RESUMEN
Chromosomal rearrangements often occur at genomic loci with DNA secondary structures, such as common fragile sites (CFSs) and palindromic repeats. We developed assays in mammalian cells that revealed CFS-derived AT-rich sequences and inverted Alu repeats (Alu-IRs) are mitotic recombination hotspots, requiring the repair functions of carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) and the Mre11/Rad50/Nbs1 complex (MRN). We also identified an endonuclease activity of CtIP that is dispensable for end resection and homologous recombination (HR) at I-SceI-generated "clean" double-strand breaks (DSBs) but is required for repair of DSBs occurring at CFS-derived AT-rich sequences. In addition, CtIP nuclease-defective mutants are impaired in Alu-IRs-induced mitotic recombination. These studies suggest that an end resection-independent CtIP function is important for processing DSB ends with secondary structures to promote HR. Furthermore, our studies uncover an important role of MRN, CtIP, and their associated nuclease activities in protecting CFSs in mammalian cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Sitios Frágiles del Cromosoma/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Secuencias Invertidas Repetidas/genética , Proteínas Nucleares/metabolismo , Ácido Anhídrido Hidrolasas , Elementos Alu/genética , Composición de Base/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas , Endonucleasas/genética , Recombinación Homóloga/genética , Humanos , Proteína Homóloga de MRE11 , Mitosis/genética , Proteínas Nucleares/genética , Recombinación GenéticaRESUMEN
CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Recombinación Homóloga , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S/fisiología , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Replicación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Rayos gamma/efectos adversos , Humanos , Fosforilación/fisiología , Fosforilación/efectos de la radiación , Proteínas Serina-Treonina Quinasas/genética , Fase S/efectos de la radiación , Proteínas Supresoras de Tumor/genéticaRESUMEN
Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.
Asunto(s)
Cisteamina/metabolismo , Úlcera Duodenal/metabolismo , Duodeno/metabolismo , Hierro/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células CACO-2 , Cistamina/metabolismo , Cisteamina/análogos & derivados , Cisteamina/farmacología , Deferoxamina/farmacología , Úlcera Duodenal/patología , Duodeno/efectos de los fármacos , Duodeno/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Hierro/farmacología , Quelantes del Hierro/farmacología , Ratones , Especificidad de Órganos , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/deficiencia , Proteínas de Transporte de Catión Orgánico/genética , Ratas , Especies Reactivas de Oxígeno/metabolismo , Sodio/metabolismoRESUMEN
BACKGROUND: Despite recent advances and better understanding of the etiology and the pathogenesis of gastrointestinal ulcer diseases, e.g., duodenal ulcer, the molecular events leading to ulcer development, delayed healing, and recurrence remain poorly elucidated. AIMS: After we found that duodenal ulcers did not heal despite increased levels of vascular endothelial growth factor (VEGF), we tested the hypothesis that an imbalance in angiogenic VEGF and anti-angiogenic endostatin and angiostatin might be important in the development and delayed healing of experimental duodenal ulcers. METHODS: Levels of VEGF, endostatin, and angiostatin, and the expression and activity of related matrix metalloproteinases (MMP) 2 and 9 were measured in scrapings of rat proximal duodenal mucosa in the early and late stages of chemically induced duodenal ulceration. Furthermore, animals were treated with recombinant endostatin and MMP 2 inhibitor to test the relationship between MMP2 and endostatin and their involvement in healing of experimental duodenal ulcers. RESULTS: A concurrent increase of duodenal VEGF, endostatin, and angiostatin was noted during duodenal ulceration. Endostatin treatment aggravated duodenal ulcer. Levels of MMP2, but not MMP9, were increased. Inhibition of MMP2 reduced levels of endostatin and angiostatin, and attenuated duodenal ulcers. CONCLUSIONS: Increased levels of endostatin and angiostatin induced by MMP2 delayed healing of duodenal ulcers despite concurrently increased VEGF. Thus, an inappropriate angiogenic response or "angiogenic imbalance" may be an important new mechanism in ulcer development and impaired healing.
