RESUMO
Primary hyperparathyroidism is a common disorder with an annual incidence of approximately 0.5 in 1,000 (ref. 1). In more than 95% of cases, the disease is caused by sporadic parathyroid adenoma or sporadic hyperplasia. Some cases are caused by inherited syndromes, such as multiple endocrine neoplasia type 1 (MEN1; ref. 2). In most cases, the molecular basis of parathyroid neoplasia is unknown. Parathyroid adenomas are usually monoclonal, suggesting that one important step in tumour development is a mutation in a progenitor cell. Approximately 30% of sporadic parathyroid tumours show loss of heterozygosity (LOH) for polymorphic markers on 11q13, the site of the MEN1 tumour suppressor gene. This raises the question of whether such sporadic parathyroid tumours are caused by sequential inactivation of both alleles of the MEN1 gene. We recently cloned the MEN1 gene and identified MEN1 germline mutations in fourteen of fifteen kindreds with familial MEN1 (ref. 10). We have studied parathyroid tumours not associated with MEN1 to determine whether somatic mutations in the MEN1 gene are present. Among 33 tumours we found somatic MEN1 gene mutation in 7, while the corresponding MEN1 germline sequence was normal in each patient. All tumours with MEN1 gene mutation showed LOH on 11q13, making the tumour cells hemi- or homozygous for the mutant allele. Thus, somatic MEN1 gene mutation for the mutant allele. Thus, somatic MEN1 gene mutation contributes to tumorigenesis in a substantial number of parathyroid tumours not associated with the MEN1 syndrome.
Assuntos
Neoplasia Endócrina Múltipla Tipo 1/genética , Mutação , Proteínas de Neoplasias/genética , Neoplasias das Paratireoides/genética , Proteínas Proto-Oncogênicas , Cromossomos Humanos Par 11 , Impressões Digitais de DNA , Análise Mutacional de DNA , DNA de Neoplasias/análise , Deleção de Genes , Heterozigoto , HumanosRESUMO
Thrombospondin induces the migration of human melanoma and carcinoma cells. Using a modified Boyden chamber assay, tumor cells migrated to a gradient of soluble thrombospondin (chemotaxis). Checkerboard analysis indicated that directional migration was induced 27-fold greater than stimulation of random motility. Tumor cells also migrated in a dose-dependent manner to a gradient of substratum-bound thrombospondin (haptotaxis). A series of human melanoma and carcinoma cells were compared for their relative motility stimulation by thrombospondin haptotaxis vs. chemotaxis. Some cell lines exhibited a stronger haptotactic response compared to their chemotactic response while other lines exhibited little or no migration response to thrombospondin. Human A2058 melanoma cells which exhibit a strong haptotactic and chemotactic response to thrombospondin were used to study the structural domains of thrombospondin required for the response. Monoclonal antibody C6.7, which binds to the COOH-terminal region of thrombospondin, inhibited haptotaxis in a dose-dependent optimal manner. C6.7 had no significant effect on thrombospondin chemotaxis. In contrast, monoclonal antibody A2.5, heparin, and fucoidan, which bind to the NH2-terminal heparin-binding domain of thrombospondin, inhibited thrombospondin chemotaxis but not haptotaxis. Monoclonal antibody A6.1 directed against the internal core region of thrombospondin had no significant effect on haptotaxis or chemotaxis. Synthetic peptides GRGDS (50 micrograms/ml), but not GRGES, blocked tumor cell haptotaxis on fibronectin, but had minimal effect on thrombospondin or laminin haptotaxis. The 140-kD fragment of thrombospondin lacking the heparin-binding amino-terminal region retained the property to fully mediate haptotaxis but not chemotaxis. When the COOH region of the 140-kD fragment, containing the C6.7-binding site, was cleaved off, the resulting 120-kD fragment (which retains the RGDA sequence) failed to induce haptotaxis. Separate structural domains of thrombospondin are therefore required for tumor cell haptotaxis vs. chemotaxis. This may have implications during hematogenous cancer metastases formation.
Assuntos
Plaquetas/fisiologia , Quimiotaxia , Glicoproteínas/fisiologia , Células Tumorais Cultivadas/fisiologia , Neoplasias da Mama , Carcinoma , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Laminina/farmacologia , Trombospondinas , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin receptor points to a role for the integrins in the interaction of endothelial cells with TSP. We also found that TSP modulates endothelial cell growth. When added to quiescent LE-II cells, it inhibited the mitogenic effects of serum and the angiogenic factor bFGF, in a dose-dependent manner. The inhibition of DNA synthesis detected in the mitogenic assay resulted in a true inhibition of BAEC and LE-II cell growth, as assessed by proliferation assay. This work indicates that TSP affects endothelial cell adhesion, spreading, motility and growth. TSP, therefore, has the potential to modulate the angiogenic process.
