RESUMO
We measured the tissue concentration of human urokinase receptor (uPAR) in 22 breast carcinomas and 9 benign breast lesions using in vitro quantitative autoradiography. Tissue sections were incubated with increasing concentrations of 125I-pro-urokinase in the presence or absence of unlabeled competitor. Breast carcinomas were found to contain 5 times more uPAR than benign breast lesions with respect to their protein content [523 +/- 72 versus 108 +/- 20 (SE) fmol/mg (P < 0.001)]. Simultaneous quantitation of urokinase (uPA) by immunoenzymatic assay on tissue extracts from the same specimens showed that breast carcinomas also contain 19 times more uPA than benign tumors (611 +/- 134 versus 32 +/- 8 fmol/mg) (P < 0.01). The reliability of quantitative autoradiography measurements was confirmed by uPAR cross-linking assay on membrane fraction from either U937 histiocytic lymphoma cells or breast carcinomas and immunoperoxidase staining with an anti-uPAR antibody on tumor sections. Also, immunoperoxidase staining with an anti-uPA monoclonal antibody showed that uPA is indeed localized on the plasma membrane of epithelial tumor cells in confined areas of breast carcinomas whereas cells from benign breast lesions were devoid of uPA under the same experimental conditions. In conclusion, our findings support the hypothesis that uPAR plays a central role in the acquisition of an invasive phenotye and favor its potential use as a prognostic factor in patients with breast carcinoma.
Assuntos
Doenças Mamárias/enzimologia , Doenças Mamárias/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/ultraestrutura , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Idoso , Autorradiografia , Neoplasias da Mama/irrigação sanguínea , Divisão Celular , Membrana Celular/ultraestrutura , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Neovascularização Patológica , Prognóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Current evidence regarding the regulation of urokinase-dependent extracellular proteolysis indicates that plasminogen activation is a surface-associated process. We have compared the histological localization of urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) in breast cancer sections using a panel of monoclonal antibodies. First, the ability of six different anti-uPA monoclonal antibodies to recognize pro-uPA, uPA, and in vitro-formed complexes of uPA with either soluble uPAR or with plasminogen activator inhibitor type 1 was compared. Then the reactivity of the anti-uPAR antibodies was tested, and the occurrence of an uPA receptor of about M(r) 55,000 in samples from breast carcinoma was assessed by immunoprecipitating the uPA receptor from an in vitro 125I-labeled tumor extract. Immunocytochemical data from adjacent sections of 10 tumor specimens showed that antibodies recognizing free and bound uPA mostly stain the cytoplasm and the membrane of epithelial tumor cells in confined areas of the tumor and some fibroblast-like stromal cells. Acid pretreatment of tumor sections, which removes receptor-bound uPA, causes a strong reduction of the immunocytochemical reactivity of epithelial tumor cells, whereas staining of fibroblast-like cells is not considerably affected. Consistent with these results, epithelial tumor cells were mostly unreactive to anti-uPAR antibodies unless pretreated with acidic buffer, whereas fibroblast-like stromal cells showed a faint but acid-resistant staining with all anti-uPARs. In conclusion, these results show that occupied uPA receptors are definitely present on the membrane of epithelial tumor cells and suggest the occurrence of uPA-uPAR-dependent proteolytic activity on the surface of breast cancer cells.
Assuntos
Anticorpos Monoclonais , Neoplasias da Mama/química , Mama/química , Carcinoma Ductal de Mama/química , Carcinoma Lobular/química , Receptores de Superfície Celular/análise , Ativador de Plasminogênio Tipo Uroquinase/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
The regulatory mechanisms underlying the overexpression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in highly invasive breast carcinomas remain poorly understood. In this study, we have simultaneously determined the level of uPAR and the activity of the transcription factor Sp1 in 14 breast carcinomas and 5 benign lesions. We found that uPAR levels and Sp1-binding activity are coordinately elevated in malignant tumors (r, 0.94; P < 0.001). On the contrary, undetectable or only barely detectable levels of uPAR and Sp1 activity were found in benign breast lesions. Finally, the engagement of uPAR by catalytically inactive uPA in the MDA-MB-231 breast carcinoma cell line results in a rapid up-regulation of Sp1-binding activity followed by an increase of uPAR protein. These results, taken together, suggest the existence of a uPA-dependent positive regulatory loop that may progressively enhance malignant breast cell invasiveness.
