Growth Factor-stimulated Cell Proliferation Research Articles

Selected contribution: estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappaB activation in VSM cells from female rats.

Epidemiological studies have demonstrated that hormone replacement therapy with estrogen (E2) or E2 plus progesterone in postmenopausal women decreases the age-associated risk of cardiovascular disease by 30-50%. Treatment of vascular smooth muscle (VSM) cells with physiological concentrations of E2 has been shown to inhibit growth factor-stimulated cell proliferation. In this study, we tested the hypothesis that E2 inhibits the age-associated increase in VSM cell proliferation by inhibiting nuclear factor (NF)-kappaB pathway. We investigated the effects of E2 treatment and adenovirus-mediated estrogen receptor (ER)-alpha gene transfer on cell proliferation and NF-kappaB activation using VSM cells cultured from 3-mo-old and 24-mo-old Fischer 344 female rats. Our results demonstrate that VSM cell proliferation was significantly increased (P < 0.05) in aged compared with young adult female rats. Treatment of VSM cells with physiological concentrations of E2 inhibited VSM cell proliferation, and this inhibition was significantly greater (P < 0.05) in cells from aged female rats compared with young adults. The inhibitory effects of E(2) on cell proliferation in aged female rats were significantly potentiated by overexpression of the human ER-alpha gene into VSM cells. Constitutive and interleukin (IL)-1beta-stimulated NF-kappaB activation was significantly greater (P < 0.05) in VSM cells from aged compared with young female rats. E2 treatment of VSM cells from aged female rats inhibited both constitutive and IL-1beta-stimulated NF-kappaB activation. ER-alpha gene transfer into VSM cells from aged female rats further augmented the inhibitory effects of E2. In conclusion, our data demonstrate that constitutive and IL-1beta-stimulated NF-kappaB activation is increased in VSM cells from aged female rats due to loss of E2 and this can be restored back to normal levels by ER-alpha gene transfer and E2 treatment. In addition, increased NF-kappaB signaling may be responsible for increased incidence of cardiovascular disease in postmenopausal females.

CYR61 Stimulates Human Skin Fibroblast Migration through Integrin αvβ5 and Enhances Mitogenesis through Integrin αvβ3, Independent of Its Carboxyl-terminal Domain

CYR61, an angiogenic factor and a member of the CCN protein family, is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, promotes cell migration, and enhances growth factor-stimulated cell proliferation. CYR61 induces angiogenesis and promotes tumor growth in vivo and is expressed in dermal fibroblasts during cutaneous wound healing. It has been demonstrated recently that adhesion of primary skin fibroblasts to CYR61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, resulting in adhesive signaling and up-regulation of matrix metalloproteinases 1 and 3. CYR61 is composed of four discrete structural domains that bear sequence similarities to the insulin-like growth factor-binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a carboxyl-terminal (CT) domain that resembles cysteine knots found in some growth factors. In this study, we show that a CYR61 mutant (CYR61DeltaCT) that has the CT domain deleted is unable to support adhesion of primary human skin fibroblasts but is still able to stimulate chemotaxis and enhance basic fibroblast growth factor-induced mitogenesis similar to wild type. In addition, fibroblast migration to CYR61 is mediated through integrin alpha(v)beta(5) but not integrins alpha(6)beta(1) or alpha(v)beta(3). Furthermore, we show that CYR61 binds directly to purified integrin alpha(v)beta(5) in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin alpha(v)beta(3), a known receptor for CYR61 that mediates CYR61-dependent cell adhesion and chemotaxis in vascular endothelial cells. Thus, CYR61 promotes primary human fibroblast adhesion, migration, and mitogenesis through integrins alpha(6)beta(1), alpha(v)beta(5), and alpha(v)beta(3), respectively. Together, these findings establish CYR61 as a novel ligand for integrin alpha(v)beta(5) and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner.

Effect of short-term ethanol on the proliferative response of Swiss 3T3 cells to mitogenic growth factors.

