TrkA overexpression enhances growth and metastasis of breast cancer cells.

The Trk family of neurotrophin tyrosine kinase receptors is emerging as an important player in carcinogenic progression in non-neuronal tissues. Here, we show that breast tumors present high levels of TrkA and phospho-TrkA compared to normal breast tissues. To further evaluate the precise functions of TrkA overexpression in breast cancer development, we have performed a series of biological tests using breast cancer cells that stably overexpress TrkA. We show that (1) TrkA overexpression promoted cell growth, migration and invasion in vitro; (2) overexpression of TrkA per se conferred constitutive activation of its tyrosine kinase activity; (3) signal pathways including PI3K-Akt and ERK/p38 MAP kinases were activated by TrkA overexpression and were required for the maintenance of a more aggressive cellular phenotype; and (4) TrkA overexpression enhanced tumor growth, angiogenesis and metastasis of xenografted breast cancer cells in immunodeficient mice. Moreover, recovered metastatic cells from the lungs exhibited enhanced anoikis resistance that was abolished by the pharmacological inhibitor K252a, suggesting that TrkA-promoted breast tumor metastasis could be mediated at least in part by enhancing anoikis resistance. Together, these results provide the first direct evidence that TrkA overexpression enhances the tumorigenic properties of breast cancer cells and point to TrkA as a potential target in breast cancer therapy.

Tamoxifen and TRAIL synergistically induce apoptosis in breast cancer cells.

Tamoxifen (TAM), is widely used as a single agent in adjuvant treatment of breast cancer. Here, we investigated the effects of TAM in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in estrogen receptor-alpha (ER-alpha)-positive and -negative breast cancer cells. We showed that cotreatment with TAM and TRAIL synergistically induced apoptosis regardless of ER-alpha status. By contrast, cotreatment did not affect the viability of normal breast epithelial cells. Cotreatment with TAM and TRAIL in breast cancer cells decreased the levels of antiapoptotic proteins including FLIPs and Bcl-2, and enhanced the levels of proapoptotic proteins such as FADD, caspase 8, tBid, Bax and caspase 9. Furthermore, cotreatment-induced apoptosis was efficiently reduced by FADD- or Bid-siRNA, indicating the implication of both extrinsic and intrinsic pathways in synergistic apoptosis induction. Importantly, cotreatment totally arrested tumor growth in an ER-alpha-negative MDA-MB-231 tumor xenograft model. The abrogation of tumor growth correlated with enhanced apoptosis in tumor tissues. Our findings raise the possibility to use TAM in combination with TRAIL for breast cancers, regardless of ER-alpha status.

ST6GalNAc I expression in MDA-MB-231 breast cancer cells greatly modifies their O-glycosylation pattern and enhances their tumourigenicity.

Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers, including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac:GalNAcalpha2,6-sialyltransferase: CMP-Neu5Ac: R-GalNAcalpha1-O-Ser/Thr alpha2,6-sialyltransferase (EC 2.4.99.3) (ST6GalNAc I), which transfers a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. However, established breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn. We have previously shown that stable transfection of MDA-MB-231, a human breast cancer cell line, with ST6GalNAc I cDNA induces sialyl-Tn antigen (STn) expression. We report here the modifications of the O-glycosylation pattern of a MUC1-related recombinant protein secreted by MDA-MB-231 sialyl-Tn positive cells. We also show that sialyl-Tn expression and concomitant changes in the overall O-glycan profiles induce a decrease of adhesion and an increase of migration of MDA-MB-231. Moreover, STn positive clones exhibit an increased tumour growth in severe combined immunodeficiency (SCID) mice. These observations suggest that modification of the O-glycosylation pattern induced by ST6GalNAc I expression are sufficient to enhance the tumourigenicity of MDA-MB-231 breast cancer cells.

Synergistic induction of apoptosis in breast cancer cells by cotreatment with butyrate and TNF-alpha, TRAIL, or anti-Fas agonist antibody involves enhancement of death receptors' signaling and requires P21(waf1).