Asunto(s)
Angiostatinas/metabolismo , Úlcera Duodenal/metabolismo , Endostatinas/metabolismo , Neovascularización Patológica/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/fisiología , Animales , Cisteamina/efectos adversos , Modelos Animales de Enfermedad , Úlcera Duodenal/inducido químicamente , Úlcera Duodenal/fisiopatología , Inhibidores Enzimáticos/farmacología , Femenino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Mucosa Intestinal/fisiopatología , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Neovascularización Patológica/fisiopatología , Nitrilos/efectos adversos , Ratas , Ratas Sprague-Dawley , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteína de Replicación A/metabolismo , Fase S/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Núcleo Celular/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/genética , Genes cdc , Humanos , Proteína Homóloga de MRE11 , Datos de Secuencia Molecular , Complejos Multiproteicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Alineación de SecuenciaRESUMEN
Mesalamine (5-aminosalicylate acid, 5-ASA) is an effective treatment for ulcerative colitis (UC). The mechanisms of its actions are not fully understood. Because angiogenesis is critical for healing UC, we examined whether 5-ASA alters the angiogenic balance between angiogenic factors [e.g., vascular endothelial growth factor (VEGF)] and antiangiogenic factors (e.g., endostatin and angiostatin) in the colon in experimental UC. Rats were treated with saline or 5-ASA (100 mg/kg) twice daily and euthanized 3 or 7 days after iodoacetamide-induced UC. Clinical signs (e.g., lethargy, diarrhea) and UC lesions were measured. Expression of VEGF, endostatin, angiostatin, tissue necrosis factor alpha (TNF-alpha), and matrix metalloproteinases (MMPs) 2 and 9 was determined by Western blots, enzyme-linked immunosorbent assay, and zymography in the distal colon. 5-ASA treatment reduced lethargy and diarrhea and significantly decreased colonic lesions (by approximately 50%) compared with saline treatment in UC (both, P < 0.05). 5-ASA did not reverse the increased levels of VEGF, but it significantly reduced expression of endostatin and angiostatin in UC compared with vehicle treatment (both, P < 0.05). Furthermore, 5-ASA treatment significantly diminished increased activity of TNF-alpha and MMP9 in UC. This is the first demonstration that 5-ASA treatment reverses an imbalance between the angiogenic factor VEGF and antiangiogenic factors endostatin and angiostatin in experimental UC. The effect of 5-ASA in UC may be caused by the down-regulation of expression of endostatin and angiostatin by modulation of MMP2 and MMP9 via inhibition of TNFalpha. The inhibition of antiangiogenic factors may represent a novel molecular mechanism of the therapeutic action of 5-ASA.
Asunto(s)
Angiostatinas/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Endostatinas/antagonistas & inhibidores , Mesalamina/uso terapéutico , Neovascularización Fisiológica/efectos de los fármacos , Angiostatinas/biosíntesis , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Western Blotting , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/fisiopatología , Colon/irrigación sanguínea , Colon/efectos de los fármacos , Colon/enzimología , Electroforesis en Gel de Poliacrilamida , Endostatinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Mesalamina/administración & dosificación , Mesalamina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
UNLABELLED: In ulcerative colitis (UC), an increased expression of vascular endothelial growth factor (VEGF) correlates with disease activity, but a causal relationship is unknown. We tested the hypothesis that VEGF plays a mechanistic role in the pathogenesis of experimental UC and that VEGF neutralization may exert therapeutic effect. UC was induced in Sprague-Dawley rats by 6% iodoacetamide given intracolonically. Neutralizing anti-VEGF antibody (50 microg/rat), nonspecific IgG, or saline (0.1 ml/rat) was injected intramuscularly on the 3rd and 5th days after iodoacetamide enema. Rats were euthanized on the 7th day. We examined the extent of macroscopic, histologic, and clinical features of colitis and colonic vascular permeability. Colonic VEGF mRNA and protein expressions increased as early as 0.5 h after iodoacetamide enema and remained elevated in the active phase of colitis. Treatment with anti-VEGF antibody markedly improved the clinical and morphologic features of UC. Colonic lesion area was significantly reduced from 370 +/- 140 or 311 +/- 170 mm(2) in saline- or IgG-treated groups to 122 +/- 57 mm(2) in the anti-VEGF-group (p < 0.05). Increased colonic vascular permeability was decreased by the anti-VEGF antibody (p < 0.05) and the Src inhibitor PP1 [pyrazolopyrimidine, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine] (p < 0.01). The number of acute and chronic inflammatory cells in the lesion area was significantly reduced in anti-VEGF-treated rats. In the anti-VEGF-treated group, mucosal levels of VEGF, platelet-derived growth factor, and basic fibroblast growth factor were also reduced. IN CONCLUSION: 1) Neutralizing anti-VEGF antibody significantly ameliorates experimental UC in rats in part by reducing excessive vascular permeability and decreasing inflammatory cells infiltration; and 2) VEGF seems to mediate increased colonic vascular permeability in experimental UC via the Src-dependent mechanism.