Both adaptive and deleterious responses of cells to ethanol are likely triggered by short-term interactions of the cells with ethanol. Many studies have demonstrated the direct effect of ethanol on growth factor-stimulated cell proliferation. Using Swiss 3T3 cells whose growth was inhibited by ethanol in a concentration-dependent manner, we further investigated the molecular mechanisms of acute ethanol treatment by examining its effect on EGF- and PDGF-mediated cellular signaling systems for the mitogenic function. Tyrosine autophosphorylation of the growth factor receptors was partially prevented by ethanol in intact cells. When ethanol was included before or after EGF stimulation, no effect on the receptor signaling was observed. Here we also report that ethanol inhibits activation of ERK induced by both EGF and PDGF. EGF-induced JNK activation was reduced but PDGF-induced rapid JNK activation was delayed by the addition of ethanol. The balance between its inhibitory and stimulatory effect on the signaling molecules might determine the rate of cell growth.

Inhibition of human endometrial cancer cell growth in vitro and in vivo by somatostatin analog RC-160

Objective: Our purpose was to investigate the effect of the somatostatin analog RC-160 on the growth of the HEC-1 human endometrial cancer cell line in vivo and in vitro. Study Design: Nude mice bearing subcutaneous implanted HEC-1 tumors were treated for 25 days with RC-160 (100 μg/d) delivered by osmotic minipumps. In cultured HEC-1 cells radioreceptor assay of somatostatin was performed, and the expression of messenger ribonucleic acid for somatostatin receptor subtypes (somatostatin receptors 1-5) was analyzed by reverse transcription–polymerase chain reaction. The effects of RC-160 on epidermal growth factor–stimulated cell proliferation and tyrosine phosphorylation of epidermal growth factor receptor were examined by colorimetric assay and Western blotting, respectively. Results: The treatment with RC-160 resulted in a significant decrease in tumor volume, tumor weight, and serum insulin-like growth factor I levels compared with those values in control animals. The presence of high-affinity somatostatin binding sites and the expression of somatostatin receptor 2 and somatostatin receptor 3 messenger ribonucleic acid were demonstrated in HEC-1 cells by radioreceptor assay and reverse transcription–polymerase chain reaction, respectively. Epidermal growth factor–stimulated proliferation of HEC-1 cells was inhibited by RC-160 in a dose-dependent manner. Western blotting revealed that epidermal growth factor–induced tyrosine phosphorylation of epidermal growth factor receptor was inhibited by RC-160, which suggests that the direct inhibitory effect of RC-160 on HEC-1 cell growth might be mediated in part by interference with epidermal growth factor receptor phosphorylation. Conclusion: These results indicate that somatostatin analog RC-160 inhibits the growth of HEC-1 human endometrial cancer cells, thus implying its potential clinical utility in treating endometrial cancer. (Am J Obstet Gynecol 1999;181:583-90.)

Proteolysis of human prolactin: resistance to cathepsin D and formation of a nonangiostatic, C-terminal 16K fragment by thrombin.

The N-terminal 16K fragments of rat and human PRLs possess angiostatic activity. 16K PRL has also been detected in vivo in both humans and rats. Based on an in vitro study, cathepsin D, an acid protease, has been implicated in the generation of rat 16K PRL. However, the proteolytic cleavage of human PRL has not been demonstrated. Our objective was to identify an enzyme that is capable of forming an angiostatic human 16K PRL. To confirm the angiostatic action of rat 16K PRL, the fragment was generated by incubating 23K PRL with rat mammary microsomal fraction at pH 3.2. Upon incubation with human umbilical vein endothelial cells (HUVEC), rat 16K PRL, but not 23K PRL, inhibited basal- and basic fibroblast growth factor-stimulated cell proliferation. Intact rat and human PRLs were then incubated with cathepsin D or acidified microsomal pellets of MCF-7 human breast cancer cells. Analysis by SDS-PAGE showed cleavage of rat, but not human, PRL. Next, hormones were incubated with thrombin at pH 7.4. As shown by SDS-PAGE, digestion of both human and rat PRL by thrombin resulted in the formation of 16K fragments. PRL contained within human amniotic fluid was also cleaved by thrombin. Enzyme specificity was supported by prevention of cleavage by the thrombin inhibitor hirudin. When tested with HUVEC, the human 16K PRL was devoid of angiostatic activity. The activity of this fragment in the Nb2 lymphoma bioassay was 10- to 15-fold lower than that of 23K PRL. Mass spectrometry revealed that the fragment has a mass of 16,878.30+/-15.8 Daltons. Subsequent N-terminal sequencing showed that the thrombin cleavage occurred between amino acid residues 53 (Lys) and 54 (Ala), resulting in the formation of a C-terminal, not an N-terminal, 16K fragment. We conclude that, unlike rat PRL, human PRL is resistant to cleavage by cathepsin D. Thrombin at a physiological pH can generate a C-terminal 16K fragment of human PRL that is not angiostatic and retains little mitogenic activity. We suggest that the precise nature of endogenous 16K PRL fragments that are present in human tissues and body fluids should be carefully examined.