Assuntos
Plaquetas/fisiologia , Endotélio Vascular/fisiologia , Glicoproteínas de Membrana/farmacologia , Neovascularização Patológica , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Cinética , TrombospondinasRESUMO
A new in vitro model has been developed for studying migration of human polymorphonuclear leukocytes (PMN) through living native cellular and matrix barriers. Human amnion membrane consists of a single layer of epithelium bound to a continuous basement membrane interfacing an avascular collagenous stroma. Living amnion was placed in plastic chambers with separate compartments on each side of the membrane. PMN were introduced on the epithelial side of the amnion, and a Millipore filter (Millipore Corp., Bedford, Mass.) was placed against the stromal side. In response to N-formylmethionyl-leucyl- phenylanlanine (FMLP) chemoattractant, PMN penetrated the full thickness of the amnion and were collected and counted on the filter. The rate of PMN traversal of the amnion was dependent on the concentration of FMLP (optimal at 10(-8)M) as well as the slope of the FMLP gradient across the amnion. The route of PMN migration was studied by transmission electron microscopy. PMN first attached to the epithelial surface, then infiltrated between intercellular junctions. PMN migrated around or through tight junction and hemidesmosome attachments. The PMN then penetrated the basement membrane and migrated through the dense collagenous stroma. The present amnion migration system has characteristics of the in vivo inflammatory state not described in any previous method for monitoring PMN migration in vitro. Prior methods have not used native epithelium, whole basement membrane, or collagenous stroma. PMN penetration of these barriers occurs in the normal inflammatory response and probably involves biochemical mechanisms not required for simple migration through the pores of an artificial filter. The amnion system can be useful for future biochemical and morphological studies of PMN penetration of these barriers and possible repair processes that may follow.
Assuntos
Âmnio/fisiologia , Quimiotaxia de Leucócito , Neutrófilos/fisiologia , Âmnio/ultraestrutura , Membrana Basal/fisiologia , Epitélio/fisiologia , Epitélio/ultraestrutura , Humanos , Junções Intercelulares/fisiologia , Cinética , N-Formilmetionina/análogos & derivados , N-Formilmetionina/farmacologia , N-Formilmetionina Leucil-Fenilalanina , Oligopeptídeos/farmacologiaRESUMO
Transduction of signals initiating motility by extracellular matrix (ECM) molecules differed depending on the type of matrix molecule and whether the ligand was in solution or bound to a substratum. Laminin, fibronectin, and type IV collagen stimulated both chemotaxis and haptotaxis of the A2058 human melanoma cell line. Peak chemotactic responses were reached at 50-200 nM for laminin, 50-100 nM for fibronectin, and 200-370 nM for type IV collagen. Checkerboard analysis of each attractant in solution demonstrated a predominantly directional (chemotactic) response, with a minor chemokinetic component. The cells also migrated in a concentration-dependent manner to insoluble step gradients of substratum-bound attractant (haptotaxis). The haptotactic responses reached maximal levels at coating concentrations of 20 nM for laminin and type IV collagen, and from 30 to 45 nM for fibronectin. Pretreatment of cells with the protein synthesis inhibitor, cycloheximide (5 micrograms/ml), resulted in a 5-30% inhibition of both chemotactic and haptotactic responses to each matrix protein, indicating that de novo protein synthesis was not required for a significant motility response. Pretreatment of cells with 50-500 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence GRGDS resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis and haptotaxis (70-80% inhibition compared to control motility); negative control peptide GRGES had only a minimal effect. Neither GRGDS nor GRGES significantly inhibited motility to laminin or type IV collagen. Therefore, these results support a role for the RGD-directed integrin receptor in both types of motility response to fibronectin. After pretreatment with pertussis toxin (PT), chemotactic responses to laminin, fibronectin, and type IV collagen were distinctly different. Chemotaxis to laminin was intermediate in sensitivity; chemotaxis to fibronectin was completely insensitive; and chemotaxis to type IV collagen was profoundly inhibited by PT. In marked contrast to the inhibition of chemotaxis, the hepatotactic responses to all three ligands were unaffected by any of the tested concentrations of PT. High concentrations of cholera toxin (CT; 10 micrograms/ml) or the cAMP analogue, 8-Br-cAMP (0.5 mM), did not significantly affect chemotactic or haptotactic motility to any of the attractant proteins, ruling out the involvement of cAMP in the biochemical pathway initiating motility in these cells. The sensitivity of chemotaxis induced by laminin and type IV collagen, but not fibronectin, to PT indicates the involvement of a PT-sensitive G protein in transduction of the signals initiating motility to soluble laminin and type IV collagen.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Movimento Celular , Quimiotaxia , Transdução de Sinais , Células Tumorais Cultivadas/fisiologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Colágeno , Cicloeximida/farmacologia , Matriz Extracelular/fisiologia , Fibronectinas , Humanos , Laminina , Melanoma , Toxina Pertussis , Células Tumorais Cultivadas/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.