Asunto(s)
Anticuerpos/uso terapéutico , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/prevención & control , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colitis Ulcerosa/inducido químicamente , Colon/efectos de los fármacos , Colon/patología , Modelos Animales de Enfermedad , Femenino , Yodoacetamida/toxicidad , Microcirculación/efectos de los fármacos , Microcirculación/fisiología , Pruebas de Neutralización , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/toxicidadRESUMEN
Stress is a major etiologic factor in the pathogenesis of gastric and duodenal ulceration, as first described in rats by Hans Selye. In patients with "peptic ulcers" duodenal ulcers are more frequent than gastric ulcers (except in Japan). Thus, our research during the last three decades focused on the molecular mechanisms of duodenal ulcer in rodent models of chemically induced duodenal ulceration, and here we review our three recent findings: Endothelins (ET-1), the immediate early gene egr-1 and imbalance of angiogenic/antiangiogenic molecules. Namely, we found an enhanced expression and release of ET-1 within 15-30 min after the administration of duodenal ulcerogen cysteamine, resulting in local ischemia that triggers the expression of hypoxia-inducible factors (HIF-1alpha). Our gene expression studies also revealed an early (0.5-2 h) increase in the expression of egr-1 that is followed (12-24 h) by upregulation of angiogenic growth factors (e.g., VEGF, bFGF, PDGF). Surprisingly, this event is also associated with an enhanced production of angiostatin and endostatin that probably counteract the beneficial effect of angiogenic molecules. Thus, the initial injury to endothelial and epithelial cells in duodenal ulceration seems to be aggravated (and not initiated) by HCl and proteolytic enzymes. The resulting mucosal necrosis does not rapidly heal because of the imbalance of VEGF and angiostatin/endostatin, hence duodenal ulcers develop. The experimental ulcers Selye described morphologically are now characterized at the molecular and genome level, involving unexpected mediators like ET-1, egr-1 and angiogenesis-related molecules.
Asunto(s)
Úlcera Duodenal/etiología , Úlcera Duodenal/metabolismo , Inhibidores de la Angiogénesis/fisiología , Proteínas Angiogénicas/fisiología , Animales , Úlcera Duodenal/inducido químicamente , Úlcera Duodenal/patología , Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Endotelina-1/biosíntesis , Endotelina-1/genética , Endotelina-1/metabolismo , HumanosRESUMEN
The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in both suppression and promotion of tumorigenesis. It remains unclear how these 2 opposite functions of p38 operate in vivo to impact cancer development. We previously reported that a p38 downstream kinase, p38-regulated/activated kinase (PRAK), suppresses tumor initiation and promotion by mediating oncogene-induced senescence in a murine skin carcinogenesis model. Here, using the same model, we show that once the tumors are formed, PRAK promotes the growth and progression of skin tumors. Further studies identify PRAK as a novel host factor essential for tumor angiogenesis. In response to tumor-secreted proangiogenic factors, PRAK is activated by p38 via a vascular endothelial growth factor receptor 2 (VEGFR2)-dependent mechanism in host endothelial cells, where it mediates cell migration toward tumors and incorporation of these cells into tumor vasculature, at least partly by regulating the phosphorylation and activation of focal adhesion kinase (FAK) and cytoskeletal reorganization. These findings have uncovered a novel signaling circuit essential for endothelial cell motility and tumor angiogenesis. Moreover, we demonstrate that the tumor-suppressing and tumor-promoting functions of the p38-PRAK pathway are temporally and spatially separated during cancer development in vivo, relying on the stimulus, and the tissue type and the stage of cancer development in which it is activated.