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Superfície Celular/biossíntese , Fator de Transcrição Sp1/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Mama/química , Mama/patologia , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HeLa , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Oligonucleotídeos/metabolismo , Ligação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Regulação para Cima/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
Perturbation of adhesive interactions at cell-substratum and cell-cell contact sites is a critical event in the multistep process of cancer invasion. Recent studies indicate that the urokinase receptor (uPAR) is associated in large molecular complexes with other molecules, such as integrins. To test the possibility that uPAR may physically and functionally interact with vitronectin (Vn) receptors, we determined the expression level of uPAR, alpha(v)beta3, and alpha(v)beta5 Vn receptors in 10 human breast carcinomas. Here, we show the ability of uPAR to physically associate with alpha(v)beta5 in the breast carcinomas examined. The functional effects of this interaction were studied using HT1080 human fibrosarcoma and MCF-7 human breast carcinoma cell lines, both exhibiting a urokinase-dependent physical association between uPAR and alpha(v)beta5. Both cell lines respond to urokinase or to its noncatalytic amino-terminal fragment by exhibiting remarkable cytoskeletal rearrangements that are mediated by alpha(v)beta5 and require protein kinase C activity. On the contrary, binding of Vn to alpha(v)beta5 results in the protein kinase C-independent formation of F-actin containing microspike-type structures. Furthermore, alpha(v)beta5 is required for urokinase-directed, receptor-dependent MCF-7 and HT1080 cell migration. These data show that uPAR association with alpha(v)beta5 leads to a functional interaction of these receptors and suggest that uPAR directs cytoskeletal rearrangements and cell migration by altering alpha(v)beta5 signaling specificity.
Assuntos
Neoplasias da Mama/patologia , Integrinas/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores de Vitronectina , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Movimento Celular , Feminino , Humanos , Pessoa de Meia-Idade , Proteína Quinase C/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Células Tumorais Cultivadas , Vitronectina/fisiologiaRESUMO
BACKGROUND: The mechanisms of increased neointimal hyperplasia after coronary interventions in diabetic patients are still unknown. METHODS AND RESULTS: Glucose and insulin effects on in vitro vascular smooth muscle cell (VSMC) proliferation and migration were assessed. The effect of balloon injury on neointimal hyperplasia was studied in streptozotocin-induced diabetic rats with or without adjunct insulin therapy. To study the effect of balloon injury in nondiabetic rats with hyperinsulinemia, pancreatic islets were transplanted under the kidney capsule in normal rats. Glucose did not increase VSMC proliferation and migration in vitro. In contrast, insulin induced a significant increase in VSMC proliferation and migration in cell cultures. Furthermore, in VSMC culture, insulin increased MAPK activation. A reduction in neointimal hyperplasia was consistently documented after vascular injury in hyperglycemic streptozotocin-induced diabetic rats. Insulin therapy significantly increased neointimal hyperplasia in these rats. This effect of hyperinsulinemia was totally abolished by transfection on the arterial wall of the N17H-ras-negative mutant gene. Finally, after experimental balloon angioplasty in hyperinsulinemic nondiabetic islet-transplanted rats, a significant increase in neointimal hyperplasia was observed. CONCLUSIONS: In rats with streptozotocin-induced diabetes, balloon injury was not associated with an increase in neointimal formation. Exogenous insulin administration in diabetic rats and islet transplantation in nondiabetic rats increased both blood insulin levels and neointimal hyperplasia after balloon injury. Hyperinsulinemia through activation of the ras/MAPK pathway, rather than hyperglycemia per se, seems to be of crucial importance in determining the exaggerated neointimal hyperplasia after balloon angioplasty in diabetic animals.