Assuntos
Adesão Celular , Endotélio Vascular/fisiologia , Matriz Extracelular/fisiologia , Integrinas/fisiologia , Oligopeptídeos/farmacologia , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Animais , Aorta , Bovinos , Adesão Celular/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Fibrinogênio/fisiologia , Fibronectinas/fisiologia , Imunofluorescência , Laminina/fisiologia , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismoRESUMO
One of the first steps in neovascularization is dissolution of the basement membrane at the point of endothelial outgrowth. An assay was developed to determine whether basement membrane collagens (types IV and V) are degraded by endothelial cells migrating toward a chemotactic stimulus. Fetal bovine endothelial cells were placed on one side of a filter containing the collagen substrate, and a chemoattractant derived from retinal extracts was placed on the opposite side. Degradation of both type IV and type V collagens was observed when the retinal factor was placed on the side of the filter opposite the endothelial cells. Metalloproteinases that cleaved type IV and type V collagens could be extracted from the endothelial cells with detergents. Such endothelial cell-associated (possibly membrane-bound) proteinases may locally disrupt the basement membrane and facilitate the outgrowth of capillary sprouts toward the angiogenic stimulus.
Assuntos
Membrana Basal/metabolismo , Colágeno/metabolismo , Animais , Bovinos , Movimento Celular , Quimiotaxia , Endotélio/metabolismo , Neovascularização Patológica , Retina/fisiologiaRESUMO
Metastatic mouse melanoma cells have a high affinity for the basement membrane and the ability to degrade it; these properties may allow tumor cells to invade the membrane and disseminate. In this study it was found that the metastatic potential of mouse melanoma cells varied when the cells were exposed in culture to fibronectin or laminin. After removal of fibronectin or exposure to laminin, the cells had an increased affinity for basement membrane collagen, were more invasive of basement membranes in vitro, and produced more lung colonies in vivo. These changes are correlated with and may be due to an increase in the laminin-binding capacity of the tumor cell surface.
Assuntos
Fibronectinas/farmacologia , Laminina/farmacologia , Melanoma/patologia , Metástase Neoplásica/patologia , Âmnio/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Soros Imunes , Cinética , Camundongos , GravidezRESUMO
Laser capture microdissection (LCM) under direct microscopic visualization permits rapid one-step procurement of selected human cell populations from a section of complex, heterogeneous tissue. In this technique, a transparent thermoplastic film (ethylene vinyl acetate polymer) is applied to the surface of the tissue section on a standard glass histopathology slide; a carbon dioxide laser pulse then specifically activates the film above the cells of interest. Strong focal adhesion allows selective procurement of the targeted cells. Multiple examples of LCM transfer and tissue analysis, including polymerase chain reaction amplification of DNA and RNA, and enzyme recovery from transferred tissue are demonstrated.
Assuntos
Separação Celular/métodos , Técnicas Histológicas , Lasers , DNA/análise , Dissecação , Secções Congeladas , Humanos , Inclusão em Parafina , Reação em Cadeia da Polimerase , Polivinil , Lesões Pré-Cancerosas/patologia , RNA/análise , Fixação de TecidosRESUMO
Multiple endocrine neoplasia-type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized by tumors in parathyroids, enteropancreatic endocrine tissues, and the anterior pituitary. DNA sequencing from a previously identified minimal interval on chromosome 11q13 identified several candidate genes, one of which contained 12 different frameshift, nonsense, missense, and in-frame deletion mutations in 14 probands from 15 families. The MEN1 gene contains 10 exons and encodes a ubiquitously expressed 2.8-kilobase transcript. The predicted 610-amino acid protein product, termed menin, exhibits no apparent similarities to any previously known proteins. The identification of MEN1 will enable improved understanding of the mechanism of endocrine tumorigenesis and should facilitate early diagnosis.
