an important role in normal and tumoral cell growth regulation, displays an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. However, the underlying molecular mechanisms remain obscure. In this study we have examined the regulation of amphiregulin (AR) gene expression by growth inhibitory (10(-9) M) and stimulatory (10(-12) M) EGF concentrations in A431 cells. The time course of AR messenger RNA (mRNA) accumulation was different with 10(-12) and 10(-9) M EGF; AR induction by 10(-9) M EGF peaked between 1 and 1.5 h, then decreased to the basal level within 2 h. Conversely, the induction by 10(-12) M EGF was slightly delayed, but persisted for 4 h. The involvement of tyrosine phosphorylation in AR induction by EGF was suggested by the ability of the tyrosine phosphatase inhibitor sodium orthovanadate to prolong AR expression induced by 10(-12) or 10(-9) M EGF. In the presence of the protein phosphatase 2A inhibitor, okadaic acid, 10(-9) M EGF induced a persistent accumulation of AR mRNA. On the contrary, okadaic acid abrogated the stimulation of AR mRNA level induced by a low EGF concentration, suggesting that both EGF concentrations activated distinct regulatory mechanisms. The signaling components involved in the differential activities of EGF in A431 cells were then examined. We previously reported a relationship between the ambivalent activity of EGF and the p42-mitogen-activated protein (MAP) kinase activity. Thus, 10(-12) M EGF induced a sustained MAP kinase activation, whereas 10(-9) M EGF led to a sharp, but transitory, activation. The MAP kinases are activated by MAP kinase kinases (MEK1 and MEK2). Whereas no significant effect of 10(-12) M EGF could be detected, 10(-9) M EGF was shown to activate MEK1 and, to a lesser extent, MEK2. Also, both MAP kinase activation and AR induction by 10(-9) M, but not by 10(-12) M, EGF were inhibited by the MEK1 inhibitor PD98059. Moreover, the involvement of c-Raf-1 in the signaling pathway induced by EGF was verified. A concentration of 10(-9) M EGF induced stimulation of c-Raf-1 kinase activity, whereas 10(-12) M EGF not only failed to activate c-Raf-1, but led to a moderate decrease in its kinase activity. These results demonstrate that in EGF receptor-overexpressing cells, EGF may differently affect gene expression and cell proliferation through distinct mechanisms of regulation.

Positive regulation of normal and tumoral mammary epithelial cell proliferation by fibroblasts in coculture.

In the mammary gland, mesenchymal-epithelial interactions are of paramount importance during normal and tumoral developments. We have studied the paracrine growth regulation of a variety of breast epithelial cells in coculture with normal or pathological breast fibroblasts. Two models of coculture were used in which the two cell types were seeded and grown, either together in microchamber slides or separated by a microporous membrane. Under these two conditions, all fibroblasts were shown to stimulate the proliferation of the hormono-responsive breast carcinoma MCF-7 cell line, suggesting that cell contacts were not indispensable for the paracrine stimulation of MCF-7 cell growth by fibroblasts. Moreover, in the Transwell coculture system, the proliferation of a variety of other breast carcinoma cells (MDA-MB231, T47D, and BT-20) was also stimulated by fibroblasts. However, the amplitude of the proliferative response seemed to be dependent on the carcinoma cell line considered. Moreover, the proliferative response of normal mammary epithelial cells to the presence of fibroblasts was shown to be significantly higher than the tumor cell response. The nature of the tissue of fibroblast origin, normal or pathological, did not influence the growth response of the epithelial cells. In this study, we thus demonstrate that fibroblasts are able to stimulate the proliferation of normal and carcinoma cells through paracrine exchange mechanisms. We also conclude that the target epithelial cell phenotype will essentially determine the extent of the proliferative response.

A minimal model for calcium signal generated by tyrosine kinase and G protein linked receptors; a stochastic computer simulation with CALSIM.