Assuntos
Angioplastia com Balão , Doenças das Artérias Carótidas/patologia , Diabetes Mellitus Experimental/patologia , Hiperinsulinismo/patologia , Hiperplasia/patologia , Transplante das Ilhotas Pancreáticas , Túnica Íntima/patologia , Angioplastia com Balão/efeitos adversos , Animais , Glicemia , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Glucose/farmacologia , Hiperinsulinismo/induzido quimicamente , Hiperinsulinismo/metabolismo , Hiperplasia/etiologia , Hiperplasia/genética , Insulina/sangue , Insulina/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Mutagênese Sítio-Dirigida , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Estreptozocina , Transfecção , Túnica Íntima/metabolismo , Proteínas ras/antagonistas & inibidores , Proteínas ras/genéticaRESUMO
PURPOSE: Since we have previously shown that the efflux rate of technetium 99m (99mTc) sestamibi, a transport substrate of P-glycoprotein (Pgp), is directly correlated with Pgp levels in untreated breast carcinoma, we tested whether tumor clearance of 9mTc-sestamibi may be predictive of therapeutic response to neoadjuvant chemotherapy in patients with locally advanced breast cancer. PATIENTS AND METHODS: Thirty-nine patients with stage III disease, median tumor diameter 5.8 cm (range, 3 to 10) were enrolled onto this prospective clinical trial and underwent 99mTc-sestamibi scan before neoadjuvant chemotherapy. Patients were injected intravenously (i.v.) with 740 MBq of 99mTc-sestamibi; a 15-minute dynamic study was performed, and static planar images were obtained at 0.5, 1, 2, and 4 hours. The time to half clearance of 99mTc-sestamibi was calculated in each patient from decay corrected time-activity curves using a monoexponential fitting. Patients were treated with epirubicin 150 mg/m2 i.v. every 2 weeks for three courses and then underwent surgery within 3 weeks from the completion of chemotherapy. Residual tumor was assessed by pathologic examination of mastectomy specimens. RESULTS: Seventeen of 39 patients showed a rapid tumor clearance of 9mTc-sestamibi (time to half clearance [t1/2] < or = 204 minutes) and 15 of these 17 (88%) showed a highly cellular macroscopic residual tumor at histology that indicated lack of tumor response to neoadjuvant chemotherapy. In contrast, only eight of 22 (36%) with prolonged retention of 99mTc-sestamibi (t1/2 > 204 minutes) showed residual macroscopic tumor at histology (Fisher's exact test, P < .01). CONCLUSION: A rapid tumor clearance of 99mTc-sestamibi may predict lack of tumor response to neoadjuvant chemotherapy with drugs affected by the multidrug-resistant phenotype in patients with locally advanced breast carcinoma.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/tratamento farmacológico , Compostos Radiofarmacêuticos , Tecnécio Tc 99m Sestamibi , Adulto , Idoso , Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Epirubicina/uso terapêutico , Feminino , Humanos , Excisão de Linfonodo , Mastectomia Radical Modificada , Pessoa de Meia-Idade , Neoplasia Residual , Valor Preditivo dos Testes , Estudos Prospectivos , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio Tc 99m Sestamibi/farmacocinéticaRESUMO
Functional assembly of the plasminogen-dependent proteolytic system on the cell surface requires multiple interactions involving urokinase (uPA), urokinase receptor (uPAR), plasminogen activator inhibitors, and other molecules that mediate cell migration and adhesion. We analyzed the in vitro interaction of uPAR-containing particulate cell fractions with the amino-terminal fragment (ATF) of human urokinase and the matrix-like form of vitronectin. Binding and cross-linking of 125I-labeled ATF to crude membrane extracts from LB6-19 mouse cells overexpressing human uPARs in the presence of 25 nM urea-denatured vitronectin led to the formation of Mr 137,000, 92, 000, and 82,000 covalent complexes. Immunoprecipitation of the preformed cross-linked 125I-labeled complexes with anti-vitronectin, anti-uPA, or anti-uPAR antibodies revealed that the Mr 82,000 and 92, 000 species do contain ATF and vitronectin and identified the Mr 137, 000 species as a ternary complex formed by ATF, uPAR, and vitronectin. A similar electrophoretic pattern was displayed by acid-pretreated membranes extracted from MCF-7 breast carcinoma or HT1080 fibrosarcoma cell lines, as well as a ductal breast carcinoma specimen; the latter exhibited complex formation at concentrations of vitronectin lower than 10 nM. Finally, uPAR-vitronectin interaction was further documented by the decreased reactivity of an anti-uPAR polyclonal antibody to acid-pretreated sections of 10 breast carcinomas that had been preincubated with vitronectin. Our findings highlight the ability of uPAR to interact simultaneously with vitronectin and uPA in breast cancer, supporting a dynamic coupling of the molecular mechanisms underlying plasminogen-dependent matrix degradation and cell adhesion.
