RESUMEN
Monocyte chemoattractant protein (MCP)-1 plays a critical role in innate immunity by directing the migration of monocytes into inflammatory sites. Recent data indicated a function for this chemokine in adaptive immunity as a regulator of T cell commitment to T helper cell type 2 (Th2) effector function. Studies in a Th1-dependent animal model, experimental autoimmune encephalomyelitis (EAE), showed that MCP-1 was highly expressed in the central nervous system (CNS) of affected rodents, and MCP-1 antibodies could block relapses of the disease. Mice deficient for the major MCP-1 receptor, CC chemokine receptor (CCR)2, did not develop EAE after active immunization but generated effector cells that could transfer the disease to naive wild-type recipients. We analyzed EAE in mice deficient for MCP-1 to define the relevant ligand for CCR2, which responds to murine MCP-1, MCP-2, MCP-3, and MCP-5. We found that C57BL/6 MCP-1-null mice were markedly resistant to EAE after active immunization, with drastically impaired recruitment of macrophages to the CNS, yet able to generate effector T cells that transferred severe disease to naive wild-type recipients. By contrast, adoptive transfer of primed T cells from wild-type mice into naive MCP-1-null recipients did not mediate clinical EAE. On the SJL background, disruption of the MCP-1 gene produced a milder EAE phenotype with diminished relapses that mimicked previous findings using anti-MCP-1 antibodies. There was no compensatory upregulation of MCP-2, MCP-3, or MCP-5 in MCP-1-null mice with EAE. These results indicated that MCP-1 is the major CCR2 ligand in mice with EAE, and provided an opportunity to define the role of MCP-1 in EAE. Compared with wild-type littermates, MCP-1-/- mice exhibited reduced expression of interferon gamma in draining lymph node and CNS and increased antigen-specific immunoglobulin G1 antibody production. Taken together, these data demonstrate that MCP-1 is crucial for Th1 immune responses in EAE induction and that macrophage recruitment to the inflamed CNS target organ is required for primed T cells to execute a Th1 effector program in EAE.
Asunto(s)
Quimiocina CCL2/inmunología , Citocinas , Encefalomielitis Autoinmune Experimental/inmunología , Macrófagos/inmunología , Células TH1/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD11/genética , Complejo CD3/genética , Antígenos CD4/genética , Antígenos CD8/genética , División Celular , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Quimiocina CCL2/genética , Quimiocina CCL4 , Quimiocina CCL7 , Quimiocina CCL8 , Quimiocina CXCL10 , Quimiocinas CXC/biosíntesis , Expresión Génica , Inmunidad Innata , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-10/biosíntesis , Interleucina-10/genética , Interleucina-4/biosíntesis , Interleucina-4/genética , Proteínas Inflamatorias de Macrófagos/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Quimioatrayentes de Monocitos/genética , Proteínas de la Mielina , Proteína Proteolipídica de la Mielina/farmacología , Glicoproteína Asociada a Mielina/farmacología , Glicoproteína Mielina-Oligodendrócito , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Infiltrating leukocytes may be responsible for autoimmune disease. We hypothesized that the chemokine monocyte chemoattractant protein (MCP)-1 recruits macrophages and T cells into tissues that, in turn, are required for autoimmune disease. Using the MRL-Fas(lpr) strain with spontaneous, fatal autoimmune disease, we constructed MCP-1-deficient MRL-Fas(lpr) mice. In MCP-1-intact MRL-Fas(lpr) mice, macrophages and T cells accumulate at sites (kidney tubules, glomeruli, pulmonary bronchioli, lymph nodes) in proportion to MCP-1 expression. Deleting MCP-1 dramatically reduces macrophage and T cell recruitment but not proliferation, protects from kidney, lung, skin, and lymph node pathology, reduces proteinuria, and prolongs survival. Notably, serum immunoglobulin (Ig) isotypes and kidney Ig/C3 deposits are not diminished in MCP-1-deficient MRL-Fas(lpr) mice, highlighting the requirement for MCP-1-dependent leukocyte recruitment to initiate autoimmune disease. However, MCP-1-deficient mice are not completely protected from leukocytic invasion. T cells surrounding vessels with meager MCP-1 expression remain. In addition, downstream effector cytokines/chemokines are decreased in MCP-1-deficient mice, perhaps reflecting a reduction of cytokine-expressing leukocytes. Thus, MCP-1 promotes MRL-Fas(lpr) autoimmune disease through macrophage and T cell recruitment, amplified by increasing local cytokines/chemokines. We suggest that MCP-1 is a principal therapeutic target with which to combat autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/genética , Quimiocina CCL2/genética , Macrófagos/inmunología , Linfocitos T/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Quimiocina CCL2/deficiencia , Quimiocina CCL2/inmunología , Eliminación de Gen , Regulación de la Expresión Génica/inmunología , Ratones , Ratones Endogámicos MRL lprRESUMEN
Interstitial fluid is constantly drained into lymph nodes (LNs) via afferent lymph vessels. This conduit enables monocyte-derived macrophages and dendritic cells to access LNs from peripheral tissues. We show that during inflammation in the skin, a second recruitment pathway is evoked that recruits large numbers of blood-borne monocytes to LNs via high endothelial venules (HEVs). Inhibition of monocyte chemoattractant protein (MCP)-1 blocked this inflammation-induced monocyte homing to LNs. MCP-1 mRNA in inflamed skin was over 100-fold upregulated and paralleled MCP-1 protein levels, whereas in draining LNs MCP-1 mRNA induction was much weaker and occurred only after a pronounced rise in MCP-1 protein. Thus, MCP-1 in draining LNs was primarily derived from inflamed skin. In MCP-1(-/-) mice, intracutaneously injected MCP-1 accumulated rapidly in the draining LNs where it enhanced monocyte recruitment. Intravital microscopy showed that skin-derived MCP-1 was transported via the lymph to the luminal surface of HEVs where it triggered integrin-dependent arrest of rolling monocytes. These findings demonstrate that inflamed peripheral tissues project their local chemokine profile to HEVs in draining LNs and thereby exert "remote control" over the composition of leukocyte populations that home to these organs from the blood.
Asunto(s)
Presentación de Antígeno/inmunología , Quimiocina CCL2/inmunología , Endotelio Linfático/inmunología , Ganglios Linfáticos/inmunología , Monocitos/inmunología , Traslado Adoptivo , Animales , Antígenos/inmunología , Transporte Biológico , Quimiocina CCL2/administración & dosificación , Quimiocina CCL2/metabolismo , Quimiotaxis/inmunología , Femenino , Adyuvante de Freund , Hemocianinas/inmunología , Leucocitos Mononucleares/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/inmunología , Piel/inmunologíaRESUMEN
Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1-deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1(-/-) mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1(-/-) mice, as was expression of IL-4, IL-5, and interferon gamma in splenocytes. In contrast, MCP-1(-/-) mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.