A software was designed to simulate the calcium signal following hormone or growth factor stimulation in epithelial cells. The software written in C runs on a PC under Windows environment. It is based on a Markov process where the dynamic of the system is characterised by phenomenological transition probabilities. Moreover a minimal model is proposed to analyse the role of plasma channels and IP3 receptors, together with the opposite action of the CaATPase pumps, in the cytosolic and endoplasmic reticulum (ER) calcium signal control. The simulation is applied on the calcium response following stimulation by carbacol (protein G coupled receptors) or epidermal growth factor (tyrosine kinase type receptors) in A431 epithelial cells. The experimental calcium signals can be grouped in three classes; a spike and a return to the basal level (signal A), a spike and a decrease to a plateau level (signal B) or a slow increase to a plateau (signal C). Epidermal growth factor induces signal A and B while carbacol gives signal B and C. When a 'pseudo' steady state is reached oscillations occur. Computer simulations show that signal A can result from the activation of IP3 receptors while signal C would result from the activation of the plasma channels; signal B appears as the additive contribution of both channels, while oscillations are compatible with a calcium induced calcium release mechanism. Simulations suggest that the calcium dynamic in the ER is a mirror of cytosolic calcium but that a simple way to produce similar calcium elevation in these two compartments is to activate plasma channels. Implications of such a mechanism is discussed.

[Contribution of the topographical analysis to quantitative microscopy in cultured cell systems: application to the evaluation of hormone therapy]. / Apport de l'analyse topographique à la microscopie quantitative dans l'étude de systèmes cellulaires en culture: application à l'évaluation des traitements hormonaux.

Quantitative microscopy by image analysis allows not only to measure various parameters on each cell but also to consider the global population as a whole. In the hypothesis that cell position is reflecting the relational and dynamical structure of the system, spatial arrangement analysis may help to show up intercellular communication (interactions and control systems via contact or diffusible factors). We describe a topographical analysis method used to study these neighbour relationships, and thus the sociological behaviour of the cells. It is applied to the study of the effect of estrogenic and antiestrogenic treatments on a breast cancer cell line (MCF-7). It shows up that estrogens increase proliferation and induce an unusual topographical behaviour, notably in cell cycle phases: cells in S phases are very randomly distributed. It points out the role of estrogens on the cells neighbour relationships inducing the way to a permissive proliferation context. This effect is reversed by antiestrogenic treatment after a few days. Antiestrogenic treatment alone increases the proliferation constraint.

Effect of stromal and epithelial cells derived from normal and tumorous breast tissue on the proliferation of human breast cancer cell lines in co-culture.

Stromal and epithelial components surrounding neoplastic cells are believed to be important in tumor regulation. We have studied the effects of stromal and epithelial cells on the proliferation of a variety of breast-cancer epithelial cell lines. Co-culture experiments were performed in which the 2 cell types were separated by a microporous membrane. Under these conditions, fibroblasts from normal breast tissues inhibited the proliferation of MCF-7 cells, but not that of immortalized normal S2T2 cells. In contrast, fibroblasts from cancerous breast tissues did not influence the proliferation of the 2 cell lines tested. Conditioned media (CM) of breast fibroblasts derived from normal tissues were not able to affect MCF-7 cell growth, suggesting complex paracrine interactions between both cell types. Normal breast epithelial cells (NBEC) have also been tested for their ability to regulate the proliferation of breast-cancer epithelial cell lines. Co-culture experiments demonstrated that NBEC inhibited a variety of breast-cancer cell lines. CM from NBEC induced similar results and the inhibitory effect appeared to be specific for epithelial cells from tumorous breast. Moreover, CM from NBEC and normal fibroblasts were shown to contain more TGF beta 1 and amphiregulin than those of MCF-7 cells. We conclude that both the tissue origin and the target tumor cell's phenotype will determine the extent of proliferative response. More important, the tumor-cell growth inhibition induced by fibroblasts and epithelial cells of normal breast tissue may constitute a tumor-growth-regulatory mechanism.

Distribution of estrogen receptor heterogeneity in growing MCF-7 cells measured by quantitative microscopy.

The existence of interactive subpopulations is a biological feature that can modulate the proliferation of tumor cells. The hormone-responsive breast cancer cell line MCF-7 has been described as heterogeneous in terms of density. In this study we describe a quantitative image analysis methodology that we developed for the in situ detection of different subpopulations in MCF-7 cell cultures. Using this technology, we demonstrate the heterogeneity of the MCF-7 cell line in terms of both nuclear size and estrogen-receptor content. Analysis of the organization (topography) of the different subpopulations in culture reveals a nonrandom distribution of cells. When studying the development of these cell subpopulations as a function of time of culture, we observe modifications of their topography associated with an increase of estrogen-receptor-expressing cells. Moreover, the use of cluster analysis allows study of the local organization of these subpopulations. These changes appear to be independent of cell proliferation.

