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1.
Cancer Res ; 61(11): 4337-40, 2001 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-11389056

RESUMO

Nerve growth factor (NGF) has been shown recently to be mitogenic for human breast cancer cells. In the present study, we have assayed the expression of NGF receptors (NGFRs: TrkA and p75) mRNAs in 363 human primary breast cancers, using real-time quantitative reverse transcription-PCR. NGFRs were found in all of the tumor biopsies. TrkA and p75 were positively correlated and were respectively associated with the histoprognostic grading and the tumor type. NGFRs were both related to progesterone receptors. In univariate analyses, TrkA (>upper quartile) was associated with longer overall survival. Histoprognostic grading, tumor size, node involvement, and steroid receptors were also prognostic factors. In Cox multivariate analyses, TrkA was not a prognostic parameter. This study demonstrates the expression of NGFRs in breast cancer and points out that patients with high levels of TrkA have a more favorable overall survival prognosis.


Assuntos
Neoplasias da Mama/metabolismo , Receptor trkA/biossíntese , Receptores de Fator de Crescimento Neural/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor de Fator de Crescimento Neural , Receptor trkA/genética , Receptores de Fator de Crescimento Neural/genética
2.
J Biol Chem ; 276(21): 17864-70, 2001 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-11359788

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fator de Crescimento Neural/farmacologia , Transdução de Sinais/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , NF-kappa B/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Células Tumorais Cultivadas
3.
Cancer Res ; 61(1): 76-80, 2001 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-11196201

RESUMO

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.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/metabolismo , Exonucleases , Proteínas de Neoplasias , Proteínas 14-3-3 , Autorradiografia , Biomarcadores Tumorais/genética , Mama/metabolismo , Neoplasias da Mama/genética , Regulação para Baixo , Eletroforese em Gel Bidimensional , Células Epiteliais/metabolismo , Exorribonucleases , Regulação Neoplásica da Expressão Gênica , Humanos , Biossíntese de Proteínas , Isoformas de Proteínas , Proteínas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais Cultivadas
4.
Exp Cell Res ; 262(1): 59-68, 2001 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-11120605

RESUMO

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.


Assuntos
Biomarcadores Tumorais , Proteínas de Ligação ao Cálcio/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteoma/metabolismo , Neoplasias da Mama , Divisão Celular , Eletroforese em Gel Bidimensional/métodos , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Células Tumorais Cultivadas , Proteína Tumoral 1 Controlada por Tradução
5.
Biochem J ; 352 Pt 2: 449-63, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11085939

RESUMO

Eggs from Xenopus laevis are surrounded by several layers of jelly that are needed for proper fertilization. Jelly coat is composed of high-molecular-mass glycoconjugates to which are bound many globular proteins. O-glycans released from the jelly coat of X. laevis have been partially described in previous studies. In this study, we compared the glycosylation pattern of the egg jelly coat isolated from six specimens of X. laevis. The O-glycans were released from jelly coats by alkali/borohydride treatment. Structural characterization was performed through a combination of one- and two-dimensional (1)H-NMR and methylation analysis. This allowed the description of a new family of sulphated O-glycans present in jelly coats of all X. laevis. However, the jelly O-glycans showed a low extent of polymorphism between specimens. This intra-specific variability was restricted to the terminal substitution of O-linked oligosaccharides. The differential expression of two glycosyltransferase [an alpha-(1-->4) galactosyltransferase and an alpha-(1-->3) fucosyltransferase] activities resulted in the characterization of four phenotypes of X. laevis. Furthermore, electrophoretic analysis suggested that the high-molecular-mass fraction of jelly coat was mostly composed of mucin-type glycoproteins. Blot analysis with lectins confirmed that the glycan variability was borne by these mucin-type components. However, fertilization assays suggested that the glycan polymorphism had no repercussion on egg fertilizability.