Asunto(s)
Quimiocina CCL2/fisiología , Citocinas/biosíntesis , Inflamación/inmunología , Monocitos/fisiología , Animales , Antígenos Helmínticos/inmunología , Quimiocina CCL2/deficiencia , Quimiocina CCL2/genética , Dermatitis por Contacto/inmunología , Modelos Animales de Enfermedad , Femenino , Marcación de Gen , Granuloma del Sistema Respiratorio/inmunología , Granuloma del Sistema Respiratorio/parasitología , Hipersensibilidad Tardía/inmunología , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Interleucina-5/biosíntesis , Masculino , Ratones , Monocitos/inmunología , Mycobacterium bovis/inmunología , Fenotipo , Esquistosomiasis mansoni/inmunología , Tuberculosis/inmunologíaRESUMEN
Footpad adhesion sites pinch off from the rest of the cell surface during EGTA-mediated detachment of normal or virus-transformed murine cells from their tissue culture substrates. In these studies, highly purified trypsin and testicullar hyaluronidase were used to investigate the selective destruction or solubilization of proteins and polysaccharides in this substrate-attached material (SAM). Trypsin-mediated detachment of cells or trypsinization of SAM after EGTA-mediated detachment of cells resulted in the following changes in SAM composition: (a) solubilization of 50-70% of the glycosaminoglycan polysaccharide with loss of only a small fraction of the protein, (b) selective loss of one species of glycosaminoglycan-associated protein in longterm radiolabeled preparations, (c) no selective loss of the LETS glycoprotein or cytoskeletal proteins in longterm radiolabeled preparations, and (d) selective loss of one species of glycosaminoglycan-associated protein, a protion of the LETS glycoprotein, and proteins Cd (mol wt 47,000 and Ce' (mol wt 39,000) in short term radiolabeled preparations. Digestion of SAM with testicular hyaluronidase resulted in: (a) almost complete solubilization of the hyaluronate and chondroitin sulfate moieties from long term radiolabeled SAM with minimal loss of heparan sulfate, (b) solubilization of a small portion of the LETS glycoprotein and the cytoskeletal proteins from longterm radiolabeled SAM, (c) resistance to solubilization of protein and polysaccharide in reattaching cell SAM which contains principally heparan sulfate, and (d) complete solubilization of the LETS glycoprotein in short term radiolabeled preparations with no loss of cytoskeletal proteins. Thus, there appear to be two distinct pools of LETS in SAM, one associated in some unknown fashion with hyaluronate-chondroitin sulfate complexes, and a second associated with some other component in SAM, perhaps heparan sulfate. These data, together with other results, suggest that the cell-substrate adhesion process may be mediated principally by a heparan sulfate--LETS complex and that hyaluronate-chondroitin sulfate complexes may be important in the detachability of cells from the serum-coated substrate by destabilizing LETS matrices at posterior footpad adhesion sites.
Asunto(s)
Adhesión Celular , Glicosaminoglicanos/análisis , Animales , Línea Celular , Separación Celular , Transformación Celular Neoplásica , Transformación Celular Viral , Sulfatos de Condroitina/análisis , Glicoproteínas/análisis , Heparitina Sulfato/análisis , Ácido Hialurónico/análisis , Ratones , Proteínas de Neoplasias/análisis , SolubilidadRESUMEN
In density-arrested monolayer cultures of Balb/c 3T3 cells, platelet-derived growth factor (PDGF) stimulates expression of the c-myc and c-fos proto-oncogenes, as well as the functionally uncharacterized genes, JE, KC, and JB. These genes are not coordinately regulated. Under ordinary conditions, c-fos, JE, KC, and JB respond to PDGF only when the cells are in a state of G0 growth arrest at the time of PDGF addition. The c-myc gene is regulated in opposition to the other genes, responding best to PDGF in cycling cultures.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proto-Oncogenes/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Interfase , Ratones , Ratones Endogámicos BALB CRESUMEN
Monocyte chemoattractant protein-1 (MCP-1) is upregulated in renal parenchymal cells during kidney disease. To investigate whether MCP-1 promotes tubular and/or glomerular injury, we induced nephrotoxic serum nephritis (NSN) in MCP-1 genetically deficient mice. Mice were analyzed when tubules and glomeruli were severely damaged in the MCP-1-intact strain (day 7). MCP-1 transcripts increased fivefold in MCP-1-intact mice. MCP-1 was predominantly localized within cortical tubules (90%), and most cortical tubules were damaged, whereas few glomerular cells expressed MCP-1 (10%). By comparison, there was a marked reduction (>40%) in tubular injury in MCP-1-deficient mice (histopathology, apoptosis). MCP-1-deficient mice were not protected from glomerular injury (histopathology, proteinuria, macrophage influx). Macrophage accumulation increased adjacent to tubules in MCP-1-intact mice compared with MCP-1-deficient mice (70%, P < 0.005), indicating that macrophages recruited by MCP-1 induce tubular epithelial cell (TEC) damage. Lipopolysaccharide-activated bone marrow macrophages released molecules that induced TEC death that was not dependent on MCP-1 expression by macrophages or TEC. In conclusion, MCP-1 is predominantly expressed by TEC and not glomeruli, promotes TEC and not glomerular damage, and increases activated macrophages adjacent to TEC that damage TEC during NSN. Therefore, we suggest that blockage of TEC MCP-1 expression is a therapeutic strategy for some forms of kidney disease.