Assuntos
Clonagem Molecular , Genes Supressores de Tumor , Neoplasia Endócrina Múltipla Tipo 1/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , DNA Complementar/genética , Éxons , Mutação da Fase de Leitura , Humanos , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/químicaRESUMO
Until recently, the low-abundance (LA) range of the serum proteome was an unexplored reservoir of diagnostic information. Today it is increasingly appreciated that a diagnostic goldmine of LA biomarkers resides in the blood stream in complexed association with more abundant higher molecular weight carrier proteins such as albumin and immunoglobulins. As we now look to the possibility of harvesting these LA biomarkers more efficiently through engineered nano-scale particles, mathematical approaches are needed in order to reveal the mechanisms by which blood carrier proteins act as molecular 'mops' for LA diagnostic cargo, and the functional relationships between bound LA biomarker concentrations and other variables of interest such as biomarker intravasation and clearance rates and protein half-lives in the bloodstream. Here we show, by simple mathematical modeling, how the relative abundance of large carrier proteins and their longer half-lives in the bloodstream work together to amplify the total blood concentration of these tiny biomarkers. The analysis further suggests that alterations in the production of biomarkers lead to gradual rather than immediate changes in biomarker levels in the blood circulation. The model analysis also points to the characteristics of artificial nano-particles that would render them more efficient harvesters of tumor biomarkers in the circulation, opening up possibilities for the early detection of curable disease, rather than simply better detection of advanced disease.
Assuntos
Biomarcadores/sangue , Proteínas de Transporte/sangue , Modelos Biológicos , Biomarcadores/metabolismo , Meia-Vida , Humanos , Rim/metabolismo , Ligação Proteica , ProteomaRESUMO
As the list of expressed human genes expands, a major scientific challenge is to understand the molecular events that drive normal tissue morphogenesis and the evolution of pathological lesions in actual tissue. Laser capture microdissection (LCM) has been developed to provide a reliable method to procure pure populations of cells from specific microscopic regions of tissue sections, in one step, under direct visualization. The cells of interest are transferred to a polymer film that is activated by laser pulses. The exact morphology of the procured cells (with intact DNA, RNA and proteins) is retained and held on the transfer film. With the advent of LCM, cDNA libraries can be developed from pure cells obtained directly from stained tissue, and microhybridization arrays of thousands of genes can now be used to examine gene expression in microdissected human tissue biopsies. The fluctuation of expressed genes or alterations in the cellular DNA that correlate with a particular disease stage can ultimately be compared within or between individual patients. Such a fingerprint of gene-expression patterns can provide crucial clues for etiology and might, ultimately, contribute to diagnostic decisions and therapies tailored to the individual patient. Molecules found to be associated with a defined pathological lesion might serve as imaging ot therapeutic targets.
Assuntos
Microscopia/métodos , DNA , Técnicas Genéticas , Humanos , Lasers , Microscopia/instrumentação , Patologia , RNARESUMO
Human and murine tumor cells contain cell surface receptors for the basement membrane glycoprotein laminin. Since a biologic role for the receptor had not previously been demonstrated, we explored the possibility that the laminin receptor may be involved in hematogenous metastases formation. Preincubation of metastatic murine melanoma cells with syngeneic whole laminin followed by tail vein injection increased tumor cell retention in the lung and strongly stimulated metastases formation. The domain of the laminin molecule responsible for stimulating metastases was identified. Laminin is a cross-shaped molecule with three short arms and one long arm. All arms have globular end regions. Purified protease-derived fragments of laminin were prepared which (a) lacked only the long arm of the molecule (alpha fragment) or, (b) lacked both the long arm and the globular end regions of the short arms (C1 fragment). Both types of fragments contained the laminin receptor binding region. The fragments had opposite effects on metastases. The alpha fragment stimulated metastases formation to the same extent as whole laminin. In contrast, the C1 fragment greatly reduced or abolished metastases formation in a dose-dependent manner. The C1 fragment also inhibited tumor cell attachment to whole amnion basement membrane in vitro. We conclude that intact globular end regions on the short arms (but not the long arm) of the cell surface receptor-bound laminin molecule are necessary for stimulating metastases by the intravenous route.
