RESUMO
Platelet activation has long been postulated to contribute to the development of atherosclerotic plaques, although the mechanism by which this might occur remains unknown. Thrombin is a potent platelet activator and transfusion of thrombin-activated platelets into mice increases plaque formation, suggesting that thrombin-induced platelet activation might contribute to platelet-dependent atherosclerosis. Platelets from protease-activated receptor 4-deficient (Par4-/-) mice fail to respond to thrombin. To determine whether thrombin-activated platelets play a necessary role in a model of atherogenesis, we compared plaque formation and progression in Par4+/+ and Par4-/- mice in the atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) background. Littermate Par4+/+ and Par4-/- mice, all ApoE-/-, were placed on a Western diet (21% fat, 0.15% cholesterol) for 5 or 10 weeks. The percent of aortic lumenal surface covered by plaques in Par4+/+ and Par4-/- mice was not different at either time point (2.2+/-0.3% vs. 2.5+/-0.2% and 5.1+/-0.4% vs. 5.6+/-0.4% after 5 and 10 weeks, respectively). Further, no differences were detected in the cross-sectional area of plaques measured at the aortic root (1.53+/-0.17 vs. 1.66+/-0.16x10(5)microm(2) and 12.56+/-1.23 vs. 13.03+/-0.55x10(5)microm(2) after 5 and 10 weeks, respectively). These findings indicate that thrombin-mediated platelet activation is not required for the early development of atherosclerotic plaques in the ApoE-/- mouse model and suggest that, if platelet activation is required for plaque formation under these experimental conditions, platelet activators other than thrombin suffice.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/sangue , Ativação Plaquetária/efeitos dos fármacos , Trombina/farmacologia , Animais , Apolipoproteínas E/fisiologia , Aterosclerose/metabolismo , Plaquetas/metabolismo , Cruzamentos Genéticos , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Ativação Plaquetária/fisiologia , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Trombina/metabolismo , Fatores de TempoRESUMO
Platelets from protease-activated receptor 4 (PAR4)-deficient mice are unresponsive to thrombin, and Par4-/- mice have prolonged bleeding times and are protected against thrombosis. However, in addition to its role in platelets, PAR4 contributes to thrombin signaling in cells in the blood vessel wall that might participate in hemostasis and thrombosis, such as endothelial cells. To determine whether the hemostatic and thrombotic phenotypes of Par4-/- mice were due to loss of PAR4 function in hematopoietic vs. other cell types, tail bleed times and thromboplastin-induced pulmonary embolism were examined in lethally irradiated mice reconstituted with Par4+/+ or Par4-/- bone marrow. In Par4+/+ and Par4-/- mice reconstituted with Par4+/+ marrow, the median tail bleed times were 2.0 and 1.7 min, respectively, vs. > 10 min for both Par4+/+ and Par4-/- mice reconstituted with Par4-/- marrow. In the pulmonary embolism model, Par4+/+ and Par4-/- mice reconstituted with Par4+/+ marrow survived a median of 3.7 and 2.8 min, respectively, after administration of thromboplastin, vs. > 20 min for both Par4+/+ and Par4-/- mice reconstituted with Par4-/- marrow. Further, the phenotype of mice reconstituted with Par4-/- marrow was almost as dramatic as that seen in Nf-e2-/- mice, which lack platelets. These data strongly suggest that increased tail bleed times and protection against thrombosis in Par4-/- mice are accounted for by lack of PAR4 function in platelets, emphasize the importance of thrombin signaling in platelets among the multiple pathways and cell types that govern hemostasis and thrombosis.