Asunto(s)
Quimiocina CCL2/genética , Glomérulos Renales/metabolismo , Túbulos Renales/patología , Macrófagos/metabolismo , Nefritis/patología , Animales , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Histocitoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Nefritis/inmunología , Proteinuria , ARN Mensajero/análisis , Linfocitos T/metabolismoRESUMEN
The earliest recognizable atherosclerotic lesions are fatty streaks composed of lipid-laden macrophages (foam cells). Circulating monocytes are the precursors of these foam cells, but the molecular mechanisms that govern macrophage trafficking through the vessel wall are poorly understood. Monocyte chemoattractant protein-1 (MCP-1), a member of the chemokine (chemotactic cytokine) family, is a potent monocyte agonist that is upregulated by oxidized lipids. Recent studies in hypercholesterolemic mice lacking apo E or the low-density lipoprotein receptor have suggested a role for MCP-1 in monocyte recruitment to early atherosclerotic lesions. To determine if MCP-1 is critically involved in atherogenesis in the setting of elevated physiological plasma cholesterol levels, we deleted the MCP-1 gene in transgenic mice expressing human apo B. Here we report that the absence of MCP-1 provides dramatic protection from macrophage recruitment and atherosclerotic lesion formation in apo B transgenic mice, without altering lipoprotein metabolism. Taken together with the results of earlier studies, these data provide compelling evidence that MCP-1 plays a critical role in the initiation of atherosclerosis.
Asunto(s)
Apolipoproteínas B/biosíntesis , Arteriosclerosis/etiología , Quimiocina CCL2/deficiencia , Células Espumosas/metabolismo , Lipoproteínas/biosíntesis , Animales , Aorta/patología , Apolipoproteínas B/genética , Arteriosclerosis/patología , Quimiocina CCL2/genética , Colesterol/sangre , HDL-Colesterol/sangre , Células Espumosas/citología , Humanos , Lipoproteínas/genética , Ratones , Ratones Transgénicos , Triglicéridos/sangreRESUMEN
The early growth response gene JE encodes a monocyte chemoattractant, MCP-1. The JE/MCP-1 protein attracts and stimulates human monocytes and induces monocyte-mediated inhibition of tumor cell growth in vitro. Expression of human or murine JE/MCP-1 in Chinese hamster ovary (CHO) cells completely suppressed their ability to form tumors in nude mice. Coinjection of JE/MCP-1-expressing cells with nonexpressing CHO cells or with HeLa cells also prevented tumor formation. Since JE/MCP-1 expression had no discernible effect on the tranformed phenotype of these cells in vitro, the suppressive effect depends on host animal factors. These factors are likely to be components of the inflammatory response, because JE/MCP-1-expressing cells elicited a predominantly monocytic infiltrate at the site of injection. Our results suggest that JE/MCP-1 protein may be useful in cancer therapy.
Asunto(s)
Factores Quimiotácticos/genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Animales , División Celular , Línea Celular , Quimiocina CCL2 , Factores Quimiotácticos/biosíntesis , Factores Quimiotácticos/fisiología , Humanos , Metionina/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Transfección , Trasplante HeterólogoRESUMEN
Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of proinflammatory cytokines, all of which share a high degree of amino acid sequence similarity. Aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component, such as atherosclerosis. Although structural analyses indicate that chemokines form homodimers, there is controversy about whether dimerization is necessary for activity. To address this question for MCP-1, we obtained evidence in four steps. First, coprecipitation experiments demonstrated that MCP-1 forms dimers at physiological concentrations. Second, chemically cross-linked MCP-1 dimers attract monocytes in vitro with a 50% effective concentration of 400 pM, identical to the activity of non-cross-linked MCP-1. Third, an N-terminal deletion variant of MCP-1 (called 7ND) that inhibits MCP-1-mediated monocyte chemotaxis specifically forms heterodimers with wild-type MCP-1. Finally, although 7ND inhibits wild-type MCP-1 activity, it has no effect on cross-linked MCP-1. These results indicate that 7ND is a dominant negative inhibitor, implying that MCP-1 activates its receptor as a dimer. In addition, chemical cross-linking restores activity to an inactive N-terminal insertional variant of MCP-1, further supporting the need for dimerization. Since the reported Kd for MCP-1 monomer dissociation is much higher than its 50% effective concentration or Kd for receptor binding, active dimer formation may require high local concentrations of MCP-1. Our data further suggest that the dimer interface can be a target for MCP-1 inhibitory drugs.