Assuntos
Neoplasias da Mama/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vitronectina/metabolismo , Animais , Adesão Celular , Feminino , Humanos , Cinética , Células L , Camundongos , Desnaturação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
UNLABELLED: The multidrug-resistant phenotype is characterized by the reduced intracellular retention of several structurally and functionally unrelated cytotoxic compounds due to the energy-dependent pump activity of P-glycoprotein (Pgp). Because 99mTc-sestamibi is a suitable transport substrate of Pgp, we tested whether the time-dependent fractional retention of this tracer could be used as an index of Pgp expression in untreated breast carcinomas. METHODS: Twenty-seven patients with histologically confirmed breast carcinoma were intravenously injected with 740 MBq (20 mCi) of 99mTc-sestamibi, and static planar images of the breast were obtained at 10, 60 and 240 min. The fractional retention of 99mTc-sestamibi was then calculated as the ratios between 60 and 10 min (R60/10) and between 240 and 10 min (R240/10) of decay-corrected counts/pixel registered in the region of interest drawn around the tumor. Surgically excised tumors were then obtained from each patient, and Pgp levels were determined using 125I-labeled MRK16 monoclonal antibody and in vitro quantitative autoradiography. RESULTS: The fractional retention of 99mTc-sestamibi at 60 and 240 min was significantly higher in tumors with low Pgp levels (Group I, n = 18) as compared to that measured in tumors with high Pgp expression (Group II, n = 9) (p < 0.001). In particular, R60/10 values were 0.86 and 0.59 in breast carcinomas of Groups I and II, respectively, whereas the values of R240/10 were 0.56 and 0.25 in low- and high-Pgp-expressing tumors, respectively. CONCLUSION: The determination of fractional retention of 99mTc-sestamibi may be used as a simple functional test for Pgp expression in untreated breast cancer. A preliminary estimate of the sensitivity and the specificity of the test indicates its potential use in clinical practice to identify patients with a high probability of developing multidrug resistance.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Proteínas de Neoplasias/análise , Tecnécio Tc 99m Sestamibi , Autorradiografia , Resistência a Múltiplos Medicamentos , Feminino , Humanos , Técnicas Imunoenzimáticas , Cintilografia , Fatores de Risco , Tecnécio Tc 99m Sestamibi/farmacocinéticaRESUMO
UNLABELLED: Folate binding protein (FBP) GP38 is a membrane-associated glycoprotein that mediates the intracellular transport of folates. The enhanced expression of FBP in ovarian carcinomas provided a rational basis for clinical studies with specific monoclonal antibodies and some newly synthesized antifolate drugs. Because the outcome of these clinical studies ultimately depends on the degree of FBP expression, we measured the local concentration of FBP using 125I-MOv18 monoclonal antibody and quantitative autoradiography. METHODS: Tissue sections from 37 specimens of ovarian carcinoma and 13 nonmalignant ovaries were incubated with increasing concentrations of 125I-MOv18 with and without an excess of unlabeled antibody. Tissue-bound radioactivity was measured by quantitative autoradiography. RESULTS: Folate binding protein was found to be overexpressed in 91% of nonmucinous ovarian carcinomas, with local concentrations ranging between 1.14 and 82.84 pmole/g. Adjacent tumor sections simultaneously studied with 125I-MOv18 and a 125I-labeled folic acid derivative showed matching and superimposable regional distributions of bound radioactivity of the two radioligands, indicating that the antigen, specifically recognized by 125I-MOv18 in nonmucinous ovarian carcinomas, is capable of binding folates. Nonmalignant ovaries did not contain measurable amounts of antigen when assayed with 125MOv18 but showed a limited and specific binding of the 125I-folic acid derivative to tissue sections. The autoradiographic findings were confirmed by testing sections from mixtures of antigen-positive and antigen-negative cells, by immunoperoxidase staining with MOv18 on tumor sections and by biochemical identification of FBP in membrane fractions from tissue samples. CONCLUSION: Folate binding protein is overexpressed up to 80-90-fold in nonmucinous ovarian carcinomas compared with nonmalignant ovaries. Quantitation of FBP may provide a useful tool in the design of clinical studies with specific monoclonal antibodies and certain antifolate drugs that enter the cell through FBP.
