RESUMEN
BACKGROUND: Severe liver steatosis is a known risk factor for increased ischemia-reperfusion injury (IRI) and poor outcomes after liver transplantation (LT). This study aimed to identify steatosis-related molecular mechanisms associated with IRI exacerbation after LT. METHODS: Paired graft biopsies (n = 60) were collected before implantation (L1) and 90 minutes after reperfusion (L2). The LT recipients (n = 30) were classified by graft macrosteatosis: without steatosis (WS) of 5% or less (n = 13) and with steatosis (S) of 25% or greater (n = 17). Plasma samples were collected at L1, L2, and 1 day after LT (postoperative [POD]1) for cytokines evaluation. Tissue RNA was isolated for gene expression microarrays. Probeset summaries were obtained using robust multiarray average algorithm. Pairwise comparisons were fit using 2-sample t test. P values 0.01 or less were significant (false discovery rate <5%). Molecular pathway analyses were conducted using Ingenuity Pathway Analysis tool. RESULTS: Significantly differentially expressed genes were identified for WS and S grafts after reperfusion. Comprehensive comparison analysis of molecular profiles revealed significant association of S grafts molecular profile with innate immune response activation, macrophage production of nitric oxide and reactive oxygen species, IL-6, IL-8, IL-10 signaling activation, recruitment of granulocytes, and accumulation of myeloid cells. Postreperfusion histological patterns of S grafts revealed neutrophilic infiltration surrounding fat accumulation. Circulating proinflammatory cytokines after reperfusion and 24 hours after LT concurred with intragraft-deregulated molecular pathways. All tested cytokines were significantly increased in plasma of S grafts recipients after reperfusion when compared with WS group at same time. CONCLUSIONS: Increases of graft steatosis exacerbate IRI by exacerbation of innate immune response after LT. Preemptive strategies should consider it for safety usage of steatotic livers.
Asunto(s)
Biomarcadores/metabolismo , Hígado Graso/cirugía , Inmunidad Innata/inmunología , Trasplante de Hígado , Hígado/patología , Daño por Reperfusión/etiología , Donantes de Tejidos , Biopsia , Citocinas/biosíntesis , Citocinas/genética , Hígado Graso/complicaciones , Hígado Graso/patología , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/inmunología , Daño por Reperfusión/patologíaRESUMEN
BACKGROUND: Ex vivo lung perfusion (EVLP) enables assessment and rehabilitation of marginal donor lungs before transplantation. We previously demonstrated that adenosine A2A receptor (A2AR) agonism attenuates lung ischemia-reperfusion injury. The current study utilizes a novel murine EVLP model to test the hypothesis that A2AR agonist enhances EVLP-mediated rehabilitation of donation after circulatory death (DCD) lungs. METHODS: Mice underwent euthanasia and 60 minutes warm ischemia, and lungs were flushed with Perfadex and underwent cold static preservation (CSP, 60 minutes). Three groups were studied: no EVLP (CSP), EVLP with Steen solution for 60 minutes (EVLP), and EVLP with Steen solution supplemented with ATL1223, a selective A2AR agonist (EVLP + ATL1223). Lung function, wet/dry weight, cytokines and neutrophil numbers were measured. Microarrays were performed using the Affymetrix GeneChip Mouse Genome 430A 2.0 Array. RESULTS: Ex vivo lung perfusion significantly improved lung function versus CSP, which was further, significantly improved by EVLP + ATL1223. Lung edema, cytokines, and neutrophil counts were reduced after EVLP and further, significantly reduced after EVLP + ATL1223. Gene array analysis revealed differential expression of 1594 genes after EVLP, which comprise canonical pathways involved in inflammation and innate immunity including IL-1, IL-8, IL-6, and IL-17 signaling. Several pathways were uniquely regulated by EVLP + ATL1223 including the downregulation of genes involved in IL-1 signaling, such as ADCY9, ECSIT, IRAK1, MAPK12, and TOLLIP. CONCLUSIONS: Ex vivo lung perfusion modulates proinflammatory genes and reduces pulmonary dysfunction, edema, and inflammation in DCD lungs, which are further reduced by A2AR agonism. This murine EVLP model provides a novel platform to study rehabilitative mechanisms of DCD lungs.