Asunto(s)
Factores Quimiotácticos/metabolismo , Quimiotaxis de Leucocito/fisiología , Citocinas/metabolismo , Secuencia de Bases , Unión Competitiva , Quimiocina CCL2 , Factores Quimiotácticos/genética , Reactivos de Enlaces Cruzados , Citocinas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Eliminación de Secuencia , SolucionesRESUMEN
The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein.
Asunto(s)
Glicoproteínas/genética , Monocitos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Quimiocina CCL2 , ADN/genética , Fibroblastos , Expresión Génica/efectos de los fármacos , Genes , Glicoproteínas/análisis , Glicoproteínas/biosíntesis , Humanos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Members of the INK4 protein family specifically inhibit cyclin-dependent kinase 4 (cdk4) and cdk6-mediated phosphorylation of the retinoblastoma susceptibility gene product (Rb). p16INK4A, a prototypic INK4 protein, has been identified as a tumor suppressor in many human cancers. Inactivation of p16INK4A in tumors expressing wild-type Rb is thought to be required in order for many malignant cell types to enter S phase efficiently or to escape senescence. Here, we demonstrate another mechanism of tumor suppression by implicating p16INK4A in a G1 arrest checkpoint in response to DNA damage. Calu-1 non-small cell lung cancer cells, which retain Rb and lack p53, do not arrest in G1 following DNA damage. However, engineered expression of p16INK4A at levels compatible with cell proliferation restores a G1 arrest checkpoint in response to treatment with gamma-irradiation, topoisomerase I and II inhibitors, and cisplatin. A similar checkpoint can be demonstrated in p53-/- fibroblasts that express p16INK4A. DNA damage-induced G1 arrest, which requires the expression of pocket proteins such as Rb, can be abrogated by overexpression of cdk4, kinase-inactive cdk4 variants capable of sequestering p16INK4A, or a cdk4 variant incapable of binding p16INK4A. After exposure to DNA-damaging agents, there was no change either in overall levels of p16INK4A or in amounts of p16INK4A found in complex with cdks 4 and 6. Nonetheless, p16INK4A expression is required for the reduction in cdk4- and cdk6-mediated Rb kinase activity observed in response to DNA damage. During tumor progression, loss of p16INK4A expression may be necessary for cells with wild-type Rb to bypass this G1 arrest checkpoint and attain a fully transformed phenotype.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Daño del ADN , Fase G1/genética , Genes Supresores de Tumor , Humanos , Células Tumorales CultivadasRESUMEN
Platelet-derived growth factor (PDGF) and the synthetic double-stranded RNA poly(I).poly(C) [poly(I.C)] stimulate transcription of the JE gene in BALB/c-3T3 fibroblasts. The response of JE to poly(I.C) does not appear to be channeled through any known component of the PDGF receptor signal transduction apparatus. In addition, JE sequences upstream of the transcription start site are devoid of previously identified poly(I.C)-responsive elements, such as those found in the beta-interferon gene. These data suggest that a novel signal transduction pathway regulates the JE response to PDGF and double-stranded RNA. The c-myc and c-fos proto-oncogenes also respond to this pathway but with poor efficiency. However, this pathway operates very efficiently on other PDGF-inducible genes that encode the secretory proteins KC and M-CSF.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Poli I-C/farmacología , Transducción de Señal/efectos de los fármacos , 1-Fosfatidilinositol 4-Quinasa , Animales , Línea Celular , Quimiocina CCL2 , Glicoproteínas/genética , Ratones , Fosfotransferasas/metabolismo , Proteína Quinasa C/metabolismo , Proto-Oncogenes/efectos de los fármacos , Receptores de Superficie Celular/efectos de los fármacos , Receptores del Factor de Crecimiento Derivado de PlaquetasRESUMEN
p16INK4A and p15INK4B were initially identified as potent inhibitors of activated cyclin/cyclin-dependent kinase complexes. These genes were colocalized to chromosome 9p21, and p16 was subsequently found to be mutated in familial melanoma and deleted in a wide variety of sporadic cancers. We recently found that de novo methylation of a 5' CpG island led to transcriptional block of full-length p16 in many neoplasms. However, the presence of a truncated p16 transcript in methylated cell lines led us to investigate the presence of an alternative promoter or initiation site. We have now identified an abundant alternative p16 transcript in both methylated and unmethylated cell lines generated from a novel sequence (exon 1 beta) potentially involved in the complex regulation of these critical cell cycle genes.