Assuntos
Mucinas/química , Óvulo/química , Polissacarídeos/química , Animais , Western Blotting , Configuração de Carboidratos , Sequência de Carboidratos , Eletroforese em Gel de Poliacrilamida , Fertilização , Glicosilação , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fenótipo , Polissacarídeos/metabolismo , Xenopus laevis
6.
Mol Cell Biol Res Commun ; 3(6): 338-44, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11032755

RESUMO

Cancer development depends not only on the nature of the tumor cells themselves but also on the regulatory effects of various normal cells. The present study was performed to better understand the mechanism by which normal breast epithelial cells (NBEC) can control the growth of MCF-7 breast cancer cells. When MCF-7 cells were treated with NBEC conditioned medium, cell growth was inhibited in a concentration-dependent manner. This inhibition was due to an induction of apoptosis without any change in cell cycle progression. The induction of apoptosis was correlated with increased levels of p53, p21(waf1) and decreased levels of bcl-2. Transient transfections of MCF-7 cells with two p53 cDNA constructs demonstrated the induction of apoptosis was mediated by endogenous p53. Taken together, our results indicate that NBEC inhibit the growth of MCF-7 breast cancer cells by inducing apoptosis in them via endogenous p53.


Assuntos
Apoptose , Neoplasias da Mama/patologia , Mama/metabolismo , Células Epiteliais/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Western Blotting , Mama/citologia , Neoplasias da Mama/metabolismo , Divisão Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Mutação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2
7.
Cytokine Growth Factor Rev ; 11(4): 295-302, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10959077

RESUMO

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.


Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Animais , Divisão Celular , Movimento Celular , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/genética , Modelos Biológicos , Mutação , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais
8.
FEBS Lett ; 478(3): 209-15, 2000 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-10930570

RESUMO

Fibroblast growth factor-2 (FGF-2) is mitogenic for the human breast cancer cell line MCF-7; here we investigate some of the signaling pathways subserving this activity. FGF-2 stimulation of MCF-7 cells resulted in a global increase of intracellular tyrosine phosphorylation of proteins, particularly FGF receptor substrate-2, the protooncogene product Src and the mitogen-activated protein kinase (MAP kinase) cascade. A major increase in the tyrosine phosphorylation of a 30-kDa protein species was also found. This protein was identified as cyclin D2 by mass spectrometry after trypsin digestion. Immunoprecipitation of cyclin D2 and immunoblotting with anti-phosphotyrosine antibodies confirmed that the tyrosine phosphorylation of cyclin D2 was indeed induced by FGF-2 stimulation. In addition, pharmacological inhibition of Src (with herbimycin A and PP2), and of the MAP kinase cascade (with PD98059), confirmed that Src activity is required for the FGF-2-induced phosphorylation of cyclin D2 whereas MAP kinase activity is not. Thus, tyrosine phosphorylation of cyclin D2 may be a key regulatory target for FGF-2 signaling.


Assuntos
Ciclinas/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Mitógenos/farmacologia , Fosfotirosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Western Blotting , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclina D2 , Ciclinas/química , Ativação Enzimática/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/antagonistas & inibidores , Dados de Sequência Molecular , Peso Molecular , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais Cultivadas
9.
J Physiol ; 527 Pt 1: 71-83, 2000 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10944171

RESUMO

1. In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+-ATPase inhibitor thapsigargin (TG), in androgen-sensitive human prostate cancer LNCaP cells. 2. Exposure of fura-2-loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store-operated Ca2+ channels (SOCs). 3. For the first time we have identified and characterized the SOC-mediated membrane current (Istore) in prostate cells using whole-cell, cell-attached, and perforated patch-clamp techniques, combined with fura-2 microspectrofluorimetric and Ca2+-imaging measurements. 4. Istore in LNCaP cells lacked voltage-dependent gating and displayed an inwardly rectifying current-voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 +/- 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5-3 mM) and La3+ (1 microM). 5. Treatment of LNCaP cells with TG (0.1 microM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG-induced apoptosis. 6. The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA-AM). 7. These results indicate that in androgen-sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store-activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.