Assuntos
Proteínas de Transporte/análise , Radioisótopos do Iodo , Neoplasias Ovarianas/química , Receptores de Superfície Celular/análise , Adenocarcinoma Mucinoso/química , Adulto , Idoso , Anticorpos Monoclonais , Autorradiografia/métodos , Cistadenoma Seroso/química , Feminino , Receptores de Folato com Âncoras de GPI , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Ovário/química , Células Tumorais CultivadasRESUMO
An immunoperoxidase staining technique was used for detecting three major iron binding proteins (ferritin, transferrin and lactoferrin) in 40 breast carcinoma cases and six benign breast proliferative lesions. Ferritin staining was detected mainly in connectival stroma and in histiocytes surrounding neoplastic cells. Few and faint ferritin positivities were also detected in neoplastic cells of 20 carcinoma cases. Transferrin was found inconsistently in myoepithelial cells surrounding normal ductules, or around neoplastic ducts of ductal in situ carcinoma. In eight carcinoma cases, transferrin staining was also positive in neoplastic cells. Lactoferrin was detected only in normal breast epithelial cells and in benign breast proliferative lesions. These immunohistochemical findings may suggest that raised serum ferritin concentrations in breast carcinoma patients might be attributed to stromal reaction rather than to tumour synthesis. Transferrin staining of neoplastic cells in these carcinoma cases appears to be very intriguing, particularly since transferrin is considered an obligate requirement for growing cells, and transferrin receptors have been demonstrated only in dividing cells. On the basis of the immunohistochemical data, lactoferrin might be used as a pointer to benign lesions.
Assuntos
Neoplasias da Mama/análise , Ferritinas/análise , Lactoferrina/análise , Lactoglobulinas/análise , Transferrina/análise , Adenocarcinoma Mucinoso/análise , Mama/análise , Doenças Mamárias/metabolismo , Carcinoma/análise , Carcinoma in Situ/análise , Carcinoma Intraductal não Infiltrante/análise , Feminino , Humanos , Técnicas ImunoenzimáticasRESUMO
Since many of the factors involved in tumor growth and progression act through a receptor-mediated mechanism, we applied in vitro receptor imaging techniques to study the intratumoral distribution and concentration of receptor-molecules having experimental biological relevance in such processes. Here we summarize the results of a study concerning the role of urokinase receptor (uPAR) in the acquisition of an invasive phenotype by tumor cells. Independently of the system studied, we demonstrated that in vitro receptor imaging techniques can be used to define the biological characteristics of human solid tumors and can contribute to clarify the complex network of ligand/receptor interactions leading to tumor spread.
Assuntos
Doenças Mamárias/diagnóstico , Neoplasias da Mama/diagnóstico , Receptores de Superfície Celular/metabolismo , Autorradiografia , Doenças Mamárias/enzimologia , Doenças Mamárias/metabolismo , Doenças Mamárias/patologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Humanos , Técnicas Imunoenzimáticas , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Metastasis is the leading cause of death by cancer. Non-small-cell lung cancer (NSCLC) represents nearly 85% of primary malignant lung tumours. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Transforming growth factor-ß1 (TGF-ß1) is the major inductor of EMT. The aim of this study is to investigate TGF-ß1's effect on cancer stem cells (CSCs) identified as cells positive for CD133, side population (SP) and non-cancer stem cells (non-CSCs) identified as cells negative for CD133, and SP in the A549 cell line. We demonstrate that TGF-ß1 induces EMT in both CSC and non-CSC A549 sublines, upregulating the expression of mesenchymal markers such as vimentin and Slug, and downregulating levels of epithelial markers such as e-cadherin and cytokeratins. CSC and non-CSC A549 sublines undergoing EMT show a strong migration and strong levels of MMP9 except for the CD133(-) cell fraction. OCT4 levels are strongly upregulated in all cell fractions except CD133(-) cells. On the contrary, wound size reveals that TGF-ß1 enhances motility in wild-type A549 as well as CD133(+) and SP(+) cells. For CD133(-) and SP(-) cells, TGF-ß1 exposure does not change the motility. Finally, assessment of growth kinetics reveals major colony-forming efficiency in CD133(+) A549 cells. In particular, SP(+) and SP(-) A549 cells show more efficiency to form colonies than untreated corresponding cells, while for CD133(-) cells no change in colony number was observable after TGF-ß1 exposure. We conclude that it is possible to highlight different cell subpopulations with different grades of stemness. Each population seems to be involved in different biological mechanisms such as stemness maintenance, tumorigenicity, invasion and migration.