Assuntos
Hemostasia , Receptores de Trombina/genética , Receptores de Trombina/fisiologia , Trombina/metabolismo , Trombose/prevenção & controle , Animais , Plaquetas/metabolismo , Células da Medula Óssea , Transplante de Medula Óssea , Separação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Feminino , Citometria de Fluxo , Genótipo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Fenótipo , Embolia Pulmonar/sangue , Embolia Pulmonar/metabolismo , Transdução de Sinais , Tromboplastina/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: In 50-60 per cent of patients who undergo hepatic resection for metastasis of colorectal cancer the first site of tumour recurrence is extrahepatic, indicating the presence of more extensive disease at the time of resection. The aim of this study was to evaluate whether the presence of disseminated tumour cells in blood and bone marrow could predict extrahepatic tumour recurrence. METHODS: Cytokeratin 20 (CK20) reverse transcriptase-polymerase chain reaction was used to study the presence of tumour cells in preoperative peripheral blood and bone marrow samples from 41 patients with liver metastasis scheduled for surgical resection. RESULTS: CK20 expression was detected in six of 41 peripheral blood samples and in eight of 32 bone marrow samples. There was no correlation between CK20-positive samples and subsequent extrahepatic recurrence. Positive blood samples did, however, correlate with high serum carcinoembryonic antigen level and large tumour volume. None of the 14 patients previously treated with chemotherapy had CK20-positive samples, whereas six of 27 blood and eight of 20 bone marrow samples were positive in the chemotherapy-naive group. CONCLUSION: Although the number of patients in this study is limited, the presence of disseminated tumour cells did not predict subsequent extrahepatic recurrence. The results strongly suggest that the presence of circulating tumour cells in peripheral blood may reflect transient shedding of tumour cells related to large tumour volume.
Assuntos
Neoplasias da Medula Óssea/secundário , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/secundário , Células Neoplásicas Circulantes/patologia , Adulto , Idoso , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Proteínas de Filamentos Intermediários/sangue , Proteínas de Filamentos Intermediários/metabolismo , Queratina-20 , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Reação em Cadeia da Polimerase/métodosRESUMO
Factor X (FX)-deficient embryos suffer partial embryonic lethality with approximately 30% of the embryos arresting at midgestation. The remaining animals survive to term but die perinatally mainly from abdominal or intracranial hemorrhage. We have rescued FX-deficient mice by transplanting fetal liver cells from FX+/+, Rosa26 fetuses into midgestation embryos derived from FX+/- heterozygous crosses. FX-/- embryos were born at the expected frequency and approximately 50% of the FX-/- neonates survived longer than 4 months. FX-/- embryos receiving saline injections that survived to term died perinatally similar to untreated FX-deficient mice. The plasma levels of FX in the rescued 16-week-old FX-/- mice were approximately 1-6% of wild-type levels. beta-Galactosidase-staining cells derived from the donor Rosa26 fetal liver cells were detected in 47% of the livers of adult mice. In addition, donor-derived cells were also recovered in the bone marrow, spleen, lung, and occasionally in the brain and testis. These results suggest that in utero cell transplantation could be an effective therapeutic strategy to treat pathologies resulting from the deficiency of hepatic-expressed factors.
Assuntos
Deficiência do Fator X/terapia , Feto/cirurgia , Transtornos Hemorrágicos/genética , Transtornos Hemorrágicos/prevenção & controle , Hepatócitos/transplante , Animais , Animais Recém-Nascidos , Tempo de Sangramento , Fator X/análise , Deficiência do Fator X/complicações , Deficiência do Fator X/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Quimeras de Transplante , Transplante Homólogo , Útero/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: In order to develop a multi-marker RT-PCR, which as such may be more sensitive than a single marker assay for the detection of disseminated tumor cells, we evaluated six RT-PCR markers: cytokeratin 20 (CK20), carcinoembryonic antigen (CEA), guanylyl cyclase C (GCC), epidermal growth factor receptor (EGFR), matrilysin (MMP-7) and HeLa metastatic gene (HLM). MATERIALS AND METHODS: The expression was studied in human colon tumor cell lines, in colon cancer tissues, and in blood and/or bone marrow samples of colorectal cancer patients and control subjects. RESULTS: The cell lines showed a differential expression pattern. The expression of all markers was detected in control blood samples with the lowest frequency for CK20 and EGFR. Semiquantitative analysis, which was performed to study threshold setting, demonstrated that GCC expression was elevated in patient compared to control samples. However, the reproducibility was questionable. CONCLUSION: The results presented in this study suggest an enhanced sensitivity for a combination of RT-PCR markers. Due to limited specificity however, the development of a multi-marker RT-PCR by using conventional PCR does not seem feasible. Future studies should focus on the potential of quantitative RT-PCR.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/sangue , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Perfilação da Expressão Gênica , Humanos , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