Regulation of p42 mitogen-activated-protein kinase activity by protein phosphatase 2A under conditions of growth inhibition by epidermal growth factor in A431 cells.

Epidermal growth factor (EGF), which plays an important role in the growth regulation of a large variety of normal and tumor cells, has been shown to display an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. In a previous study aimed at dissecting the biochemical events leading to this dual action in A431 cells which over express EGF receptor, we have reported a relationship between the dual stimulator/inhibitor effect of EGF and the activity of the serine/threonine p42 mitogen-activated protein (MAP) kinase. Indeed, a growth stimulatory concentration of EGF is shown to lead to a moderate but persistent activation of p42 MAP kinase. Conversely, an early peak of MAP kinase activation, that rapidly falls below the basal level, is observed in the presence of a growth-inhibitory concentration of EGF. To assess the mechanism of the p42 MAP kinase inactivation under circumstances of negative growth regulation by EGF, we have investigated the role of the serine/threonine phosphatase 2A in this process. A constitutive phosphatase 2A activity was observed in untreated cells, that decreases rapidly in response to both high and low EGF concentrations. However, after this early inactivation, the phosphatase 2A activity was completely reversed concurrently with MAP kinase inactivation, after 40 min of treatment with 10 nM EGF. Conversely, in cells treated with 1 pM EGF, phosphatase 2A activity remained below the control level during all the time of the treatment, in association with a sustained MAP kinase activation. These results suggest that MAP kinase inactivation is closely related to phosphatase 2A activation. We then investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on the MAP kinase inactivation and observed that okadaic acid, at a concentration reported to specifically inhibit phosphatase 2A activity, totally reverses the MAP kinase inactivation induced by long-term treatment with 10 nM EGF. Additionally, we have shown that the protein synthesis inhibitor cycloheximide fails to affect the EGF-induced MAP kinase regulation, indicating that mitogen-induced protein phosphatases are not, or are only slightly, required in this regulation. In conclusion, our data demonstrate that the ambivalent action of EGF on the proliferation of A431 cells is associated with differential mechanisms of p42 MAP kinase regulation catalysed by the serine/threonine phosphatase 2A.

Relationship between the MAP kinase activity and the dual effect of EGF on A431 cell proliferation.

EGF is involved in the regulation of cell proliferation in normal as well as in neoplastic tissues. The A431 cells that over-express EGFR, display in vitro ambivalent growth properties in response to EGF, since stimulation induced by low concentrations (10(-12) M-10(-10) M) is reversed with increasing concentrations (10(-9) M-10(-8) M). To assess differential mechanisms of signal transduction that determine growth stimulatory and inhibitory activity, we have studied the MAP kinase activation induced by mitotic and antimitotic concentrations of EGF. We demonstrate that the presence of a growth stimulatory concentration of EGF (10(-12) M) leads to a moderate but persistent activation of p42 MAP kinase during all the time of the EGF treatment. Conversely, an early peak of kinase activation that rapidly falls down below the basal level, is observed when a growth inhibitory concentration of EGF (10(-8) M) is used. Moreover, the addition of Na-orthovanadate in 10(-8) M EGF-treated cells leads to the rescue of the MAP kinase activity, suggesting that the loss of kinase activity induced by growth inhibitory EGF concentrations involves the dephosphorylation of the MAP kinase. In conclusion, our data demonstrate that the dual effect (stimulator/inhibitor) of EGF on the proliferation of A431 cells is associated to differential mechanisms of p42 MAP kinase regulation.

Agonist-antagonist activity of anti-estrogens in the human breast cancer cell line MCF-7: an hypothesis for the interaction with a site distinct from the estrogen binding site.

Non-steroidal anti-estrogens exhibit an extremely complex pharmacology because of their estrogenic and anti-estrogenic effects in different species. Recently, we have reported evidence for an immunochemical difference in the estrogen receptor (ER) when it is occupied with anti-estrogens as compared to estrogens (Martin et al., 1988). In this study, we have compared immunoreactivity of MCF-7 cell estrogen receptor when bound to anti-estrogen versus estrogen. We show that the occupation of ER with antiproliferative concentrations of various anti-estrogens leads to the appearance of additional antigenic determinants for the H222 monoclonal anti-estrogen receptor antibody. When performing ER immunoassay after sedimentation of estrogen receptors on sucrose gradients, we show that exposure of new epitopes induced by anti-estrogens can occur on a 4 s molecular form related to the 66 kDa monomeric estrogen receptor. Also, when ER are previously occupied by estradiol, the addition of low anti-estrogen concentrations, which are unable to displace estradiol from the estrogen receptor, leads to a significant increase of H222 epitopes. Our results led us to propose a molecular model for anti-estrogen-receptor interaction in which their dual agonist/antagonist activity may be due to the occupation of distinct binding sites on the estrogen receptor.