Assuntos
Antígenos CD/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glicoproteínas/metabolismo , Peptídeos/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Antígeno AC133 , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metaloproteinase 9 da Matriz/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Vimentina/metabolismoRESUMO
BACKGROUND: The urokinase plasminogen activator receptor (u-PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u-PA) on the endothelial cell surface, thus promoting angiogenesis in a protease-dependent manner. The u-PAR may exist in a glycophosphatidylinositol-anchored and in a soluble form (soluble u-PAR [Su-PAR]), both including the chemotactic Ser88 -Arg-Ser-Arg-Tyr9² internal sequence. OBJECTIVE: To investigate whether Su-PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser88 -Arg-Ser-Arg-Tyr9² sequence. METHODS AND RESULTS: In this study, the formation of vascular-like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su-PAR protein promotes the formation of cord-like structures, and that this ability is retained by the isolated Ser(88) -Arg-Ser-Arg-Tyr9² chemotactic sequence, the maximal effect being reached at 10 nmol L⻹ SRSRY peptide (SRSRY). This effect is mediated by the α(v) ß3 vitronectin receptor, is independent of u-PA proteolytic activity, and involves the internalization of the G-protein-coupled formyl-peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su-PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su-PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2. CONCLUSIONS: Our results show a new protease-independent activity of Su-PAR that stimulates in vivo angiogenesis through its Ser88 -Arg-Ser-Arg-Tyr9² chemotactic sequence.
Assuntos
Quimiotaxia , Neovascularização Fisiológica/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Técnicas de Cocultura , Humanos , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Transdução de Sinais , SolubilidadeRESUMO
BACKGROUND: Recent evidence assigns the vitronectin receptors (VnRs) an important role in regulating tumour cell invasion and dissemination. In vivo and in vitro studies document that all trans-retinoid acids (ATRAs) inhibit growth-inducing apoptosis in melanomas. OBJECTIVES: We have analysed the effects of ATRA treatment on melanoma cell adhesion and motility. METHODS: Human M14 melanoma cells were treated with 10 micromol L-1 ATRA for different times and stained with rhodamine-phalloidin to analyse the effect of treatment on cytoskeleton organization. Cell adhesion and cell migration assays were performed to analyse the role of VnRs in the ATRA-induced early stages of apoptosis. VnR expression was evaluated by Western blot, immunoprecipitation and immunocytochemistry assays. RESULTS: First, using an annexin V assay, we found that apoptosis was triggered by 48 h with 10 micromol L-1 ATRA exposure. At this time point, decrease in the F-actin polymerization as well as inhibition of cell adhesive ability to vitronectin (Vn) was exerted by ATRA treatment. In the presence of serum, exposure to 10 micromol L-1 ATRA for 48 h produced a dramatic inhibition of the cell adhesion ability that was comparable with that exerted by untreated cells preincubated with anti-alpha(v)beta(3) or anti-alpha(v)beta(5) VnR monoclonal antibodies. Functionally, the treatment of melanoma cells with 10 micromol L-1 ATRA for 48 h causes an inhibition of directional cell migration towards Vn-coated filters. Therefore, we analysed the effect of ATRA on the VnR expression. Both alpha(v)beta(3) and alpha(v)beta(5) VnR levels were reduced upon exposure to 10 micromol L-1 ATRA for 48 h as shown by Western blot, immunoprecipitation and immunocytochemistry assays. CONCLUSIONS: Altogether, our data indicate that treatment of M14 melanoma cells with ATRA downregulates VnR expression and that this reduction is closely correlated with the ATRA-dependent inhibition of actin-fibre organization, cell adhesion and migration. Although the mechanism by which ATRA regulates the expression of VnR in M14 melanoma cells needs further elucidation, this system