Asunto(s)
Carcinoma/irrigación sanguínea , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neovascularización Patológica/metabolismo , Papiloma/irrigación sanguínea , Proteínas Serina-Treonina Quinasas/fisiología , Neoplasias Cutáneas/irrigación sanguínea , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Carcinógenos/toxicidad , Carcinoma/inducido químicamente , Movimiento Celular/fisiología , Transformación Celular Neoplásica/inducido químicamente , Citoesqueleto/metabolismo , Progresión de la Enfermedad , Células Endoteliales/fisiología , Proteína-Tirosina Quinasas de Adhesión Focal , Sistema de Señalización de MAP Quinasas , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Neovascularización Patológica/inducido químicamente , Papiloma/inducido químicamente , Forboles/toxicidad , Neoplasias Cutáneas/inducido químicamente , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Inflammatory bowel disease (IBD): ulcerative colitis (UC) and Crohn disease (CD) are characterized by recurrent inflammation and ulceration of intestinal and/or colonic mucosa and an inappropriate and delayed healing. Current therapies with, e.g., anti-TNFα antibody (infliximab) and other anti-inflammatory drugs (e.g., mesalamine) do not induce sustained remission, complete healing or prevent recurrence of UC. Although the pathogenesis of UC is not fully understood, pathologic angiogenesis has been postulated as a critical pathogenic component in UC. Recent studies demonstrated that the poor healing, chronic inflammation in colon of UC could be the result of microvascular dysfunction and endothelial barrier defect, resulting in sustained tissue hypoperfusion and ischemia in the colon. Previously, regeneration of injured endothelium and neovascularization were believed to rely solely on the migration and proliferation of neighboring endothelial cells from existing blood vessels. However, accumulating evidence shows that additional mechanisms may exist, and may be mediated by the circulating pool of bone marrow-derived endothelial progenitor cells (BMD-EPC). Furthermore, stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 have been demonstrated to play an important role in the "homing" of BMD-EPC to injured sites and neovascularization in tissue repair. Recent studies by others and us showed reduced BMD-EPC levels in the circulation of IBD patients and rats with experimental UC. However, the potential therapeutic effect of BMD-EPC on neovascularization and colonic mucosal repair in UC has not been elucidated. In this review, we discussed the possibility that impaired contribution of BMD-EPC (i.e., decreased release of BMD-EPC from bone marrow to circulation and/or blocked/impaired homing of BMD-EPC to colonic lesions) may be a critical component of mechanisms in the incomplete/delayed healing of UC, and may offer a novel form of cell therapy for IBD.
Asunto(s)
Trasplante de Células , Colitis Ulcerosa/terapia , Endotelio Vascular/trasplante , Trasplante de Células Madre Hematopoyéticas , Neovascularización Patológica , Cicatrización de Heridas , Animales , Colitis Ulcerosa/patología , Humanos , RatasRESUMEN
Gastrointestinal (GI) ulcers are essentially internal wounds that resist normal healing processes. Since their pathogenesis is poorly understood, and the etiologic (e.g., gastric acid, aspirin-like drugs, stress) and aggravating factors (e.g., H. pylori) are not well characterized, the remaining therapeutic option is to accelerate healing. Superficial mucosal lesions, i.e., erosions usually heal by epithelial regeneration and restitution, but when ulcers involve the muscularis propria, smooth muscle cells do not divide/regenerate. These deep lesions are filled by granulation tissue, i.e., angiogenesis followed by proliferation of connective tissue fibroblasts that deposit collagen over which adjacent surviving and dividing epithelial cells migrate to complete the healing. Our laboratory was the first to postulate that stimulation of angiogenesis alone might be sufficient to accelerate ulcer healing in the GI tract. Indeed, daily treatment of rats with bFGF, PDGF or VEGF markedly improved the healing of cysteamine-induced chronic duodenal ulcers, without any reduction in gastric acid secretion. These results were reproduced by a single dose of gene therapy by adenoviral vectors encoding PDGF or VEGF genes. The molar potency of angiogenic growth factors was 2-7 million times better than the antiulcerogenic effect of antisecretory H2 antagonists. Since histologically & pathologically gastroduodenal ulcers look similar to ulcers in the lower GI tract, we also predicted that the healing of experimental ulcerative colitis might be also improved by these angiogenic growth factors. Rectal enemas containing bFGF or PDGF indeed accelerated the healing of chemically induced ulcerative colitis in rats. VEGF, also known as VPF (vascular permeability factor), however, had no