Mode of EGF action on cell cycle kinetics in human breast cancer cell line MCF-7: some evidence that EGF acts as a "progression factor".

EGF is known to play a very important role in the growth regulation of tumor cells. We have determined the effect of EGF in the absence and in the presence of serum on the cell cycle of MCF-7 cells synchronized in the G1 phase by serum deprivation. In the presence of 1% serum, EGF was found to increase DNA synthesis to 120% of control (P < 0.02), but did not modify the transition time from G1 into S phases, nor the cell doubling time during the first generation following the cell synchronization. The autoradiography analysis of 3H-thymidine labeled cells indicated that, following 24 h of EGF treatment, a constant additional number of cells (11 +/- 1.5%, P < 0.002) were recruited into the S phase in the presence as well as in the absence of serum. These data indicate that EGF exerts its mitogenic effect on MCF-7 cells by increasing the percent of S phase cells without modulating the cell doubling time. However, in the absence of serum a significant increase of thymidine incorporation in whole cells required 12 h of EGF treatment, whereas a 6 h-incubation with EGF was sufficient to stimulate DNA synthesis when synchronized cells were pretreated with serum for 6 h, suggesting that EGF sensitivity is dependent on the cell advance into the G1 phase at the moment of EGF addition. Topographical analysis of 3H-thymidine-labeled cells aimed at determining the spatial distribution of cells in culture revealed that EGF-stimulated cells were disposed near proliferative cells, indicating the local influence on cell proliferation. Taken together, our results suggest that in the MCF-7 cell line, EGF acts in the G1 phase by increasing the proportion of S cells without affecting the duration of the cell cycle. In our model, EGF seems to act as a "progression factor", in that it stimulates only cells already traversing a certain stage in the G1 phase under the action of serum factors, cell secreted diffusible products and cell-cell contact.

Cell cycle phase dependence of estrogen and epidermal growth factor (EGF) receptor expression in MCF-7 cells: implications in antiestrogen and EGF cell responsiveness.

In this report the effect of epidermal growth factor (EGF) and the antiestrogen hydroxytamoxifen (OH-TAM) on the cell cycle of the breast cancer cell line MCF-7 was investigated as a function of the presence of their respective receptors. For this study synchronized cells were obtained by cell incubation in the presence of 2 mM thymidine for 24 h at 37 C. The treatment led to a partial synchronization, since at the end of thymidine treatment, 80% of cells were accumulated in the G1 phase. The removal of thymidine allowed the cells to progress through the cell cycle, since between 6-9 h after the arrest of the treatment, about 50% of cells were found in the S phase. By 9-12 h, most of the cells entered the G2 phase, and by 24 h, the cells returned to the G1 phase. When MCF-7 cells were incubated in the presence of OH-TAM for various periods of time before thymidine exposure, the progression of the cells through the cell cycle was dramatically inhibited. Also, a short term antiestrogen treatment (2 h) before or immediately after the addition of thymidine led to an accumulation of MCF-7 cells in the G1 phase. However, when the cells were treated for 2 h with OH-TAM 22 h after thymidine addition or shortly after its removal from the cell culture, no effect of the antiestrogen on the cell cycle could be observed. In parallel, the effect of thymidine on the level of estrogen receptor was studied. Although low affinity estrogen-binding sites were maintained, high affinity ER were found to be dramatically reduced during the thymidine treatment. The comparison between the effect of OH-TAM on the cell cycle and the expression of ER revealed that the antiestrogen OH-TAM was effective only in the presence of ER. EGF was found to have no effect on the cell cycle of thymidine-synchronized cells, although it did partially reverse the G1 phase block induced by OH-TAM when added simultaneously to cell culture 24 h before thymidine exposure. The parallel analysis of EGF receptor level demonstrated that thymidine treatment also reduced EGF receptors that were found to reappear after the synchronization, during the S and G2 phases of the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)