Assuntos
Apoptose , Canais de Cálcio/metabolismo , Cálcio/fisiologia , Neoplasias da Próstata/fisiopatologia , Androgênios/farmacologia , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Condutividade Elétrica , Humanos , Lantânio/farmacologia , Masculino , Microscopia de Fluorescência , Níquel/farmacologia , Técnicas de Patch-Clamp , Neoplasias da Próstata/metabolismo , Tapsigargina/farmacologia , Células Tumorais Cultivadas
10.
Breast Cancer Res Treat ; 60(3): 251-8, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10930113

RESUMO

Breast epithelial cells produce both mitogens and growth inhibitors which are involved in the control of mammary gland development through autocrine and paracrine pathways. While the mechanisms of action of several growth factors have been well established and related strategies proposed for breast cancer therapy, little is known concerning growth inhibitors. In this review, we present an overview of current information about major autocrine and paracrine growth inhibitors of breast epithelial cells, and we discuss their potential functions in the control of breast cancer development.


Assuntos
Comunicação Autócrina/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Substâncias de Crescimento/farmacologia , Comunicação Parácrina/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Proteoglicanas/metabolismo
11.
J Biol Chem ; 275(39): 30009-18, 2000 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-10862617

RESUMO

To explore how heparan sulfate (HS) controls the responsiveness of the breast cancer cell lines MCF-7 and MDA-MB-231 to fibroblast growth factors (FGFs), we have exposed them to HS preparations known to have specificity for FGF-1 (HS glycosaminoglycan (HSGAG A)) or FGF-2 (HSGAGB). Proliferation assays confirmed that MCF-7 cells were highly responsive to FGF-2 complexed with GAGB, whereas migration assays indicated that FGF-1/HSGAGA combinations were stimulatory for the highly invasive MDA-MB-231 cells. Quantitative polymerase chain reaction for the levels of FGF receptor (FGFR) isoforms revealed that MCF-7 cells have greater levels of FGFR1 and that MDA-MB-231 cells have greater relative levels of FGFR2. Cross-linking demonstrated that FGF-2/HSGAGB primarily activated FGFR1, which in turn up-regulated the activity of mitogen-activated protein kinase; in contrast, FGF-1/HSGAGA led to the phosphorylation of equal proportions of both FGFR1 and FGFR2, which in turn led to the up-regulation of Src and p125(FAK). MDA-MB-231 cells were particularly responsive to vitronectin substrates in the presence of FGF-1/HSGAGA, and blocking antibodies established that they used the alpha(v)beta(3) integrin to bind to it. These results suggest that the clustering of particular FGFR configurations on breast cancer cells induced by different HS chains leads to distinct phenotypic behaviors.


Assuntos
Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Heparitina Sulfato/farmacologia , Meios de Cultura Livres de Soro , Interações Medicamentosas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Fator 1 de Crescimento de Fibroblastos , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fenótipo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Vitronectina/metabolismo
12.
Biochem Biophys Res Commun ; 267(3): 770-6, 2000 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-10673367

RESUMO

The growth of the malignant human mammary MDA-MB-231 cells is stimulated by fibroblast growth factor-1 (FGF-1) but not by FGF-2. When these cells are cultured in the presence of chlorate, an inhibitor of heparan sulfate (HS) sulfation, their proliferation is stimulated by both FGF-1 and FGF-2. We analyzed the interactions of FGF-1 and FGF-2 with HS purified from the cell layer and the culture medium of control and chlorate-treated MDA-MB-231 cells. The HS from the cell layer bound FGF-1 with faster association kinetics than the HS from the culture medium, and so had a higher affinity for FGF-1. Chlorate treatment had no significant effect on the FGF-1 binding kinetics of the HS. In contrast to FGF-1, chlorate treatment of the cells significantly altered the FGF-2 binding kinetics. The HS from untreated cells possessed two binding sites for FGF-2, one with fast association kinetics (k(ass) 470,000 to 610,000 M(-1) s(-1)) and a high affinity (K(d) 46 to 70 nM) and one with slower association kinetics (k(ass) 74,000 to 100,000 M(-1) s(-1)) and a lower affinity (K(d) 290 to 400 nM). HS from chlorate-treated cells possessed just a single binding site for FGF-2 with fast association kinetics (k(ass) 270,000 to 290,000 M(-1) s(-1)) and a high affinity (K(d) 41 to 57 nM). These results show that there is a relationship between the binding kinetics of FGFs and their ability to stimulate cell growth.


Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Heparitina Sulfato/metabolismo , Artefatos , Neoplasias da Mama , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cloratos/farmacologia , Feminino , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Cinética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Células Tumorais Cultivadas
13.
Brain Res Dev Brain Res ; 119(1): 47-54, 2000 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-10648871

RESUMO

The effects of limb blastemas of Pleurodeles waltl on axon growth from fragments of spinal cord were studied in vitro. Cultured in a defined medium, spinal cord fragments regenerated sparse, short axons. The culture of spinal fragments in the presence of blastemas greatly enhanced the length, number and survival of axons. Testing separately each of the two components of the blastema showed that only the mesenchyme exerts a neurotropic effect on the spinal fragments. Other tissues such as muscle or skin had a limited neurotrophic effect. Additionally, the neurotrophic activity of blastemas seems to be dependent of its proliferation status. Compared with blastemas of regenerating limbs from young animals, irradiated blastemas (devoid of mitotic activity) and blastemas of regenerating limbs from old animals or differentiated blastemas (both characterized by a low mitotic activity), exhibited a weaker neurotrophic influence. The blastema neurotrophic factor is not an attachment molecule but a soluble one and cannot be nerve growth factor (NGF) or fibroblast growth factor (FGF). It has a relatively low molecular weight (less than 15 kDa) and its protein nature was ascertained by its sensitivity to heating and proteases. As the production of this mesenchyme-derived neurotrophic factor depends upon mesenchymal cell proliferation of the blastema, we suggest that there is loop of positive regulation between spinal nerves and blastema. Blastema tissues may stimulate nerve regeneration allowing the stimulation of proliferation of blastema cells by regenerating nerve fibers. Alternatively, blastema cells may produce a neurotrophic factor whose secretion might be dependent on cell proliferation.


Assuntos
Cotos de Amputação/inervação , Axônios/fisiologia , Botões de Extremidades/inervação , Regeneração Nervosa/fisiologia , Pleurodeles/fisiologia , Medula Espinal/citologia , Animais , Contagem de Células/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Técnicas de Cultura , Membro Anterior/inervação , Botões de Extremidades/citologia , Botões de Extremidades/crescimento & desenvolvimento , Botões de Extremidades/efeitos da radiação , Mesoderma/citologia , Peso Molecular , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia
14.
Oncogene ; 18(31): 4460-73, 1999 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-10442637

RESUMO

H19 is an imprinted and developmentally regulated gene whose product remains apparently untranslated. In a previous study on breast adenocarcinomas, we reported that overexpression of the H19 gene was significantly correlated with the presence of steroid receptors, suggesting the putative role of hormones in H19 transcription. To determine the mode of steroid action, we have detected levels of H19 RNA synthesis during mammary gland development by in situ hybridization (ISH): two peaks of H19 transcription occur during puberty and pregnancy. Furthermore, we demonstrated by ISH that in the uterus H19 RNA synthesis is high during estrus and metestrus phases. To test steroid control of H19 transcription, ovariectomized and adrenalectomized mice were supplemented, 1 week after surgery, with 17-beta-estradiol (E2, 20 microg/kg/day), progesterone (P, 1 mg/kg/day) or corticosterone (B, 0.3 mg/ kg/day) for 2 weeks. According to ISH data, E2 and to a lesser extent B stimulated H19 transcription in the uterus, whereas P inhibited it. To confirm the in vivo results, in vitro experiments were performed using cultures of MCF-7 cells (a hormone-sensitive mammary cell line). E2 stimulated the endogenous H19 gene of this cell line and tamoxifen inhibited this effect. Furthermore, we performed transient cotransfections in MCF-7, in HBL-100 (another hormone-sensitive mammary cell line) and in BT-20 (a hormone-insensitive mammary cell line) with various constructs of ERalpha (WT or mutated) and PR-A, in presence or absence of steroid hormones. We demonstrated that ERalpha up-regulated the H19 promoter in MCF-7 and in HBL-100, whereas PR-A did not have any effect per se. Moreover, in MCF-7, PR-A antagonized clearly the ERalpha-mediated promoter enhancement, but in HBL-100 this counteracting effect on the ERalpha up-regulation was not found. Interestingly, the same experiments performed in BT-20 cell line provided very similar results as those obtained in MCF-7 cells, with a clear down-regulation mediated by PR-A on the H19 promoter. All these in vitro data are in agreement with in vivo results. In addition, data obtained with ERalpha mutants indicate that H19 promoter activation is both ligand-dependent and ligand-independent. We have thus demonstrated that H19 gene expression is controlled by steroid hormones; furthermore, this gene is highly expressed in hormone-sensitive organs when the hormonal stimulation is accompanied with a morphological repair.