Inhibitors of histone deacetylase (HDAC) are considered as potential anticancer agents. We have previously demonstrated that an inhibitor of HDAC, sodium butyrate (NaB), induces apoptosis of breast cancer cells in a P53-independent and P21(waf1)-dependent manner. In this study, we showed that tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), and anti-Fas agonist antibody potentiated NaB-induced growth inhibition through synergistic induction of apoptosis in breast cancer cell lines (MCF-7, T47-D, and BT-20). In MCF-7 cells, NaB increased the expression of death receptors; NaB alone or in combination with TNF-alpha, TRAIL, and anti-Fas agonist antibody increased the levels of Bid, tBid, and that of cytosolic cytochrome c. Synergistic induction of apoptosis was strongly inhibited by dominant-negative Fas-associated death domain (FADD) and inhibitors of caspases-8 and -9, indicating that potentiation of apoptosis involved key elements of death receptors' signaling pathways. Moreover, cotreatment of NaB and ligands of death receptors up-regulated the levels of P21(waf1) and that of proliferating cell nuclear antigen (PCNA) associated with P21(waf1). Transient transfections of p21(waf1) antisense or p21(waf1) deficient for its interaction with PCNA abolished synergistic induction of apoptosis. This suggested that potentiation of apoptosis by cotreatments required P21(waf1) and its interaction with PCNA. Since breast tumors contain rarely p21 mutations, our results may open interesting prospects in the fight against breast cancer.

Changes in the K+ current-density of MCF-7 cells during progression through the cell cycle: possible involvement of a h-ether.a-gogo K+ channel.

MCF-7 cells express voltage-activated K+ channels. In the present study, we used the patch-clamp and RT-PCR techniques to investigate the involvement of these channels during the cell cycle progression. The outward rectifier current (IK) recorded during depolarization was almost completely suppressed by the classical K+ channel blocker tetraethylammonium (TEA) in MCF-7 cells. TEA also inhibited cell proliferation, as measured with 3H-thymidine incorporation. Moreover, profound changes were observed in both the resting membrane potential (RMP) and IK during the release from the G0/G1 phase of the cell cycle. MCF-7 cells arrested in G0/G1 were depolarized (-26.3 +/- 10 mV, n = 30) and IK-density was small (9.4 +/- 5.6 pA/pF, n = 60) compared to cells progressing in the G1 phase (RMP = -60 +/- 7.9 mV; n = 35 and IK-density = 30.2 +/- 8.5 pA/pF; n = 76). IK was highly sensitive to Mg2+, astemizole and TEA (10 mM). Extracellular perfusion of 5 mM Mg2+ dramatically slowed the activation and perfusion of 2 microM astemizole inhibited both IK (20 +/- 3%) and cell proliferation (23%). Moreover, the h-EAG mRNA expression was modulated during the cell cycle. Thus, these data suggested that h-EAG K+ channels play a role in controlling the proliferation and/or cell cycle.

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways.

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). In contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-kappaB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

Verapamil inhibits proliferation of LNCaP human prostate cancer cells influencing K+ channel gating.