AIMS: Despite informative staging of patients with colorectal cancer, some patients with localised disease at diagnosis will develop recurrence or metastasis. Attempts to improve staging include sensitive detection of disseminated tumour cells in blood and bone marrow by reverse transcriptase polymerase chain reaction (RT-PCR). The results of this study have been considered in relation to the controversial results in the literature to elucidate the usefulness of cytokeratin 20 (CK20) RT-PCR to detect disseminated tumour cells further. PATIENTS/METHODS: Blood and bone marrow samples from 30 patients with colorectal cancer were studied by CK20 RT-PCR. Specificity was evaluated in 47 blood and 15 bone marrow samples from non-cancer controls. In addition, the expression of CK20 mRNA and protein was studied in normal and tumour colon tissue samples. RESULTS: CK20 expression was detected in nine of 30 and nine of 19 of the blood and bone marrow samples from patients with colorectal cancer, respectively. In non-cancer control blood and bone marrow samples, CK20 expression was detected in 10 of 47 and four of 15, respectively. A difference between patient and control samples may be observed in terms of frequency of positive PCR tests. In tissue samples, CK20 mRNA expression was downregulated in tumour compared with normal colon tissue. CONCLUSIONS: CK20 expression was downregulated in tumour tissue compared with normal colon and a background expression of CK20 was seen in some control blood and bone marrow samples. Despite a lack of standardisation (which hampers comparison of studies), these results, together with other reports in the literature, suggest that CK20 may still be a suitable marker, but that background expression and threshold setting should be studied further.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Medula Óssea/secundário , Neoplasias Colorretais/patologia , Proteínas de Filamentos Intermediários/análise , Células Neoplásicas Circulantes/química , Biomarcadores Tumorais/genética , Neoplasias da Medula Óssea/diagnóstico , Regulação para Baixo , Humanos , Proteínas de Filamentos Intermediários/genética , Queratina-20 , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase/métodos , Controle de Qualidade , RNA Mensageiro/genética , RNA Neoplásico/genéticaRESUMO
Bleomycin is an antineoplastic drug commonly used for the treatment of many carcinomas and lymphomas. Its toxic side effect on lung tissue is a major limitation to its use, with approximately 3-5% of patients affected. Although the number of affected patients is small, the damage incurred by bleomycin in these patients is often irreversible and, at times, fatal. A number of therapies have been shown to be effective in animal studies to minimize damage, but to date no "magic bullet" has been identified. Many proteins of the fibrinolytic system have been implicated as playing a role in the progression of the disease, one of which is fibrinogen (Fg) acting in the context of a fibroproliferative agent. Its presence correlates with an upregulation of plasminogen activator inhibitor-1 and tissue factor in alveolar cells surrounding the lesion area. It is believed that Fg participates in the activation and migration of fibroblasts and provides a scaffold, in the form of fibrin, for cell migration following induction of acute lung injury. To further understand the mechanism of injury following bleomycin treatment and the possible role of fibrinogen therein, mice have been generated with a targeted deletion of the gamma-chain of Fg, which resulted in the absence of detectable circulating Fg. The offsprings of Fg heterozygous mice (FG+/-) mice follow Mendelian distributions indicating no embryonic lethality with this deletion. Approximately one-half of the Fg-deficient (FG-/-) neonates exhibited bleeding episodes, approximately one-half of which were fatal. For the pulmonary fibrosis study, FG-/- mice and wildtype littermates were administered a bleomycin solution intratracheally and the disease was allowed to progress for two weeks. The mice were then sacrificed, the left lung was excised for hydroxyproline analysis, the right lung was processed for histologic profiling. Examination of trichrome stained sections, surprisingly, revealed no qualitative difference between wildtype and FG-/- animals. The extent and pattern of the deposition of collagen were also similar. These results were quantitatively confirmed by hydroxyproline analysis, which revealed equivalent increases in collagen content between wildtype and FG-/- animals when compared to appropriate saline controls. Analysis of the early acute inflammatory stage of the disease showed a difference in the neutrophil population between days three and five of the disease. These studies suggest that, although fibrinogen is not required for collagen deposition at the later stage of the disease, it may play a role in the early acute inflammation stage.