Assuntos
Adrenalectomia , Corticosterona/farmacologia , Estradiol/farmacologia , Regulação da Expressão Gênica/fisiologia , Glândulas Mamárias Animais/fisiologia , Proteínas Musculares/genética , Ovariectomia , Progesterona/farmacologia , RNA não Traduzido , Útero/fisiologia , Envelhecimento , Animais , Desenvolvimento Embrionário e Fetal , Estro/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Supressores de Tumor , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Gravidez , RNA Longo não Codificante , Transcrição Gênica
15.
Exp Cell Res ; 245(2): 239-44, 1998 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-9851863

RESUMO

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.


Assuntos
Divisão Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Inibidores do Crescimento/farmacologia , Proteoglicanas de Heparan Sulfato/metabolismo , Sítios de Ligação/efeitos dos fármacos , Neoplasias da Mama , Butiratos/farmacologia , Membrana Celular/metabolismo , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais , Feminino , Humanos , Mitógenos , Sódio/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia , Células Tumorais Cultivadas
16.
J Biol Chem ; 273(27): 16659-62, 1998 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-9642218

RESUMO

We show here that nerve growth factor (NGF), the archetypal neurotrophic factor, is able to stimulate the proliferation of breast cancer cells (MCF-7 and MDA-MB-231 cell lines), although it is unable to stimulate growth of normal breast epithelial cells (NBEC). This stimulation induced cells in the G0 phase to reenter the cell cycle, as well as shortening cell cycle duration. Immunoblotting experiments revealed that both the two cancer cell lines and the NBEC express high affinity (p140(trk)) and low affinity (p75) NGF receptors. Inhibition of the NGF growth-promoting effect by the drugs K-252a and PD98059 indicated that activation of Trk-tyrosine kinase activity and the mitogen-activated protein kinase cascade are necessary to obtain the mitogenic effect. Activation of mitogen-activated protein kinase can be detected in breast cancer cells after 10 min of NGF stimulation, whereas no change was detected in NBEC. These results demonstrate that NGF is a mitogenic factor for human breast cancer cells and that it might constitute a new regulator of breast tumor growth.


Assuntos
Neoplasias da Mama/patologia , Mama/citologia , Mitógenos/fisiologia , Fatores de Crescimento Neural/fisiologia , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/fisiologia , Linhagem Celular , Ativação Enzimática , Humanos , Proteínas Tirosina Quinases/metabolismo , Células Tumorais Cultivadas
17.
Int J Oncol ; 12(6): 1397-401, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9592206

RESUMO

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.