The mechanisms of verapamil and tetraethylammonium (TEA) inhibition of voltage-gated K+ channels in LNCaP human prostate cancer cells were studied in whole-cell and outside/inside-out patch-clamp configurations. Rapidly activating outward K+ currents (I(K)) exhibited neither C-type, nor rapid (human ether á go-go-related gene-type) inactivation. With 2 mM [Mg(2+)](o), I(K) activation kinetics was independent of holding potential, suggesting the absence of ether á go-go-type K+ channels. Extracellular applications of TEA and verapamil (IC(50) = 11 microM) rapidly (12 s) inhibited I(K) in LNCaP cells. Blocking was also rapidly reversible. Intracellular TEA (1 mM), verapamil (1 mM), and membrane-impermeable N-methyl-verapamil (25 microM) did not influence whole-cell I(K), although both phenylalkylamines inhibited single-channel currents in inside-out patches. Extracellular application of N-methyl-verapamil (25 microM) had no influence on I(K). Our results are compatible with the hypothesis that, in LNCaP cells expressing C-type inactivation-deficient voltage-activated K+ channels, phenylalkylamines interact with an intracellular binding site, and probably an additional hydrophobic binding site that does not bind charged phenylalkylamines. The inhibiting effects of verapamil and TEA on I(K) were additive, suggesting independent K+-channel blocking mechanisms. Indeed, TEA (1 mM) reduced a single-channel conductance (from 7.3 +/- 0.5 to 3.2 +/- 0.4 pA at a membrane potential of +50 mV, n = 6), whereas verapamil (10 microM) reduced an open-channel probability (from 0.45 +/- 0.1 in control to 0.1 +/- 0.09 in verapamil-treated cells, n = 9). The inhibiting effects of verapamil and TEA on LNCaP cell proliferation were not multiplicative, suggesting that both share a common antiproliferative mechanism initiated through a K+ channel block.

Proteomic analysis reveals that 14-3-3sigma is down-regulated in human breast cancer cells.

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, we have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 were the same in both normal and transformed cells. The data support the idea that 14-3-3sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

Proteomic detection of changes in protein synthesis induced by fibroblast growth factor-2 in MCF-7 human breast cancer cells.

Fibroblast growth factor-2 (FGF-2) is a potent regulator of breast cancer cell growth through stimulation of tyrosine kinase receptors and activation of the mitogen-activated protein kinase cascade. In the present study, we have investigated changes in protein synthesis induced by FGF-2 stimulation of the prototypic human breast cancer cell line MCF-7. Using high-resolution two-dimensional electrophoresis of (35)S amino acid metabolically labeled proteins and computerized analysis of 2D autoradiograms, we found that four proteins were up-regulated within the first 12 h of FGF-2 stimulation. Mass spectrometry analysis (MALDI-TOF and MS-MS) of tryptic fragments and database searches allowed the identification of these FGF-2-regulated proteins as the heat shock proteins HSP90 and HSP70, the proliferating cell nuclear antigen (PCNA), and the transcriptionaly controlled tumor protein (TCTP). We then analyzed the distribution of these proteins in various cancerous and normal breast epithelial cells. Interestingly, the four FGF-2-regulated proteins were found to be constitutively up-regulated in ras-transfected MCF-7 cells, indicating their relevance to the up-regulation of cellular proliferation. Moreover, HSP90 and PCNA were found at higher levels in cancerous cells than in normal cells. The role of HSP90 was further investigated using the specific inhibitor geldanamycin. We showed that the functionality of HSP90 is strictly required in order to obtain FGF-2 mitogenic stimulation in MCF-7 cells, indicating the crucial role played by this molecular chaperone in the control of breast cancer cell growth. Finally, these results show that proteomic analysis is a valuable method for identifying potential markers or therapeutic targets related to cancer growth.

Effects of hyperprolactinemia on rat prostate growth: evidence of androgeno-dependence.

The effects of the polypeptide hormone prolactin (PRL) in the development and regulation of benign prostate hyperplasia (BPH) and also in prostate cancer are not very well characterized. This study examines the action of PRL, either alone or in association with androgens [testosterone (T) or dihydrotestosterone (DHT)], in the rat prostate gland. The effects of PRL and androgens were investigated after 30 and 60 days in control, castrated, castrated with a substitutive implant of T or DHT, and sham-operated Wistar rats. To enhance PRL release, we induced hyperprolactinemia by administering chronic injections of sulpiride (40 mg. kg(-1). day(-1)). Chronic hyperprolactinemia induces enlargement and inflammation of the lateral rat prostate without any histological changes on ventral and dorsal lobes. We also demonstrate that hyperprolactinemia induces Bcl-2 overexpression in the lateral rat prostate and that this could inhibit the level of apoptosis. The in vivo model established here is a useful in vivo approach for studying the hormonal regulation of normal and pathological prostate development.