Asunto(s)
Proteínas Portadoras/genética , Transcripción Genética , Secuencia de Bases , Cromosomas Humanos Par 9 , Inhibidor p16 de la Quinasa Dependiente de Ciclina , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/genética , Exones , Amplificación de Genes , Genes Supresores de Tumor , Humanos , Datos de Secuencia Molecular , Neoplasias/genética , Reacción en Cadena de la Polimerasa , ARN/análisis , ARN/genética , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
cdk4-mediated phosphorylation of the retinoblastoma susceptibility protein (Rb) is stimulated by cyclin D1, an oncogene, and inhibited by p16, a candidate tumor suppressor. We examined these proteins in non-small cell lung cancer (NSCLC), which is predominantly Rb positive, and small cell lung cancer (SCLC), which is Rb negative. Most NSCLC and SCLC resection specimens and cell lines overexpress cyclin D1 (indicating that cyclin D1 overexpression and Rb inactivation can coexist in SCLC). However, 9 of 9 Rb-positive NSCLC cell lines have absent or low p16, while an Rb-negative NSCLC line and 5 of 5 SCLC cell lines have high levels of p16. In primary resection specimens, p16 was undetectable in 18 of 27 NSCLC samples and abundant in 4 of 5 SCLC samples. Our data confirm the predicted reciprocity between Rb inactivation and p16 expression in a common human malignancy and define differential p16 expression as a fundamental distinction between NSCLC and SCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/biosíntesis , Expresión Génica , Genes de Retinoblastoma , Genes Supresores de Tumor , Neoplasias Pulmonares/metabolismo , Southern Blotting , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras/análisis , Línea Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , ADN de Neoplasias/análisis , Humanos , Immunoblotting , Inmunohistoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Inhibidores de Proteínas Quinasas , Proteína de Retinoblastoma/análisis , Proteína de Retinoblastoma/biosíntesis , Células Tumorales CultivadasRESUMEN
p16INK4A, a specific inhibitor of cyclin-dependent kinase (cdk)4 and cdk6, is a candidate tumor suppressor in malignancies with wild-type retinoblastoma (Rb). Loss of p16INK4A frees these cdks from inhibition, permitting constitutive phosphorylation of Rb and inactivation of its growth suppressive properties. Consistent with this model, Rb-positive non-small cell lung cancers (NSCLCs) have little or no detectable p16INK4A protein, whereas Rb-negative lung cancers have abundant p16INK4A. However, only some NSCLCs have homozygous deletions or nonsense mutations in a remaining p16INK4A allele, suggesting that other mechanisms must account for absent or low levels of p16INK4A protein. Here, we analyzed 9 Rb-positive NSCLC cell lines for the controls governing p16INK4A activity. Four lines had homozygous deletions of p16INK4A (SK-LU-1, SK-MES-1, A-427, and SW900), and three had a point mutation in a single allele. First, in H520 cells, the previously reported deletion at codon 45 results in a frameshift that produces no detectable protein. Second, in Calu-3 cells, a His to Tyr substitution at codon 83 produced a variant with a shortened half-life that was unable to form complexes with cdk4 or cdk6. Third, in H661 cells, the previously reported point mutation in the second intron splice donor site resulted in a smaller p16INK4A protein. Although this variant formed complexes with cdk4 and cdk6, it had a profoundly reduced half-life, producing low steady-state levels of p16INK4A and abundant levels of free cdks. Finally, Calu-1 and Calu-6 cells transcribed no detectable mRNA encoding authentic p16INK4A. These cell lines displayed methylation of the CpG island surrounding the first exon of p16INK4A and expressed abundant levels of a nontranslated mRNA containing an alternative first exon (E1 beta), as did all other cell lines in which the p16INK4A locus was not deleted. These data indicate that Rb-positive NSCLC cells have evolved a variety of pathways to suppress p16INK4A expression. Reintroduction of p16INK4A into these cell lines by retroviral transfer resulted in a reduced growth rate, increased abundance of hypophosphorylated Rb, accumulation of cells in G1, and a less transformed morphology in Rb-positive, but not Rb-negative cells, suggesting that loss of p16INK4A is essential for maintenance of the transformed phenotype.