Interferon-alpha is a potent inhibitor of basic fibroblast growth factor-stimulated cell proliferation in human uterine cells.

Abnormal uterine bleeding is a significant health problem for many women and is the number-one reason for performing hysterectomy in the United States. Leiomyomas (uterine fibroids) are benign neoplasms that are a frequent cause of abnormal uterine bleeding. The goal of this study was to assess the effects of the anti-angiogenic cytokine, interferon (INF)-alpha, on the proliferation of both leiomyoma and normal uterine cells. Primary cultures of leiomyoma, myometrial, and endometrial stromal cells were established for in vitro study. The effects of INF-alpha (10, 100, and 1000 U/ml) were tested on serum-stimulated and basic fibroblast growth factor-stimulated cell proliferation using the [3H]thymidine incorporation assay. INF-alpha was a potent inhibitor of cell proliferation for all three cell types, with endometrial stromal cells showing the greatest sensitivity. The antiproliferative effect did not appear to result from toxic effects on the cells. INFs may prove to be useful therapeutic agents for the treatment of leiomyoma-related abnormal uterine bleeding.

Growth Factors.

Growth factors are polypeptides that interact with specific cellular receptors leading to many different biological responses. There are various families of growth factors that have similar biochemical structures. Although many growth factors stimulate cell proliferation, a few have primarily inhibitory functions, such as transforming growth factor-p (TGFB). Growth factors regulate various modes of action of endocrine tissues, including autocrine, paracrine, and endocrine functions. Recent studies have shown that growth factors also regulate various proteins in the cell cycle, and may have a direct or indirect effect on cell proliferation. For example, TGFB regulates various inhibitory cell-cycle proteins, including p27(kip1) and p15(INK4B). Molecular analyses of growth factors, including the cloning and sequencing of specific growth factor receptors, have contributed greatly to our understanding of the role these factors play in cellular homeostasis and neoplastic development.

Regulatory interaction between calmodulin and the epidermal growth factor receptor.

Growth factor-stimulated cell proliferation is preceded by a transient increase in the cytoplasmic Ca concentration,' and calmodulin, an intracellular calcium receptor protein,' appears to intervene in the modulation of this process.3 Calmodulin binds to nuclear proteins and could therefore affect their functions! However, calmodulin does not control cell proliferation by acting exclusively at the nuclear level. Several lines of evidence obtained in our laboratory indicate that the epidermal growth factor receptor (EGFR) is a calmodulin-binding protein and that calmodulin acts as a modulator of its intrinsic tyrosine kinase activity.'.' We have demonstrated that the EGFR can be isolated from solubilized rat liver plasma membranes by calmodulin-affinity chromatography.' Binding of the EGFR to calmodulin-agarose occurs in the presence of Ca', and its elution is achieved upon addition of EGTA. The preparation obtained contains a set of calmodulinbinding proteins associated with the plasma membrane. However, the major autophosphorylated protein in this preparation corresponds to the EGFR. This autophosphorylation is stimulated by epidermal growth factor (EGF) and transforming growth factor-a (TGF-a) with essentially the same effi~iency.~ Furthermore, the isolated receptor presents both EGFand TGFa-stimulated tyrosine kinase activity towards the exogenous substrate PoIY-L-(GIu : Tyr).'.' The identity of the isolated EGFR

Epidermal growth factor stimulates cell proliferation and inhibits iodide uptake of FRTL-5 cells in vitro.

In accordance with the available data most authors conclude that epidermal growth factor (EGF) has very little or no effect on FRTL-5 cells. This has been viewed as a serious handicap of this cell line. In the present study we cultivated three strains of FRTL-5 cells from different sources and assessed their response to EGF with regard to proliferation, function and differentiation. Cell proliferation was assessed by counting in a Coulter cell counter after culturing cells at suboptimal conditions in well plates. Cell function was studied by measuring iodide uptake. Cell differentiation was examined immunocytochemically by staining monolayer cultures for thyroglobulin (Tg) and EGF receptor (EGFr) as well as morphologically by microscopical evaluation of monolayer cultures. All three FRTL-5 cell lines investigated express EGFr. In two wild type FRTL-5 cell lines EGF stimulates growth, an effect that is enhanced by the presence of TSH, and partially inhibits iodide uptake. A third mutated strain of FRTL-5 cells does not respond to EGF. Tg expression can be demonstrated immunocytochemically in EGF-treated cells as well as in controls. Morphologically, in monolayer culture EGF-treated cells cannot be distinguished from controls. Contrary to previous reports, these studies demonstrate EGF effects on FRTL-5 cells that are consistent with EGF effects established in other thyroid follicular cells.