Assuntos
Fibrinogênio/fisiologia , Fibrose Pulmonar/genética , Animais , Fibrinogênio/genética , Deleção de Genes , Camundongos , Camundongos Knockout , FenótipoRESUMO
Activated protein C functions directly as an anticoagulant and indirectly as a profibrinolytic enzyme. To determine whether the fibrin deposition previously observed in PC(-/-) murine embryos and neonates was mediated through the FXI pathway, PC(+/-)/FXI(-/-) mice were generated and crossbred to produce double-deficient progeny (PC(-/-)/FXI(-/-)). PC(-/-)/FXI(-/-) mice survived the early lethality observed in the PC(-/-)/FXI(+/+) neonates, with the oldest PC(-/-)/FXI(-/-) animal living to 3 months of age. However, the majority of these animals was sedentary and significantly growth-retarded. On sacrifice or natural death, all of these PC(-/-)/FXI(-/-) mice demonstrated massive systemic fibrin deposition with concomitant hemorrhage and fibrosis, as confirmed through histological analyses. Several of these animals also presented with enlarged lymph nodes and extensive lymphatic fluid in the thoracic cavity. Thus, although a number of the PC(-/-)/FXI(-/-) mice survived the lethal perinatal coagulopathy seen in the PC(-/-) neonates, they nonetheless succumbed to overwhelming thrombotic disease in later life. This combined deficiency state provided the first clear indication that the course of a severe thrombotic disorder could be manipulated by blocking the intrinsic pathway and provided the first opportunity to study a total protein C deficiency in an adult animal.
Assuntos
Deficiência do Fator XI/genética , Deficiência de Proteína C/genética , Animais , Animais Recém-Nascidos , Cruzamentos Genéticos , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário e Fetal/genética , Fator XI/genética , Fator XI/metabolismo , Feminino , Fibrina/metabolismo , Genótipo , Interleucina-6/sangue , Fígado/metabolismo , Fígado/patologia , Pulmão/metabolismo , Pulmão/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Inibidor 1 de Ativador de Plasminogênio/sangue , Gravidez , Proteína C/genética , Proteína C/metabolismo , Solubilidade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Clinical studies have indicated that high plasma levels of fibrinogen, or decreased fibrinolytic potential, are conducive to an increased risk of cardiovascular disease. Other investigations have shown that insoluble fibrin promotes atherosclerotic lesion formation by affecting smooth muscle cell proliferation, collagen deposition, and cholesterol accumulation. To directly assess the physiological impact of an imbalanced fibrinolytic system on both early and late stages of this disease, mice deficient for plasminogen activator inhibitor-1 (PAI-1(-/-)) were used in a model of vascular injury/repair, and the resulting phenotype compared to that of wild-type (WT) mice. A copper-induced arterial injury was found to generate a lesion with characteristics similar to many of the clinical features of atherosclerosis. Fibrin deposition in the injured arterial wall at early (7 days) and late (21 days) times after copper cuff placement was prevalent in WT mice, but was greatly diminished in PAI-1(-/-) mice. A multilayered neointima with enhanced collagen deposition was evident at day 21 in WT mice. In contrast, only diffuse fibrin was identified in the adventitial compartments of arteries from PAI-1(-/-) mice, with no evidence of a neointima. Neovascularization was observed in the adventitia and was more extensive in WT arteries, relative to PAI-1(-/-) arteries. Additionally, enhanced PAI-1 expression and fat deposition were seen only in the arterial walls of WT mice. The results of this study emphasize the involvement of the fibrinolytic system in vascular repair processes after injury and indicate that alterations in the fibrinolytic balance in the vessel wall have a profound effect on the development and progression of vascular lesion formation.