effect or slightly aggravated the colonic lesions. Injection of anti-VEGF neutralizing antibodies, however, counteracted the increased vascular permeability in the early stages of experimental ulcerative colitis and subsequently decreased the number of inflammatory cells in colonic ulcers in rats, resulting in significantly improved healing in the lower GI tract lesions. Thus, the three angiogenic growth factors tested exerted beneficial effect on gastroduodenal ulcers, and rectal enemas with bFGF or PDGF also accelerated the healing of experimental ulcerative colitis. Surprisingly, we achieved the latter effect with anti-VEGF antibodies, most likely because of the pro-inflammatory actions of VEGF in the pathogenesis of ulcerative colitis.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Diseño de Fármacos , Fármacos Gastrointestinales/uso terapéutico , Neovascularización Patológica/tratamiento farmacológico , Úlcera Gástrica/tratamiento farmacológico , Inhibidores de la Angiogénesis/química , HumanosRESUMEN
AIMS: Vascular endothelial growth factor (VEGF) and pathologic angiogenesis have been demonstrated to play a pathogenic role in the development and progression of inflammatory bowel disease. Thus, we hypothesized that the potent anti-angiogenic factor endostatin might play a beneficial role in experimental ulcerative colitis (UC). MAIN METHODS: We used three animal models of UC: (1) induced by 6% iodoacetamide (IA) in rats, or (2) by 3% dextran sulfate sodium (DSS) in matrix metalloproteinase-9 (MMP-9) knockout (KO) and wild-type mice, and (3) interleukin-10 (IL-10) KO mice. Groups of MMP-9 KO mice with DSS-induced UC were treated with endostatin or water for 5days. KEY FINDINGS: We found concomitant upregulation of VEGF, PDGF, MMP-9 and endostatin in both rat and mouse models of UC. A positive correlation between the levels of endostatin or VEGF and the sizes of colonic lesions was seen in IA-induced UC. The levels and activities of MMP-9 were also significantly increased during UC induced by IA and IL-10 KO. Deletion of MMP-9 decreased the levels of endostatin in both water- and DSS-treated MMP-9 KO mice. Treatment with endostatin significantly improved DSS-induced UC in MMP-9 KO mice. SIGNIFICANCE: 1) Concomitantly increased endostatin is a defensive response to the increased VEGF in UC, 2) MMP-9 is a key enzyme to generate endostatin which may modulate the balance between VEGF and endostatin during experimental UC, and 3) endostatin treatment plays a beneficial role in UC. Thus, anti-angiogenesis seems to be a new therapeutic option for UC.
Asunto(s)
Colitis Ulcerosa/etiología , Endostatinas/fisiología , Animales , Western Blotting , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/fisiopatología , Colon/efectos de los fármacos , Colon/metabolismo , Colon/fisiopatología , Sulfato de Dextran/farmacología , Modelos Animales de Enfermedad , Endostatinas/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Yodoacetamida/farmacología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/fisiología , Ratones , Ratones Noqueados , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/fisiología , Ratas , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Induction of apoptosis is a promising strategy that could lead to the discovery of new molecules active in cancer chemotherapy. This property is generally observed when cells are treated with agents that target microtubules, dynamic structures that play a crucial role in cell division. Small molecules such as benzo[b]furans are attractive as inhibitors of tubulin polymerization. A new class of inhibitors of tubulin polymerization based on the 2-(3',4',5'-trimethoxybenzoyl)benzo[b]furan molecular skeleton, with the amino group placed at different positions on the benzene ring, were synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell-cycle effects. The methoxy substitution pattern on the benzene portion of the benzo[b]furan moiety played an important role in affecting antiproliferative activity. In the series of 5-amino derivatives, the greatest inhibition of cell growth occurred if the methoxy substituent is placed at the C6 position, whereas C7 substitution decreases potency. The most promising compound in this series is 2-(3',4',5'-trimethoxybenzoyl)-3-methyl-5-amino-6-methoxybenzo[b]furan (3 h), which inhibits cancer cell growth at nanomolar concentrations (IC(50) =16-24 nM), and interacts strongly with tubulin by binding to the colchicine site. Sub-G(1) apoptotic cells in cultures of HL-60 and U937 cells were observed by flow cytometric analysis after treatment with 3 h in a concentration-dependent manner. We also show that compound 3 h induces apoptosis by activation of caspase-3, -8, and -9, and this is associated with cytochrome c release from mitochondria. The introduction of an α-bromoacryloyl group increased antiproliferative activity with respect to the parent amino derivatives.