936-83 Chronic Estradiol Treatment of Rabbits with Cardiac Allografts Attenuates Graft Vascular Proliferation Induced by Insulin-like Growth Factor-I (IGF-I) and Interleukin-6 (IL-6)

Male New Zealand White rabbits fed a 0.5% cholesterol diet (n = 13) received a heterotopic cardiac allograft and cyclosporine ( 10 mg/kg/day) until sacrifice 6 to 7 weeks later, at which time both the native and transplanted hearts and aorta were harvested. Six of the rabbits were treated with estradiol 100 μg/kg/day i.m., beginning 7 days prior to transplantation and continuing until sacrifice; the remaining seven rabbits served as controls and received placebo. The native aorta as well as the aorta from the grafted hearts were isolated and used for in vitro studies involving cytokine and growth factor stimulated cell proliferation. Tritiated thymidine incorporation is expressed as CPM/mg protein and the changes in cell proliferation are expressed as % changes from the control (the unstimulated vessel). IGF-I and IL-6 but not interferon-γ (INF-γ) significantly increased cell proliferation, measured as 3 H-thymidine incorporation. in explants from both native and allograft vessels and significantly more so in the graft (p &lt; 0.05). The mitogenic effect of both IGF-I and IL-6 in the graft aorta, but not in that associated with the native heart, was abrogated by chronic treatment with estradiol (p &lt; 0.02). % Change in Vascular Wall Cell Proliferation IGF-I (20 μg/ml) INF-γ (50 U/ml) IL-6 (50 U/ml) Native - Estradiol 175 ± 32 189 ± 43 162 ± 28 + Estradiol 167 ± 42 143 ± 79 159 ± 36 Allograft - Estradiol 328 ± 66 197 ± 56 366 ± 101 + Estradiol 64 ± 12 116 ± 41 72 ± 14 Mean ± SEM This inhibitory effect of estradiol treatment on the mitogenic effect of IGF-I and IL-6 on the vascular wall of the allograft supports previous studies showing that accelerated coronary transplant arteriosclerosis in this model is inhibited by estrogen. These data speak to the use of hormone replacement therapy in post menopausal women receiving a cardiac transplant.

Cytostatic Gene Therapy for Vascular Proliferative Disorders with a Constitutively Active form of the Retinoblastoma Gene Product

Vascular smooth muscle cell (SMC) proliferation in response to injury is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. The retinoblastoma gene product (Rb) is present in the unphosphorylated and active form in quiescent primary arterial SMCs, but is rapidly inactivated by phosphorylation in response to growth factor stimulation in vitro. A replication-defective adenovirus encoding a nonphosphorylatable, constitutively active form of Rb was constructed. Infection of cultured primary rat aortic SMCs with this virus inhibited growth factor-stimulated cell proliferation in vitro. Localized arterial infection with the virus at the time of balloon angioplasty significantly reduced SMC proliferation and neointima formation in both the rat carotid and porcine femoral artery models of restenosis. These results demonstrate the role of Rb in regulating vascular SMC proliferation and suggest a gene therapy approach for vascular proliferative disorders associated with arterial injury.

Cooperative roles of hepatocyte growth factor and plasminogen activator in tubular morphogenesis by human microvascular endothelial cells.