Assuntos
Vasos Sanguíneos/fisiopatologia , Cobre/administração & dosagem , Inibidor 1 de Ativador de Plasminogênio/deficiência , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Artérias Carótidas/patologia , Artérias Carótidas/fisiopatologia , Artérias Carótidas/ultraestrutura , Divisão Celular , Feminino , Genótipo , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Inibidor 1 de Ativador de Plasminogênio/genética , Fatores de Tempo , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Túnica Íntima/fisiopatologia , Túnica Média/metabolismo , Túnica Média/patologia , Túnica Média/fisiopatologiaRESUMO
In addition to their well-known roles in hemostasis, fibrinogen (Fg) and fibrin (Fn) have been implicated in a number of other physiological and pathophysiological events. One of these involves the fibroproliferative response after acute lung injury, which is the focus of the current study. Mice with a total Fg deficiency (FG(-/-)) were generated by breeding heterozygous (FG(+/-)) pairs, each of which contained an allele with a targeted deletion of its Fg-gamma-chain gene. The resulting FG(-/-) animals did not possess detectable plasma Fg. FG(-/-) mice were then used to assess the roles of Fg and Fn in a bleomycin-induced acute lung injury model. Intratracheal administration of bleomycin in wild-type and FG(-/-) mice resulted in equivalent deposition of interstitial collagen and fibrotic lesions at days 7 and 14 after administration. This indicates that Fg and/or Fn are not essential for the development of bleomycin-induced pulmonary fibrosis.
Assuntos
Afibrinogenemia/genética , Fibrinogênio/genética , Fibrose Pulmonar/genética , Afibrinogenemia/metabolismo , Afibrinogenemia/patologia , Animais , Bleomicina , Colágeno/metabolismo , DNA/análise , Modelos Animais de Doenças , Feminino , Fibrinogênio/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Marcação de Genes , Heterozigoto , Homozigoto , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologiaRESUMO
To determine whether an additional loss of the coagulation factor VII (FVII) gene influenced the coagulopathy observed in protein C gene-deficient (PC(-/-)) embryos and neonates, we crossed mice doubly heterozygous for the factor VII (FVII(+/-)) and protein C (PC(+/-)) genes to produce offspring possessing the 9 predicted genotypic combinations. FVII(-/-)/PC(-/-) embryos, although present at their expected Mendelian frequency, displayed a phenotype that had not been observed in either the FVII or PC singly deficient embryos. At E12.5 days postcoitum (dpc), FVII(-/-)/PC(-/-) embryos demonstrated an intra- and extravascular coagulopathy that progressed with substantial concomitant hemorrhage and peripheral edema by E17.5dpc, resulting in mortality immediately after birth. FVII(+/-)/PC(-/-) embryos showed a less severe phenotype, suggesting a gene dosage effect. The lack of rescue of PC(-/-) embryos and neonates and augmented coagulopathy resulting from an additional heterozygous or homozygous FVII deficiency are probably due to increased factor Xa and thrombin generation, resulting from loss of FVIIa-dependent tissue factor pathway inhibitor function and the absence of control at the levels of factors Va and VIIIa. The presence of fibrin in embryos in the absence of fetal FVII suggests that significant clot-generating potential exists outside of the embryonic factor VII-dependent pathway.
Assuntos
Deficiência do Fator VII/complicações , Deficiência de Proteína C/complicações , Doenças Uterinas/etiologia , Animais , Cruzamentos Genéticos , Desenvolvimento Embrionário e Fetal , Deficiência do Fator VII/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Deficiência de Proteína C/genéticaRESUMO
Dendritic cells (DC) capture microorganisms that enter peripheral mucosal tissues and then migrate to secondary lymphoid organs, where they present these in antigenic form to resting T cells and thus initiate adaptive immune responses. Here, we describe the properties of a DC-specific C-type lectin, DC-SIGN, that is highly expressed on DC present in mucosal tissues and binds to the HIV-1 envelope glycoprotein gp120. DC-SIGN does not function as a receptor for viral entry into DC but instead promotes efficient infection in trans of cells that express CD4 and chemokine receptors. We propose that DC-SIGN efficiently captures HIV-1 in the periphery and facilitates its transport to secondary lymphoid organs rich in T cells, to enhance infection in trans of these target cells.