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Proteínas Portadoras/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras/metabolismo , Ciclo Celular , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Quinasas Ciclina-Dependientes/metabolismo , ADN de Neoplasias/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Neoplasias Pulmonares/patología , Metilación , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Neoplásico/genética , Proteína de Retinoblastoma/metabolismo , Eliminación de Secuencia , Células Tumorales CultivadasRESUMEN
Lung cancer can lead to abnormalities of the actin cytoskeleton structure which may be important in transformation. In this study, we have investigated the expression of the cytoskeletal associated protein paxillin in lung cancer. Paxillin is a 68 kDa focal adhesion protein, with four tandem LIM domains at the C-terminus, involved in growth factor receptor, integrin and oncogenic signaling such as v-src, BCR/ABL, and E6 of the papilloma virus. In non-small cell lung cancer (NSCLC) cell lines, paxillin localized to the focal adhesions. The possible role of paxillin in lung cancer cells was assessed by overexpressing green fluorescence protein (GFP)-paxillin construct in two separate NSCLC cell lines (Calu-1 and H661). Over the course of 48 h, GFP-paxillin consistently caused the cells to become round and to decrease cell motility as compared to normal controls, GFP-N-terminus paxillin, or GFP-LIM transfected cells. Because some lung cancers may be quite aggressive and metastasize quickly, which may be related to the cytoskeleton, we determined the expression of paxillin in NSCLC and small cell lung cancer (SCLC) cell lines and patient tumor tissues. Expression of paxillin in NSCLC and SCLC cell lines were determined by Northern blot and Western blot analysis. The expression of paxillin was consistently low in SCLC cell lines, whereas there was paxillin expression in NSCLC cell lines. There was a variability of expression of paxillin in NSCLC tumor tissue as compared to normal lung tissue. In contrast, by immunohistochemistry, we show that there was no detectable expression of paxillin in 5/5 SCLC patients. This data suggests that absence or low level of paxillin protein expression may cause certain lung cancers, such as SCLC, to be more motile and possibly more aggressive.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Movimiento Celular , Proteínas del Citoesqueleto/biosíntesis , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/biosíntesis , Células 3T3 , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas , Moléculas de Adhesión Celular/genética , Proteínas del Citoesqueleto/genética , Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Ratones , Paxillin , Fosfoproteínas/genética , Proteínas Recombinantes de Fusión/genética , Células Tumorales CultivadasRESUMEN
Flavopiridol, a synthetic flavone that inhibits tumor growth in vitro and in vivo, is a potent cyclin-dependent kinase (cdk) inhibitor presently in clinical trials. In the present study, the effect of 100-500 nM flavopiridol on a panel of non-small cell lung cancer cell lines was examined. All express a wild-type retinoblastoma susceptibility protein and lack p16INK4A, and only A549 cells are known to express wild-type p53. During 72 h of treatment, flavopiridol was shown to be cytotoxic to all seven cell lines, as measured by trypan blue exclusion, regardless of whether cells were actively cycling. In most cycling cells, cytotoxicity was preceded or accompanied by cell cycle arrest. Cell death resulted in the appearance of cells with a sub-G1 DNA content, suggestive of apoptosis, which was confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by demonstration of cleavage of caspase targets including poly(ADP-ribose) polymerase, p21Waf1, and p27Kip1. At doses at or below 500 nM, maximal cytotoxicity required 72 h of exposure. Although flavopiridol resulted in the accumulation of p53 in A549 cells, flavopiridol-mediated apoptosis was p53 independent because it occurred to the same degree in A549 cells in which p53 was targeted for degradation by HPV16E6 expression. The data indicate that flavopiridol has activity against non-small cell lung cancers in vitro and is worthy of continued clinical development in the treatment of this disease.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Piperidinas/farmacología , Proteína p53 Supresora de Tumor/fisiología , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares/patología , Células Tumorales CultivadasRESUMEN
PURPOSE: Flavopiridol is a potent cyclin-dependent kinase inhibitor with preclinical activity against non-small cell lung cancer (NSCLC), inhibiting tumor growth in vitro and in vivo by cytostatic and cytotoxic mechanisms. A Phase II trial was conducted to determine the activity and toxicity of flavopiridol in untreated patients with metastatic NSCLC. EXPERIMENTAL DESIGN: A total of 20 patients were treated with a 72-h continuous infusion of flavopiridol every 14 days at a dose of 50 mg/m(2)/day and a concentration of 0.1-0.2 mg/ml. Dose escalation to 60 mg/m(2)/day was permitted if no significant toxicity occurred. Response was initially assessed after every two infusions; patients treated longer than 8 weeks were then assessed after every four infusions. Plasma levels of flavopiridol were measured daily during the first two infusions to determine steady-state concentrations. RESULTS: This study was designed to evaluate a total of 45 patients in two stages. However, because no objective responses were seen in the first 20 patients, the early-stopping rule was invoked, and patient accrual was halted. In four patients who received eight infusions, progression was documented at 15, 20, 40, and 65 weeks, respectively. The most common toxicities included grade 1 or 2 diarrhea in 11 patients, asthenia in 10 patients, and venous thromboses in 7 patients. The mean +/- SD steady-state concentration of drug during the first infusion was 200 +/- 89.9 nM, sufficient for cytostatic effects in in vitro models. CONCLUSIONS: At the current doses and schedule, flavopiridol does not have cytotoxic activity in NSCLC, although protracted periods of disease stability were observed with an acceptable degree of toxicity.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Flavonoides/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Piperidinas/uso terapéutico , Anciano , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/uso terapéutico , Femenino , Flavonoides/farmacocinética , Flavonoides/toxicidad , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Piperidinas/farmacocinética , Piperidinas/toxicidad , Factores de TiempoRESUMEN
Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes. Its receptor (CCR2) is a heptahelical G-protein-coupled receptor (GPCR) whose signal transduction pathways for chemotaxis have not been completely defined. Because other GPCRs stimulate the mitogen-activated protein kinase (MAPK) cascade, we examined this pathway's activity in response to MCP-1. MCP-1 induced rapid and transient activation of MAPK in human monocytes and in Chinese hamster ovary cells expressing CCR2B. This effect was largely insensitive to pertussis toxin and wortmannin, and was protein kinase C-dependent and protein tyrosine kinase-independent. PD 098059, an inhibitor of MEK activation, not only prevented MAPK activation but also inhibited MCP-1-induced chemotaxis. Because pertussis toxin and wortmannin also efficiently inhibit chemotaxis but do not completely inhibit MAPK activation, these data may define non-overlapping signal transduction pathways that all must be activated to produce chemokine-mediated chemotaxis.