Epidermal growth factor (EGF) or transforming growth factor‐α (TGF‐α) stimulated cell migration, chemotaxis, and the expression of tissue‐type plasminogen activator (t‐PA) in human omental microvascular endothelial (HOME) cells. Hepatocyte growth factor (HGF) stimulated cell proliferation, but had a negligible stimulatory effect on cell migration, the expression of t‐PA and tube‐like formation into collagen gel in HOME cells. Basic fibroblast growth factor stimulated cell proliferation, cell migration, tubulogenesis and the expression of urokinase‐type plasminogen activator (u‐PA) in bovine aortic endothelial (BAE) cells. HOME and BAE cells had both high‐ and low‐affinity receptors for HGF. In BAE cells, u‐PA activity and tube‐like structures in collagen gel were induced in the presence of HGF alone. In contrast, in HOME cells, t‐PA activity and tube‐like structures were induced in the presence of TGF‐a alone, but not in the presence of HGF alone. However, we observed a marked induction of tube formation by HOME cells when both t‐PA and HGF were added simultaneously. In the model system for tumor angiogenesis, when HOME cells were co‐cultured with a renal cancer cell line, KPK13, tube‐like structures were induced in the presence of HGF: KPK13 cells expressed large amounts of t‐PA mRNA. Our present study suggested that HGF in concert with active t‐PA could be angiogenic in HOME cells.

Induction of immediate early response genes by macrophage colony-stimulating factor in normal human monocytes.

A group of coordinately induced protooncogenes, cytoskeletal, and extracellular matrix genes have been termed immediate early response genes, and their induction has been associated with growth factor-stimulated cell proliferation. We have investigated the induction of these genes by macrophage-CSF (M-CSF) in human monocytes that do not proliferate in response to M-CSF but require the factor for optimal cell differentiation. Normal human monocytes were isolated, carefully washed, and incubated for 36 to 48 h in fetal bovine serum-containing medium. At the end of this incubation the resting cells were stimulated with M-CSF, and RNA was isolated for analysis by Northern blotting. RNA from control resting cells contained low to undetectable levels of c-jun, fibronectin receptor, and actin mRNA. Within 15 to 30 min of addition of M-CSF, however, there was a dramatic coordinate induction of these genes. The c-jun gene expression was very transient and was not detectable by 60 min after M-CSF addition. In contrast, the expression of actin and fibronectin receptor mRNA was more sustained, and the expression of these genes remained elevated at 24 to 48 h after M-CSF addition. We also observed the induction of the myelomonocytic specific tyrosine kinase hck gene simultaneously with the other immediate early response genes. The protein synthesis inhibitor cycloheximide did not block the induction of any of these genes, and in fact, super-induced the expression of c-jun and hck. Nuclear run on transcription of the c-jun, hck, and actin genes. Therefore, in normal human monocytes M-CSF induces immediate early response genes without inducing cell proliferation. These genes may then play a role in altering the physiologic status of the cells in response to CSF.

Regulation of Granulosa Cell Proliferation: Facilitative Roles of Platelet-Derived Growth Factor and Low Density Lipoprotein*

Recent studies suggest that epidermal growth factor (EGF) and/or transforming growth factor-alpha (TGF-alpha) and insulin-like growth factor-I (IGF-I) act synergistically to promote granulosa cell proliferation in vitro suggesting a similar role in vivo. Using a serum-restricted, monolayer culture system containing very low levels of platelet-poor plasma-derived serum (PPPDS), the facilitative roles of platelet-derived growth factor (PDGF) and low density lipoprotein (LDL) with respect to growth factor-stimulated granulosa cell proliferation were investigated. In nutrient medium containing only 0.1% PPPDS, PDGF (1-25 ng/ml) had no effect upon granulosa cell proliferation. When combined with EGF, which alone does not stimulate granulosa cell proliferation, PDGF dose-dependently increased cell proliferation to levels obtained with 10% fetal calf serum (2.4-fold increase relative to controls, P less than 0.05). When combined with EGF and IGF-I, a combination which does stimulate mitosis in granulosa cells, PDGF again dose-dependently enhanced proliferation (P less than 0.05). The extent of proliferation obtained with EGF + IGF-I + PDGF was consistently greater than that obtained with 10% fetal calf serum (P less than 0.05) but significantly less than that obtained with EGF + fetal calf serum, a treatment which stimulates rapid granulosa cell proliferation. LDL has been shown to greatly enhance granulosa cell steroidogenesis by providing exogenous cholesterol. However, cholesterol is also required for plasma membrane biosynthesis and cell growth. LDL alone, had no effect upon porcine granulosa cell proliferation relative to media controls (0.1% PPPDS) nor did it synergize with any single growth factor to induce mitosis. When combined with EGF + IGF-I, and EGF + PDGF, but not PDGF + IGF-I, LDL dose-dependently (1-25 micrograms/ml) enhanced proliferation (P less than 0.05) to levels equivalent to that obtained with 10% fetal calf serum. When combined with EGF, IGF-I, and PDGF, LDL at 10 micrograms/ml enhanced proliferation to an extent equivalent with EGF + fetal calf serum (a 5.4-fold increase relative to media controls). High density lipoprotein did not itself stimulate proliferation nor did it facilitate proliferation mediated by growth factors. When maintained in medium alone (0.1% PPPDS), the cell population doubling time was 8.0 +/- 0.5 days. In the presence of EGF, IGF-I, PDGF, and LDL (10, 10, and 5 ng/ml, and 10 micrograms/ml, respectively) the doubling time was reduced to 2.0 +/- 0.1 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidermal Growth Factor Stimulate Cell Proliferation and Inhibits Prolactin Secretion of the Human Decidual Cells in Culture.