Assuntos
Linfócitos T CD4-Positivos/virologia , Células Dendríticas/fisiologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/transmissão , HIV-1/fisiologia , Mucosa/virologia , Receptores de HIV/fisiologia , Antígenos CD4/fisiologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Moléculas de Adesão Celular/fisiologia , Movimento Celular , Células Cultivadas , Colo do Útero/citologia , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Humanos , Lectinas/fisiologia , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/virologia , Tecido Linfoide/citologia , Tecido Linfoide/virologia , Substâncias Macromoleculares , Masculino , Mucosa/citologia , Receptores CCR5/fisiologia , Reto/citologia , Transfecção , Útero/citologiaRESUMO
Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumour progression and metastasis. In this study, we investigated the in vitro and in vivo expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA and protein in a previously described human melanoma xenograft model. This model consists of eight human melanoma cell lines with different metastatic behaviour after subcutaneous (s.c.) injection into nude mice. MMP-1 mRNA was detectable in all cell lines by reverse transcription polymerase chain reaction (RT-PCR), but the expression was too low to be detected by Northern blot analysis. No MMP-1 protein could be found using Western blotting. MMP-2 mRNA and protein were present in all cell lines, with the highest expression of both latent and active MMP-2 in the highest metastatic cell lines MV3 and BLM. MMP-3 mRNA was expressed in MV3 and BLM, and in the non-metastatic cell line 530, whereas MMP-3 protein was detectable only in MV3 and BLM. None of the melanoma cell lines expressed MMP-9. TIMP-1 and TIMP-2 mRNA and protein, finally, were present in all cell lines. A correlation between TIMP expression level and metastatic capacity of cell lines, however, was lacking. MMP and TIMP mRNA and protein expression levels were also studied in s.c. xenograft lesions derived from a selection of these cell lines. RT-PCR analysis revealed that MMP-1 mRNA was present in MV3 and BLM xenografts, and to a lesser extent in 530. Positive staining for MMP-1 protein was found in xenograft lesions derived from both low and high metastatic cell lines, indicating an in vivo up-regulation of MMP-1. MMP-2 mRNA was detectable only in xenografts derived from the highly metastatic cell lines 1F6m, MV3 and BLM. In agreement with the in vitro results, the highest levels of both latent and activated MMP-2 protein were observed in MV3 and BLM xenografts. With the exception of MMP-9 mRNA expression in 530 xenografts, MMP-3, MMP-9, and TIMP-1 mRNA and protein were not detectable in any xenograft, indicating a down-regulated expression of MMP-3 and TIMP-1 in vivo. TIMP-2 mRNA and protein were present in all xenografts; interestingly, the strongest immunoreactivity of tumour cells was found at the border of necrotic areas. Our study demonstrates that of all tested components of the matrix metalloproteinase system, only expression of activated MMP-2 correlates with increased malignancy in our melanoma xenograft model, corroborating an important role of MMP-2 in human melanoma invasion and metastasis.
Assuntos
Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinases da Matriz/biossíntese , Melanoma/enzimologia , Melanoma/patologia , Transplante Heterólogo/patologia , Animais , Progressão da Doença , Ativação Enzimática , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Melanoma/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , RNA Mensageiro/metabolismo , Ratos , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Células Tumorais CultivadasAssuntos
Modelos Animais de Doenças , Fatores de Crescimento Endotelial/deficiência , Linfocinas/deficiência , Camundongos Mutantes , Isquemia Miocárdica/patologia , Miocárdio/patologia , Neovascularização Fisiológica/genética , Processamento Alternativo , Animais , Antígenos de Diferenciação , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/química , Marcação de Genes , Testes de Função Cardíaca , Linfocinas/genética , Camundongos , Músculo Esquelético/irrigação sanguínea , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We have identified a novel 3845 bp cDNA differentially expressed in a human melanoma metastasis model. Northern blot analysis showed expression in the poorly and intermediately metastasizing cell lines and a marked downregulation in the highly metastatic cell lines. Using RT-PCR expression was also seen in several other tumor cell lines and normal cell types of human origin. cDNA sequence analysis revealed an ORF of 687 amino acids containing seven putative transmembrane domains C-terminally and a long N-terminus. The gene was mapped to 16q13. Highest homology was observed with members of the EGF-TM7 subfamily of the secretin/calcitonin receptor family. We propose the delineation of a subfamily of TM7 proteins, LN-TM7, containing seven transmembrane proteins with a long N-terminal extracellular part.