Assuntos
Laminina/fisiologia , Neoplasias Pulmonares/secundário , Melanoma/patologia , Metástase Neoplásica/patologia , Receptores de Superfície Celular/fisiologia , Animais , Cinética , Laminina/farmacologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LamininaRESUMO
Polymorphonuclear neutrophils (PMN) traverse basement membrane to reach sites of infection. We have studied the role of laminin, a specific basement membrane component, in this process using three assay systems. In the Boyden chamber, laminin was found to stimulate chemotaxis of neutrophils while fibronectin did not. Co-incubation of cells with antibody to laminin blocked this chemotaxis, while antibody to fibronectin was without effect. In the human amnion system, neutrophils were shown to penetrate through the tissue when the peptide chemoattractant f-Met-Leu-Phe was placed on the opposing side. Antibody to laminin, but not to fibronectin, blocked this penetration. In an attachment assay system, laminin, but not fibronectin, was found to increase dispase-treated neutrophil attachment to type IV (basement membrane) collagen-coated plastic and to a plastic substrate itself. Electrophoretic analysis of PMN extract indicated the presence of laminin, and indirect immunofluorescence suggested that laminin is localized on the surface of the neutrophils. These data suggest that PMN can bind laminin on their cell surfaces, use laminin to attach to basement (type IV) membrane collagen, and migrate toward a gradient of laminin. These properties may be important for the passage of neutrophils from the circulation to sites of infection.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Laminina/farmacologia , Neutrófilos/citologia , Âmnio/citologia , Animais , Adesão Celular/efeitos dos fármacos , Colágeno , Relação Dose-Resposta a Droga , Feminino , Fibronectinas/farmacologia , Imunofluorescência , Humanos , Neutrófilos/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Gravidez , CoelhosRESUMO
NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.
Assuntos
DNA de Neoplasias/genética , Leucemia/genética , Metástase Neoplásica , Neoplasias Experimentais/patologia , Oncogenes , Animais , Transformação Celular Viral , Leucemia/patologia , Camundongos , Camundongos Nus , Fenótipo , TransfecçãoRESUMO
L651582 (Merck Institute for Therapeutic Research, Rahway, NJ) is a novel carboxyamide-amino-imidazole compound originally developed as a coccidiostat (U.S. patent No. 4,590,201). We studied the inhibitory effects of this compound on cancer proliferation, adhesion, and motility in vitro and in vivo in a model of ovarian cancer progression. L651582 reversibly inhibited up to 60% of the autocrine motility factor-stimulated tumor cell motility and tumor cell adhesion to tissue culture plastic. Autocrine motility factor-stimulated phosphoinositide metabolism was reduced significantly by treatment of the cells with 3 microM L651582 (P = .022). Thymidine incorporation and clonogenic growth of A2058 human melanoma, MDA-MB-231 human breast cancer, OVCAR-3 human ovarian cancer, and 5R-transformed rat embryo fibroblast cell lines were inhibited 60%-80% by 1-10 microM L651582. Intraperitoneal injection of OVCAR-3 cells causes malignant ascites, peritoneal carcinomatosis, and serosal and visceral seeding that, if left untreated, are lethal to nude mice. Intraperitoneal L651582 markedly prolonged survival of nude mice heavily laden with ovarian cancer [mean survival time of treated group divided by mean survival time of control group = 220% (P less than .03)]. The apparent mechanism of action of L651582 is via inhibition of the receptor-mediated stimulation of effector enzymes utilizing guanine nucleotide-binding protein signal transduction, which thus makes L651582 a novel anticancer agent. L651582 should be considered for further clinical development.
Assuntos
Aminoimidazol Carboxamida/farmacologia , Antineoplásicos/farmacologia , Imidazóis/farmacologia , Triazóis , Aminoimidazol Carboxamida/análogos & derivados , Animais , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Feminino , Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Camundongos , Metástase Neoplásica , Fosfatidilinositóis/metabolismo , Ratos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
A human colon adenocarcinoma cell line (LoVo) established from a lymph node metastasis was used to study properties associated with invasive tumor cells. Human amniotic membranes were used as invasion barriers to select highly invasive and noninvasive subpopulations of cells from the parent LoVo line. Enriched subpopulations were compared with respect to parameters associated with invasion. The invasive cells were more invasive in vitro and more highly sialylated than either the parental or noninvasive line. Invasive cells migrated more strongly in vitro toward gradients of soluble and insoluble laminin, and their augmented migration correlated with increased adhesion and spreading on laminin-coated substrata. Invasive cells also had the highest level of specific laminin-binding activity. Thus, the invasive cells were shown to possess specific properties that correlated with their successful traversal of the human amnion membrane.