Assuntos
Mama/citologia , Carragenina/farmacologia , Células Epiteliais/efeitos dos fármacos , Excipientes/farmacologia , Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Anticoagulantes/administração & dosagem , Anticoagulantes/farmacologia , Mama/efeitos dos fármacos , Mama/metabolismo , Carragenina/administração & dosagem , Divisão Celular/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/metabolismo , Células Epiteliais/metabolismo , Excipientes/administração & dosagem , Feminino , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/administração & dosagem , Heparina/farmacologia , Humanos , Radioisótopos do Iodo , Plasminogênio/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativadores de Plasminogênio/efeitos dos fármacos , Ativadores de Plasminogênio/metabolismo , Ligação Proteica/efeitos dos fármacos , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacos
18.
J Cell Sci ; 111 ( Pt 11): 1521-34, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9580560

RESUMO

Cell migration and invasion play a crucial role during normal and pathological development. The expression of several members of the Ets family of transcription factors has been shown to correlate with the occurrence of these processes. In the present study, we investigated the effect of the expression of Ets1-DB, the DNA-binding domain of c-Ets1, on the functional properties of NMuMG and MMT epithelial cell lines, from normal and cancerous mouse mammary tissues, respectively. We found that stable expression of this Ets1-DB mutant inhibited, in both cell types, the gene expression and activity of urokinase type-plasminogen activator (uPA), a potential target of c-Ets1. uPA is a key serine proteinase in the proteolytic cascade leading to the degradation of the extracellular matrix. In two-dimensional cultures, expression of the Ets1-DB mutant resulted in a decrease in cell migration and invasion in both cell lines. In three-dimensional collagen gels, NMuMG cells underwent tubular morphogenesis, while MMT cells developed as scattered structures. The Ets1-DB mutant impaired the capacity of NMuMG cells to form tubules and reduced the ability of MMT cells to invade these gels. Similar inhibition of cell migration, invasion and morphogenesis were observed in non-infected NMuMG and MMT cell lines treated with aprotinin, a serine proteinase inhibitor, suggesting that the inhibition of the plasmin cascade mediates in part the biological effects induced by the Ets1-DB mutant. These results demonstrate that Ets family members are involved in the control of uPA activity, cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells.


Assuntos
Movimento Celular/fisiologia , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Animais , Divisão Celular/fisiologia , Linhagem Celular , Camundongos , Morfogênese/fisiologia , Mutação , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas c-ets , Ativador de Plasminogênio Tipo Uroquinase/fisiologia
19.
Br J Cancer ; 77(3): 396-403, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9472634

RESUMO

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.


Assuntos
Neoplasias da Mama/patologia , Mama/efeitos dos fármacos , Butiratos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Ativadores de Plasminogênio/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Ácido Butírico , Divisão Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Células Tumorais Cultivadas
20.
Pathol Biol (Paris) ; 45(4): 305-11, 1997 Apr.
Artigo em Francês | MEDLINE | ID: mdl-9296079

RESUMO

Proteoglycans (PG) are complex sulphated macromolecules composed of linear polysaccharide chains of glycosaminoglycans (GAG) covalently attached to a core protein. These GAG chains contain sulphate groups at various positions, giving them a high density of negative charges, and allowing them to interact with extracellular matrix molecules, including various growth factors. In the developing mammary gland, sulphated proteoglycans participate in morphogenesis and interact with extracellular matrix components in order to constitute a functional matrix. In breast pathogenesis, qualitative or quantitative changes in PG may have important consequences on cell proliferation and/or differentiation. Thus, several studies showed large variations in the nature and distribution of PG/GAG in breast cancer. Accumulation of chondroitin sulfate proteoglycans was described in the stromal compartment of mammary biopsy sections, and content in heparan sulfate proteoglycans, which were more specifically distributed in the epithelial compartment, increased with the level of malignancy and invasiveness of breast cancer tissues. Furthermore, heparan sulfate proteoglycans seem to be involved in control of the growth-promoting activity of numerous growth factors such as fibroblast growth factors also named Heparin-Binding Growth Factors (HBGF). The implication of PG in growth factor activity suggest that PG may have prognostic value in breast cancer. In future, structural studies into the specific HS-sequences involvement in growth factors binding could allow the development of new antiproliferative strategies.


Assuntos
Neoplasias da Mama/metabolismo , Proteoglicanas/metabolismo , Divisão Celular , Células Epiteliais , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Proteoglicanas/química , Proteoglicanas/classificação
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