Assuntos
Fator de Crescimento Epidérmico/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Expressão Gênica , Humanos , Melanoma , Dados de Sequência Molecular , Metástase Neoplásica , RNA Mensageiro , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
To identify new genes that may contribute to the metastatic pathway of neoplastic cells, we compared mRNA expression of the parental human melanoma cell line 1F6 and its metastatic variant 1F6m using mRNA differential display. We isolated a cDNA clone that was exclusively expressed in 1F6m. Northern blot analysis on a broader panel of human melanoma cell lines with different metastatic capacity following s.c. inoculation into nude mice demonstrated that the gene was expressed only in the most aggressive, highly metastatic cell lines, giving a band of 0.5 kb. The isolated full length cDNA clone showed an open reading frame of 97 amino acids. To study the subcellular localization of the gene product, COS-1 cells were transfected with cDNA of the gene fused to eGFP. We found the fusion protein to be exclusively present in the nucleus. A computer search showed strong homology with human genomic clones all localized on chromosome X (Xq26.3-Xq27.1) and with several expressed sequence tags, all from testis. Localization of the gene on chromosome X was confirmed by genomic PCR on a panel of human chromosome-specific rodent/human hybrid cell lines. Northern blotting and reverse transcription-PCR on 17 different normal human tissue samples showed that the gene was only expressed in normal testis. Reverse transcription-PCR on a great number of different human tumor cell lines showed expression in 25-30% of the melanoma and bladder carcinoma cell lines. Only 2 of 29 other tumor cell lines were positive. Nested PCR analysis of a series of fresh human melanocytic tumors demonstrated expression in 7 of 10 melanomas tested. No expression was seen in benign melanocytic tumors. In addition to melanoma, some malignant tumors from other histological types were also found to be positive. Based on these data, we conclude that the described gene, CTp11 (cancer/testis-associated protein of 11 kDa), is a novel member of the family of cancer/testis antigens.
Assuntos
Antígenos de Neoplasias/genética , Melanoma/metabolismo , Proteínas de Neoplasias/genética , Testículo/metabolismo , Cromossomo X , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/biossíntese , Sequência de Bases , Células COS , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/análise , DNA de Neoplasias , Regulação da Expressão Gênica , Humanos , Masculino , Melanoma/genética , Melanoma/imunologia , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Dados de Sequência Molecular , Peso Molecular , Proteínas de Neoplasias/biossíntese , Frações Subcelulares , Células Tumorais CultivadasRESUMO
Peptides containing the integrin recognition sequence, RGD, can inhibit experimental metastasis of mouse melanoma cells, but the integrin(s) affected in these experiments is unknown. Besides "classical" RGD-binding integrins such as alpha 5 beta 1 and alpha v beta 3, RGD has been reported to bind alpha 4 beta 1, and mAbs to alpha 4 beta 1 can inhibit melanoma metastasis. We investigated the mode of action of the disintegrin eristostatin, an RGD-containing peptide isolated from snake venom, in a human melanoma experimental metastasis model. Lung colonization following i.v. injection of MV3 cells in nude mice was strongly inhibited by eristostatin. MV3 cells bound FITC-eristostatin and adhered to eristostatin-coated wells. This adhesion was partially inhibited by a GRGDSP peptide and by alpha 4 mAb. Binding of FITC-eristostatin to Jurkat cells and adhesion of Jurkat (but not K562) cells to eristostatin-coated wells further suggested that eristostatin binds alpha 4 beta 1, even though, again, alpha 4 mAb only partially inhibited adhesion. Expression of alpha 4 beta 1 was enhanced in metastatic melanoma cells compared to normal melanocytes and nonmetastatic melanoma cells. Finally, eristostatin inhibited adhesion of both MV3 and CHO alpha 4 cells to the alpha 4 beta 1-ligand VCAM-1, while adhesion to other ligands via other integrins was not affected. These findings demonstrate that inhibition of melanoma cell metastasis by RGD-containing peptides such as eristostatin, may be due to interference with alpha 4 beta 1-VCAM binding, in addition to inhibition of the classical RGD-binding integrins.