Assuntos
Adenocarcinoma/patologia , Neoplasias Colorretais/patologia , Invasividade Neoplásica/patologia , Divisão Celular , Linhagem Celular , Movimento Celular , Colágeno/fisiologia , Fibronectinas/fisiologia , Humanos , Laminina/metabolismoRESUMO
Tumor cells from the murine T241 fibrosarcoma, which rapidly and reproducibility produces pulmonary metastases, were tested in vitro for their ability to degrade isolated pulmonary basement membrane. Degradation of basement membrane substrate was quantified by the culture of the substrate with tumor cells and measurement of the solubilized hydroxyproline and hexose glycoprotein at neutral pH. It was found that tumor cells collected in the tumor venous drainage were associated with a significantly greater solubilization of basement membrane than were tumor cells obtained from the primary tumor mass. Tumor cells were also assayed for their ability to solubilize type I collagen purified from human dura. Venous effluent tumor cells solubilized collagen to a significantly greater level than primary tumor cells, spleen cells, or liver cells. These findings raised the possibility that metastasizing tumor cells may be a distinct tumor subpopulation with regard to invasive potential.
Assuntos
Membrana Basal/patologia , Neoplasias Pulmonares/patologia , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes , Animais , Membrana Basal/metabolismo , Colágeno/metabolismo , Fibrossarcoma/enzimologia , Fibrossarcoma/patologia , Técnicas In Vitro , Neoplasias Pulmonares/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Colagenase Microbiana/metabolismo , Invasividade Neoplásica , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , SolubilidadeRESUMO
The effects of estradiol and two synthetic progestins (ORG2058 and R5020) on the expression of the high-affinity, metastasis-associated laminin receptor in two human breast carcinoma cell lines were examined. The T47D cell line contains estrogen and progesterone receptors, but the MDA-MB 231 cell line lacks both receptors. Treatment of T47D cells with 10(-9) M estradiol alone results in a three-fold increase (P less than or equal to .05) in the steady-state level of laminin receptor mRNA determined by RNA blot analysis as well as in cell-surface, laminin receptor expression that is evaluated by immunofluorescence. No effects of estradiol on the receptor-negative MDA-MB 231 cells were observed. Untreated and steroid-treated MDA-MB 231 cells had higher levels of laminin receptor mRNA than did untreated or estradiol-treated T47D cells. A more dramatic increase (five-fold; P less than or equal to .005) of mRNA and cell-surface expression in T47D cells was observed after treatment with estradiol plus 10(-8) M progestin or with progestin alone. Estradiol treatment also increased chemotaxis and haptotaxis of T47D cells but not of MDA-MB 231 cells to laminin; it had no effect on the attachment of these latter cells to laminin. Interestingly, treatment with estradiol plus progestin or progestin alone significantly increased the attachment of T47D cells to laminin but did not have an effect on either haptotaxis or chemotaxis to laminin. These results suggest that the various cell-laminin interactions are mediated by different mechanisms. The augmentation of laminin receptor mRNA by estrogen and progesterone treatment in hormone receptor-positive cells, but not in cells that lack these receptors, may relate functionally to the difference in the clinical aggressiveness between classes of breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Laminina/metabolismo , Congêneres da Progesterona/farmacologia , Receptores Imunológicos/biossíntese , Movimento Celular/efeitos dos fármacos , Imunofluorescência , Humanos , Laminina/farmacologia , Pregnenodionas/farmacologia , Promegestona/farmacologia , RNA Mensageiro/metabolismo , Receptores Imunológicos/efeitos dos fármacos , Receptores de Laminina , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The energy requirements via glycolytic pathways were directly measured in migrating tumor cells. Motility in the metastatic human melanoma cell line A2058, stimulated by insulinlike growth factor I (IGF-I), depends on glycolysis in the presence of glucose as its principal source of energy. Motility in glucose-free medium was 75% reduced and utilized mitochondrial respiration (inhibited by oligomycin). With increasing (physiologic) glucose concentrations, there was a dramatic shift to anaerobic glycolysis as the energy source and 93% elimination of the oligomycin inhibition of motility. Oxamate, an inhibitor of glycolysis, inhibited motility at all glucose concentrations. CO2 production from glycolysis and from the hexose monophosphate shunt was measured in migrating tumor cells. The time course and glucose-dose dependence of glycolytic CO2 production correlated directly with motility. In contrast, mitochondrial CO2 production was inversely related to glucose concentration. A monoclonal antibody for the IGF-I receptor inhibited both motility and glycolytic CO2 production, indicating that both processes are receptor mediated.