Expression of sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase (ST6GalNac I) cDNA.

Sialyl-Tn antigen (STn) is a cancer associated carbohydrate antigen over-expressed in several cancers including breast cancer, and currently associated with more aggressive diseases and poor prognosis. However, the commonly used breast cancer cell lines (MDA-MB-231, T47-D and MCF7) do not express STn antigen. The key step in the biosynthesis of STn is the transfer of a sialic acid residue in alpha2,6-linkage to GalNAc alpha-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc alpha2,6-sialyltransferase: ST6GalNAc I. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-length human ST6GalNAc I from HT-29-MTX cells. The stable transfection of MDA-MB-231 with an expression vector encoding ST6GalNAc I induces the expression of STn antigen at the cell surface. The expression of STn short cuts the initial O-glycosylation pattern of these cell lines, by competing with the Core-1 beta1,3-galactosyltransferase, the first enzyme involved in the elongation of O-glycan chains. Moreover, we show that STn expression is associated with morphological changes, decreased growth and increased migration of MDA-MB-231 cells.

FGF signals for cell proliferation and migration through different pathways.

FGFs are pleiotropic growth factors that control cell proliferation, migration and differentiation. However, FGF transduction studies have so far focused primarily on the mitogenic effect of this growth factor family and it has been difficult to assess if the described intracellular signaling pathways are dedicated solely to cell proliferation, or whether they are equally important for the migratory activity often seen in responsive cells. We review here papers in which the migratory effects of this growth factor family were clearly discriminated from proliferative effects. In toto, these studies suggest that cells use different signaling pathways for migration, such as Src and p38 MAP kinase, from those for proliferation, which tend to upregulate the ERKs. Which signaling pathway a cell uses for proliferation or migration appears to depend on many factors, including the structure and the quantity of available FGF trapped in the basal lamina by heparan sulfate co-factors, the disposition of cognate high affinity receptors and the general environment of the cell. Thus the density of the cell population, the state of the cell cycle, the presence of other factors or receptors will modulate the migratory response of cells to FGF.

Evidence of functional ryanodine receptor involved in apoptosis of prostate cancer (LNCaP) cells.

BACKGROUND: Very little is known about the functional expression and the physiological role of ryanodine receptors in nonexcitable cells, and in prostate cancer cells in particular. Nonetheless, different studies have demonstrated that calcium is a major factor involved in apoptosis. Therefore, the calcium-regulatory mechanisms, such as ryanodine-mediated calcium release, may play a substantial role in the regulation of apoptosis. METHODS: We assessed the presence of such functional receptors in LNCaP prostate cancer cells, using fluorimetric measurements of intracellular calcium and expression assays of mRNA encoding ryanodine receptors. RESULTS: We show here that LNCaP cells responded to caffeine, a ryanodine receptor agonist, by mobilizing calcium. Another ryanodine receptor agonist, 4-chloro-m-cresol, had a similar effect and promoted calcium release. These effects were inhibited by pretreatment with ryanodine or thapsigargin. In addition to a calcium release, caffeine was able to produce a calcium entry blocked by nickel. We used a reverse transcription-polymerase chain reaction assay to investigate the expression of ryanodine receptors in LNCaP cells. Two types of ryanodine receptor mRNAs were expressed in LNCaP cells: RyR1 and RyR2 mRNAs. Finally, we show that ryanodine receptor activation by caffeine slightly stimulates apoptosis of prostate cancer cells, and that the inhibition of these receptors by ryanodine protects the cells against apoptosis. CONCLUSIONS: The combination of results showed that LNCaP cells, derived from a human prostate cancer, express functional RyRs able to mobilize Ca(2+) from intracellular stores and which might control apoptosis.