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Anisoles/síntesis química , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Benzofuranos/química , Benzofuranos/síntesis química , Benzofuranos/farmacología , Moduladores de Tubulina , Tubulina (Proteína)/química , Animales , Anisoles/química , Anisoles/toxicidad , Benzofuranos/toxicidad , Sitios de Unión , Caspasas/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Colchicina/química , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/farmacologíaRESUMEN
A series of 1-aryl-5-(3',4',5'-trimethoxyphenyl) derivatives and their related 1-(3',4',5'-trimethoxyphenyl)-5-aryl-1,2,4-triazoles, designed as cis-restricted combretastatin analogues, were synthesized and evaluated for antiproliferative activity, inhibitory effects on tubulin polymerization, cell cycle effects, and apoptosis induction. Their activity was greater than, or comparable with, that of the reference compound CA-4. Flow cytometry studies showed that HeLa and Jurkat cells treated with the most active compounds 4l and 4o were arrested in the G2/M phase of the cell cycle in a concentration dependent manner. This effect was accompanied by apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, activation of caspase-3, and PARP cleavage. Compound 4l was also shown to have potential antivascular activity, since it induced endothelial cell shape change in vitro and disrupted the sprouting of endothelial cells in the chick aortic ring assay.
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Antineoplásicos/síntesis química , Bibencilos/síntesis química , Triazoles/síntesis química , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis , Bibencilos/química , Bibencilos/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Forma de la Célula , Células Cultivadas , Embrión de Pollo , Colchicina/química , Ensayos de Selección de Medicamentos Antitumorales , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Activación Enzimática , Fase G2/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Modelos Moleculares , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-Actividad , Triazoles/química , Triazoles/farmacología , Tubulina (Proteína)/química , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
BRCA1 plays an important role in the homologous recombination (HR)-mediated DNA double-strand break (DSB) repair, but the mechanism is not clear. Here we describe that BRCA1 forms a complex with CtIP and MRN (Mre11/Rad50/Nbs1) in a cell cycle-dependent manner. Significantly, the complex formation, especially the ionizing radiation-enhanced association of BRCA1 with MRN, requires cyclin-dependent kinase activity. CtIP directly interacts with Nbs1. The in vivo association of BRCA1 with MRN is largely dependent on the association of CtIP with the BRCT domains at the C terminus of BRCA1, whereas the N terminus of BRCA1 also contributes to its association with MRN. CtIP, as well as the interaction of BRCA1 with CtIP and MRN, is critical for IR-induced single-stranded DNA formation and cellular resistance to radiation. Consistently, CtIP itself is required for efficient HR-mediated DSB repair, like BRCA1 and MRN. These studies suggest that the complex formation of BRCA1.CtIP.MRN is important for facilitating DSB resection to generate single-stranded DNA that is needed for HR-mediated DSB repair. Because cyclin-dependent kinase is important for establishing IR-enhanced interaction of MRN with BRCA1, we propose that the cell cycle-dependent complex formation of BRCA1, CtIP, and MRN contributes to the activation of HR-mediated DSB repair in the S and G(2) phases of the cell cycle.
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Proteína BRCA1/metabolismo , Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Fase G2/fisiología , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Fase S/fisiología , Ácido Anhídrido Hidrolasas , Proteína BRCA1/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas , Fase G2/efectos de la radiación , Humanos , Proteína Homóloga de MRE11 , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Estructura Terciaria de Proteína/genética , Radiación Ionizante , Fase S/efectos de la radiaciónRESUMEN
UNLABELLED: We assessed the expression of about 8,000 known or unknown genes in the preulcerogenic stages of cysteamine-induced duodenal ulceration in rats, in comparison with the toxic but nonulcerogen ethanolamine. The most prominent gene changes were confirmed by custom gene blots, reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, radio-immunoassay, Western blot, or enzyme-linked immunosorbent assay (ELISA), and the levels of their expression in other gastrointestinal organs such as ileum and colon were identified by real-time PCR. The time-course study after cysteamine showed 40 genes with marked changes, belonging to cell surface antigens, transcription factors, DNA binding proteins, ion channels, transport proteins, cellular receptors, and expressed sequence tags (i.e., unknown genes). In comparison with ethanolamine, these 40 genes changed by cysteamine only may represent ulcer-associated genes, such as endothelin receptor B, endothelin 1, caspase 3, transcription factors egr-1, Sp1, the angiogenic growth factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), and especially egr-1 and endothelin receptor B (ETRB) showed no changes in ileum and colon. CONCLUSIONS: (1) These data suggest that duodenal ulcerogenesis may require the interaction of several genes leading to endothelial and epithelial cell injury, mucosal erosion, and ulcer; (2) these new findings may offer a new approach to the identification of potential ulcerogenic genes and provide new insights into the molecular mechanisms of duodenal ulceration.