Assuntos
Integrinas/fisiologia , Melanoma/patologia , Metástase Neoplásica , Peptídeos/uso terapêutico , Receptores de Retorno de Linfócitos/fisiologia , Neoplasias Cutâneas/patologia , Venenos de Víboras/uso terapêutico , Animais , Sítios de Ligação , Humanos , Recém-Nascido , Integrina alfa4beta1 , Integrinas/química , Integrinas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Melanócitos/citologia , Melanoma/tratamento farmacológico , Melanoma/fisiopatologia , Camundongos , Camundongos Nus , Metástase Neoplásica/prevenção & controle , Oligopeptídeos , Peptídeos/farmacocinética , Inibidores da Agregação Plaquetária/uso terapêutico , Receptores de Retorno de Linfócitos/química , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Pele/citologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/fisiopatologia , Venenos de Serpentes , Venenos de Víboras/farmacocinéticaRESUMO
We studied the relation between tumour vascular density and tumour growth rate, metastatic incidence and vascular permeability factor (VPF) mRNA levels in a human xenograft model described previously. Vascular density was determined by automated image analysis. Xenografts derived from cell lines BLM and MV3 showed the highest mean vascular density (MVD), the highest in vivo growth rate, high VPF mRNA levels and rapid development of lung metastases. Xenografts of cell lines M14, Mel57 and MV1 showed a significantly lower degree of vascularization, lower in vivo growth rates and lower levels of VPF mRNA, but formed lung metastases with a similar incidence as those of BLM and MV3. Xenografts from cell line 1F6 did not form lung metastases, whereas tumours derived from a spontaneous mutant of 1F6, designated 1F6m, gave rise to lung metastases to the same extent as Mel57, M14 and MV1 tumours. MVD values in 1F6 and 1F6m xenografts, VPF mRNA levels and in vivo growth rates of 1F6 and 1 F6m xenografts, however, were similar. In conclusion, in the melanoma xenograft model vascular density is correlated with in vivo growth rate and with in vitro VPF mRNA levels, but not with the ability to metastasize.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Linfocinas/biossíntese , Melanoma/irrigação sanguínea , Melanoma/patologia , Transcrição Gênica , Animais , Divisão Celular , Linhagem Celular , Humanos , Cinética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma/metabolismo , Camundongos , Camundongos Nus , Metástase Neoplásica , Neovascularização Patológica , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Transplante Heterólogo/patologia , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Even though integrin alpha v beta 3 is thought to play a role in invasive growth of melanomas, some metastatic melanoma cell lines lack alpha v beta 3, and downmodulation of alpha v beta 3, expression can enhance the invasive capacity of certain melanoma cells. To further investigate this apparent dualistic role of alpha v beta 3, we transfected beta 3 cDNA into the highly metastatic, beta 3-negative human melanoma cell line MV3. MV3 cells adhered to fibronectin but not to fibrinogen or a synthetic RGD peptide, while MV3-beta 3 adhered to all three RGD-containing adhesive ligands, and this adhesion was inhibited by LM609 alpha v beta 3 mAb. Expression of alpha v beta 3 did not affect MV3 in vitro proliferation or in vivo tumorigenicity upon subcutaneous inoculation into nude mice. In contrast, it strongly reduced invasion in matrigel and lung colonization in nude mice of MV3 cells. Thus, certain melanoma cell lines have adopted a metastatic strategy in the absence of alpha v beta 3, and in such cells expression of this integrin leads to a less aggressive phenotype.
Assuntos
Antígenos CD/genética , Melanoma/patologia , Glicoproteínas da Membrana de Plaquetas/genética , Animais , DNA Complementar , Humanos , Integrina beta3 , Neoplasias Pulmonares/secundário , Melanoma/genética , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Transplante de Neoplasias , Transfecção , Células Tumorais CultivadasRESUMO
The highly metastatic human melanoma cell line BLM was transfected with the E1A or E1A + E1B regions of adenovirus 5 (Ad5). A series of progression markers, correlated with the malignant phenotype of parental BLM (including calcyclin, thymosin beta 10, plasminogen activator inhibitors types 1 and 2, urokinase type and tissue type plasminogen activators, vimentin, tissue type transglutaminase, and interleukin-6), was collectively repressed in the transfectants, whereas several control genes were not affected or even induced. The apparently coordinate repression of a set of markers by the same regulator gene, Ad5 E1A in this case, suggests the existence of one pathway under the control of a main switch and predicts that one or more as yet unidentified cellular master genes normally exert this function. A reduced oncogenicity was observed after subcutaneous inoculation of the E1A transfectants into nude mice and provides additional evidence in support of a tumor suppressor function of Ad5 E1A.