Pharmacological effects of the lipidosterolic extract of Serenoa repens (Permixon) on rat prostate hyperplasia induced by hyperprolactinemia: comparison with finasteride.

BACKGROUND: The growth of the prostate gland is mainly dependent on androgens. Other hormones, like prolactin (PRL), also influence prostate development. Our purpose was to analyze and compare the effects of two drugs (5alpha-reductase inhibitor) used in the therapy of benign prostatic hyperplasia: lipidosterolic extract of Serenoa repens (LSESR), and finasteride in an in vivo model of rat prostate hyperplasia induced by hyperprolactinemia. METHODS: Hyperprolactinemia was induced by 30 daily injections of sulpiride. Wistar rats received daily gavages of LSESR or finasteride. We used the following groups: control, castrated, castrated with a substitute testosterone (T), or 5alpha-dihydrotestosterone (DHT) implant. RESULTS: Hyperprolactinemia increases the wet weight and induces hyperplasia in the lateral prostate (LP). Unlike finasteride, LSESR significantly reduced LP growth and hyperplasia in castrated, DHT-implanted, and sulpiride-treated rats. CONCLUSIONS: Finasteride was only capable of inhibiting the effect of androgens on rat prostate enlargement. LSESR inhibited not only the androgenic but also the trophic effect of PRL in rat LP hyperplasia.

Potassium channels in rat prostate epithelial cells.

Voltage-dependent K(+) channels were identified and characterized in primary culture of rat prostate epithelial cells. A voltage-dependent, inactivating K(+) channel was the most commonly observed ion channel in both lateral and dorsal cells. The K(+) current exhibited a voltage threshold at -40 mV. Averaged half-inactivation potential (V(1/2)) and the slope factor (k) values were -26 mV and 6, respectively. It showed a monoexponential decay with an inactivation time constant of about 600 ms at +60 mV. The deactivation time constant at -60 mV was 30 ms and the reversal potential was estimated at -80 mV, suggesting that current was carried by potassium ions. The scorpion venom peptides charybdotoxin (5 nM) and margatoxin (1 nM), inhibited K(+) current at all membrane potentials with a rapid and a slow reversibility respectively. Both tetraethylammonium (10 mM) and 4-aminopyridine (50 microM) reduced K(+) current by approximately 40%. We conclude that plasma membranes of lateral and dorsal rat prostate epithelial cells contain Kv K(+) channels that have biophysical and pharmacological properties consistent with those of the Kv1.3 family.

Alterations in both heparan sulfate proteoglycans and mitogenic activity of fibroblast growth factor-2 are triggered by inhibitors of proliferation in normal and breast cancer epithelial cells.

Heparan sulfate proteoglycans (HSPG) are involved in the regulation of cellular proliferation, differentiation, and migration. We have studied the effect of three inhibitors of proliferation on 35S incorporation into HSPG of the breast cancer cell lines MCF-7 and MDA-MB-231 and the normal breast epithelial cells (NBEC). Transforming growth factor beta-1 (TGFbeta-1), which inhibits the proliferation of NBEC, but not of MCF-7 and MDA-MB-231, cells induced an increase in 35S incorporation of HSPG in NBEC, but had no effect on cancer cells. Sodium butyrate (NaB), which inhibits NBEC as well as cancer cell proliferation, induced an increase in 35S incorporation into HSPG in all cell types studied. In contrast, retinoic acid had no effect on HSPG of breast epithelial cells. Modification of HSPG induced by TGFbeta-1 or NaB treatments in normal and breast cancer epithelial cells resulted in an increase in 125I-fibroblast growth factor-2 (FGF-2) binding on HSPG. More importantly, NaB pretreatment resulted in an inhibition of the MCF-7 cell responsiveness to FGF-2, even though these cells remained sensitive to growth stimulation induced by serum or epidermal growth factor. These results indicate that changes in HSPG production are a key process involved in the mechanism of breast epithelial cell growth regulation.

Regulation of cell proliferation and urokinase plasminogen activation of human breast epithelial cells by carrageenans.