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Úlcera Duodenal/genética , Expresión Génica , Animales , Western Blotting , Úlcera Duodenal/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Mucosa Intestinal/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The Mre11-Rad50-Nbs1 (MRN) complex is required for mediating the S-phase checkpoint following UV treatment, but the underlying mechanism is not clear. Here we demonstrate that at least two mechanisms are involved in regulating the S-phase checkpoint in an MRN-dependent manner following UV treatment. First, when replication forks are stalled, MRN is required upstream of ataxia telangiectasia mutated and Rad3-related protein (ATR) to facilitate ATR activation in a substrate and dosage-dependent manner. In particular, MRN is required for ATR-directed phosphorylation of RPA2, a critical event in mediating the S-phase checkpoint following UV treatment. Second, MRN is a downstream substrate of ATR. Nbs1 is phosphorylated by ATR at Ser-343 when replication forks are stalled, and this phosphorylation event is also important for down-regulating DNA replication following UV treatment. Moreover, we demonstrate that MRN and ATR/ATR-interacting protein (TRIP) interact with each other, and the forkhead-associated/breast cancer C-terminal domains (FHA/BRCT) of Nbs1 play a significant role in mediating this interaction. Mutations in the FHA/BRCT domains do not prevent ATR activation but specifically impair ATR-mediated Nbs1 phosphorylation at Ser-343, which results in a defect in the S-phase checkpoint. These data suggest that MRN plays critical roles both upstream and downstream of ATR to regulate the S-phase checkpoint when replication forks are stalled.
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Proteínas de Ciclo Celular/fisiología , Enzimas Reparadoras del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Ácido Anhídrido Hidrolasas , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular , Línea Celular Tumoral , Daño del ADN , Glutatión Transferasa/metabolismo , Humanos , Proteína Homóloga de MRE11 , Fosforilación , Estructura Terciaria de Proteína , Fase S , Serina/química , Rayos UltravioletaRESUMEN
Previously, we demonstrated that cysteamine releases endothelin-1 in the rat duodenal mucosa, followed by increased expression of early growth response factor-1 (egr-1). We hypothesized that egr-1 is a key mediator gene in the multifactorial mechanisms of duodenal ulcer development and healing because its protein, transcription factor product Egr-1, regulates the expression of angiogenic growth factors. We wanted to determine the effect of egr-1 antisense oligonucleotide on cysteamine-induced duodenal ulcers as well as on the expression of bFGF, PDGF, and VEGF, of which synthesis is modulated by Egr-1. An antisense oligonucleotide to egr-1 was used to inhibit the synthesis of Egr-1 and to determine its effect on ulcer formation in the rat model of cysteamine-induced duodenal ulceration. Real-time RT-PCR and Western blot analysis were used to assess the expression of Egr-1 mRNA and protein as well as ERK, bFGF, PDGF, and VEGF. The antisense Egr-1 oligonucleotide inhibited the expression of egr-1 mRNA and protein and increased the duodenal ulcer size from 8.1 +/- 1.8 mm(2) in controls to 20.7 +/- 4.0 mm(2) (P < 0.01). Cysteamine induced phosphorylation of ERK1/2 and enhanced the synthesis of bFGF, PDGF, and VEGF in the preulcerogenic stages of duodenal ulceration, whereas egr-1 antisense oligonucleotide markedly decreased the expression of these growth factors in the duodenal mucosa. We also demonstrated that Egr-1 expression relates to the ulcerogenic effect of cysteamine because these actions were not exerted by the toxic analog ethanolamine. Thus Egr-1 seems to play a critical role in duodenal ulceration because Egr-1 downregulation aggravates experimental duodenal ulcers, most likely through the transcriptional inhibition of bFGF, PDGF, and VEGF synthesis.