may represent a model for understanding the molecular basis of ATRA therapy in melanoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Integrina alfaVbeta3/metabolismo , Melanoma Experimental/metabolismo , Proteínas de Neoplasias/metabolismo , Tretinoína/farmacologia , Western Blotting/métodos , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , DNA de Neoplasias/análise , Regulação para Baixo/efeitos dos fármacos , Humanos , Testes de Precipitina , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The present study was performed to evaluate Ki-67 and B72.3 immunostaining in 20 selected cases of breast cancer. In particular, we have examined the intracellular localization of TAG 72 and the tumour growth fraction, identified by Ki-67 antibody, on frozen sections of mammary carcinoma, by immunohistochemical technique (ABC method sec.Hsu). Immunostaining of TAG 72 and Ki-67 antigen was related to histologic subtype, diameter, nodal involvement, and number of positive axillary nodes. The preliminary results suggest that: (a) the presence of Ki-67 nuclear staining appeared to be associated with a poorer degree of differentiation, but no direct relationships were observed with diameter and nodal involvement; (b) no correlation between Ki-67 labelling rates and B72.3 intracytoplasmic immunostaining was observed; (c) myoepithelial cells show weak intracytoplasmic positivities.
Assuntos
Anticorpos Monoclonais/análise , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Neoplasias da Mama/metabolismo , Glicoproteínas/imunologia , Adulto , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Antígeno Ki-67 , Pessoa de Meia-IdadeRESUMO
Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo.
Assuntos
Movimento Celular , Macrófagos/enzimologia , Monócitos/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/metabolismo , Actinas/metabolismo , Animais , Calcitriol/farmacologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Ativação Enzimática , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Mutação , Fenótipo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-hck , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Temperatura , Transfecção , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
Technetium-99m sestamibi is a transport substrate recognised by the multidrug-resistant P-glycoprotein (Pgp). To test whether 99mTc-sestamibi efflux is enhanced in breast carcinomas overexpressing Pgp, we determined the efflux rates of 99mTc-sestamibi and Pgp levels in tumours from 30 patients with untreated breast carcinoma. Patients were intravenously injected with 740 MBq of 99mTc-sestamibi and underwent a 15-min dynamic study followed by the acquisition of static planar images at 0.5, 1, 2 and 4 h. Tumour specimens were obtained from each patient 24 h after 99mTc-sestamibi scan and Pgp levels were determined using 125I-MRK16 monoclonal antibody and in vitro quantitative autoradiography. All breast carcinomas showed high uptake of 99mTc-sestamibi and data from region of interest analysis on sequential images were fitted with a monoexponential function. The efflux rates of 99mTc-sestamibi, calculated from decay-corrected time-activity curves, ranged between 0.00121 and 0.01690 min-1 and were directly correlated with Pgp levels measured in the same tumours (r=0.62; P<0.001). Ten out of 30 breast carcinomas (33%) contained 5 times more Pgp than benign breast lesions and showed a mean concentration of 5.73+/- 1.63 pmol/g of tumour (group A). The remaining 20 breast carcinomas had a mean Pgp concentration of 1.29+/-0.64 pmol/g (group B), equivalent to that found in benign breast lesions. 99mTc-sestamibi efflux from tumours of group A was 2.7 times higher than that observed in tumours of group B (0.00686+/-0.00390 min-1 vs 0.00250+/-0.00090 min-1, P<0.001). The in vivo functional test with 99mTc-sestamibi showed a sensitivity and a specificity of 80% and 95%, respectively. In conclusion, the efflux rate of 99mTc-sestamibi may be used for the in vivo identification of the multidrug resistant (MDR1) phenotype in untreated breast cancer patients.