Carrageenans, a family of polysulphated carbohydrates, are able to inhibit the binding to cells of growth factors such as transforming growth factor 1 (TGF 1), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor (PDGF) and so modulate cell invasion and proliferation. We studied the effects of carrageenans on the proliferation and on the uPA/PAI-1 system in breast epithelial cells. Carrageenans were able to inhibit the proliferation of both normal breast epithelial cells (NBEC) and breast epithelial cancer cell lines (MDA-MB-231 and MCF-7) but could only inhibit the uPA activity in the MDA-MB-231 cells. Moreover, carrageenans inhibited FGF-2 binding in all three cell types, suggesting that they regulate cell proliferation and uPA/PAI-1 system through two distinct mechanisms. These molecules could be considered as potentially useful anti-cancer agents.

Transforming growth factor beta 1 and sodium butyrate differentially modulate urokinase plasminogen activator and plasminogen activator inhibitor-1 in human breast normal and cancer cells.

The effects of transforming growth factor beta 1 (TGF-beta1) and sodium butyrate on cell proliferation and the urokinase plasminogen activator (uPA) system were examined in normal human breast epithelial cells (HBECs) and in a breast cancer cell line, MDA-MB-231. In HBECs, TGF-beta1 inhibited cell proliferation and uPA activity, while it augmented plasminogen activator inhibitor-1 (PAI-1) antigen level. Sodium butyrate inhibited both cell proliferation and uPA activity but did not affect the level of PAI-1. In MDA-MB-231, TGF-beta1 had no effect on cell proliferation but increased uPA activity and PAI-1 antigen level; sodium butyrate inhibited both cell proliferation and uPA activity but had no effect on PAI-1 level. Moreover, in the presence of plasminogen, cell detachment could be modulated by the level of cell-associated uPA. Our results indicate (1) that the effects of TGF-beta1 on cell growth can be dissociated from its effects on the uPA/PAI system; (2) that, while TGF-beta1 is a potent inhibitor of cell proliferation and uPA activity in normal cells, it may promote invasion and metastasis of tumour cells by increasing uPA activity and PAI-1 levels; and (3) that sodium butyrate offers a potential approach to preventing tumour development by inhibiting both cell proliferation and invasion.

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.

Characterization of morphogenetic and invasive abilities of human mammary epithelial cells: correlation with variations of urokinase-type plasminogen activator activity and type-1 plasminogen activator inhibitor level.

Urokinase-type plasminogen activator (uPA) and one of its inhibitors, the PAI-1, are involved in the proteolytic cascade of matrix degradation during in vivo morphogenesis or metastasis. In the present study, we have characterized the in vitro morphological behavior of human normal and malignant mammary epithelial cells and determined the levels of uPA activity and PAI-1 during these events. Two-dimensional cultures in the presence of inductive fibroblast-conditioned medium (CM) allowed migration of HBL-100 cells and MDA-MB-231 cells. Normal human mammary epithelial cells (HMEC) and MCF-7 cells failed to migrate under these conditions. The epithelial cell migration correlated with an increase in the uPA activity whereas their immobility correlated with both increases in uPA activity and PAI-1 level. In three-dimensional cultures in collagen gel, fibroblasts or fibroblast CM induced branching tubular morphogenesis to HMEC, cord-like extensions to HBL-100 cells and a greater invasiveness ability to MDA-MB-231 cells. These events correlated with an increased uPA activity. In contrast, no morphological rearrangement was observed in MCF-7 cells and this correlated with both increases in uPA activity and PAI-1 level. Altogether, these results show that the in vitro mammary epithelial behavior is under the influence of mesenchymal inductive signals and is in agreement with modifications of uPA activity and PAI-1 levels. Our culture system gives a suitable model to study the mechanisms of mammary development and metastasis and to highlight the involvement of proteases and their inhibitors in cell-cell positioning and cell-matrix reorganization.