Epidermal growth factor (EGF) has been found to be mitogenic in a variety of tissues. We investigated the biological effect of EGF on early pregnant human decidua and the non-pregnant decidualized human endometrium in the primary cell culture. EGF had a stimulatory action on cell proliferation in the early pregnant decidual cells and an inhibitory effect on prolactin (PRL) secretion from the decidual cells. The addition of progesterone into culture medium suppressed cell proliferation of decidual cells, whereas it enhanced PRL secretion from decidua. The analysis of the specific receptor for EGF in the early pregnant decidua and non-pregnant decidualized endometrium revealed that both tissues had a single component EGF receptor with a dissociation constant of nM order. These results suggest that EGF may play a role in the growth and function of endometrial stromal cells.

Epidermal growth factor stimulates cell proliferation and inhibits functional differentiation of mouse mammary epithelial cells in culture.

Mouse mammary epithelial cells cultured on collagen gels multiplied and produced casein and alpha-lactalbumin in response to insulin, cortisol, and PRL. The addition of epidermal growth factor (EGF) at 50 ng/ml increased the total number of epithelial cells by 30-40% and thymidine incorporation into DNA 4.7-fold after 5 days of culture. In contrast, EGF inhibited hormonal induction of the synthesis of casein and alpha-lactalbumin in those cells by about 45% and 55%, respectively, without inhibiting total protein synthesis. Furthermore, EGF decreased casein mRNA activity by 55% and increased total mRNA activity by 66% in cells cultured with the three hormones. These effects of EGF were apparent at 0.1 ng/ml and were maximal at 50-100 ng/ml and could be reversed by its removal from the medium, followed by the addition of anti-EGF antibody. The inhibition of casein synthesis by EGF was unaffected by the concentrations of insulin, cortisol, and PRL. Other growth factors, such as fibroblast growth factor, multiplication-stimulating activity, nerve growth factor, and platelet-derived growth factor, did not simulate the effects of EGF. Cytarabine (1 microgram/ml), which inhibited thymidine incorporation into DNA by 94%, did not block the inhibitory action of EGF on casein synthesis. These results suggest that EGF serves as a regulator of hormone-dependent growth and differentiation of mammary epithelial cells.

Possible physiological role of epidermal growth factor in the development of the mouse mammary gland during pregnancy

Epidermal growth factor stimulated cell proliferation in a primary mammary epithelial cell culture derived from mice at different stages of pregnancy. Moreover, the peptide hormone inhibited casein production induced by the synergistic actions of insulin, cortisol and prolactin. The inhibitory effect of epidermal growth factor was influenced by the gestational stages of the mammary gland. These effects of epidermal growth factor were exerted at physiological concentrations. The dual actions of epidermal growth factor on mammary cells implicate its participation in regulation of the growth and differentiation of the mammary gland during pregnancy.

Adaptation of the human breast cancer cell line MCF-7 to serum free medium culture on extracellular matrix

The human breast cancer cell line MCF-7 has been adapted to grow in serum free medium on an extracellular matrix produced by bovine corneal endothelial cells. The cells have been continuously maintained in these conditions for more than 11 months with the same doubling time as the original population grown on plastic dishes in presence of charcoal treated fetal calf serum. The hormonal control of their growth appears intact. Insulin and epidermal growth factor stimulate cell proliferation. Estradiol-17β (E 2 ), dexamethasone, L-triiodothyronine, prolactin have no effect. Treatment with tamoxifen and estrone induces an inhibition of cell multiplication. This inhibition by tamoxifen can be rescued by 4 % charcoal treated fetal calf serum and E 2 but not by E 2 alone.