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Agonistas del Receptor de Adenosina A2/farmacología , Trasplante de Pulmón , Pulmón/irrigación sanguínea , Perfusión/métodos , Daño por Reperfusión/rehabilitación , Animales , Modelos Animales de Enfermedad , Circulación Extracorporea , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Tacrolimus and sirolimus are commonly used maintenance immunosuppressants in kidney transplantation. As their effects on immune cells and allograft molecular profiles have not been elucidated, we characterized the effects of tacrolimus to sirolimus conversion on the frequency and function of T cells, and on graft molecular profiles. Samples from renal transplant patients in a randomized trial of 18 patients with late sirolimus conversion and 12 on tacrolimus maintenance were utilized. Peripheral blood was collected at 0, 6, 12, and 24 months post randomization, with T-cell subpopulations analyzed by flow cytometry and T-cell alloreactivity tested by IFN-γ ELISPOT. Graft biopsy samples obtained 24 months post randomization were used for gene expression analysis. Sirolimus conversion led to an increase in CD4(+)25(+++)Foxp3(+) regulatory T cells. While tacrolimus-maintained patients showed a decrease in indirect alloreactivity over time post transplant, sirolimus conversion increased indirect alloreactive T-cell frequencies compared with tacrolimus-maintained patients. No histological differences were found in graft biopsies, but molecular profiles showed activation of the antigen presentation, IL-12 signaling, oxidative stress, macrophage-derived production pathways, and increased inflammatory and immune response in sirolimus-converted patients. Thus, chronic immune alterations are induced after sirolimus conversion. Despite the molecular profile being favorable to calcineurin inhibitor-based regimen, there was no impact in renal function over 30 months of follow-up.
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Inmunosupresores/uso terapéutico , Trasplante de Riñón , Sirolimus/uso terapéutico , Tacrolimus/uso terapéutico , Inmunología del Trasplante/efectos de los fármacos , Inmunología del Trasplante/inmunología , Adulto , Aloinjertos/inmunología , Aloinjertos/patología , Presentación de Antígeno/genética , Recuento de Linfocito CD4 , Sustitución de Medicamentos , Femenino , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Supervivencia de Injerto/efectos de los fármacos , Humanos , Interferón gamma/sangre , Interleucina-12/metabolismo , Macrófagos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Linfocitos T ReguladoresRESUMEN
Despite improvement on outcomes post liver transplantation (LT), complications such as HCV recurrence (HCV-rec) and acute cellular rejection (ACR) continue to be a challenge for transplant physicians. Accurate diagnostic tools to better dissect between those complications post-LT are crucial for prompt and correct diagnosis and treatment. It is well known that the overlapping features of clinical and histo-pathological characteristics between these conditions turn difficult the appropriate differential diagnosis. Recently, new technological advances had supported the field of biomarker discovery in many diseases. Disease biomarkers capable to differentiate ACR versus HCV-rec post-LT is a long waited task in the transplant community. This editorial describes and discusses potential biomarkers of disease differentiation including recent reports in the field of genomics, proteomics, immunohistochemistry among other technologies.
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Rechazo de Injerto/diagnóstico , Hepatitis C/cirugía , Trasplante de Hígado/efectos adversos , Técnicas de Diagnóstico Molecular , Enfermedad Aguda , Biomarcadores/análisis , Diagnóstico Diferencial , Marcadores Genéticos , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Hepatitis C/diagnóstico , Hepatitis C/genética , Hepatitis C/virología , Humanos , Técnicas de Diagnóstico Molecular/métodos , Valor Predictivo de las Pruebas , Recurrencia , Resultado del TratamientoRESUMEN
Noninvasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers, we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation, and the longitudinal validation studies for noninvasive monitoring of graft function. Of 1733 mature miRNAs studied using microarrays, 22 were found to be differentially expressed between groups. Ontology and pathway analyses showed inflammation as the principal biological function associated with these miRNAs. Twelve selected miRNAs were longitudinally evaluated in urine samples of an independent set of 66 patients, at two time points after kidney transplant. A subset of these miRNAs was found to be differentially expressed between groups early after kidney transplant before histological allograft injury was evident. Thus, a panel of urine miRNAs was identified as potential biomarkers for monitoring graft function and anticipating progression to CAD in kidney transplant patients.
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Perfilación de la Expresión Génica , Pruebas Genéticas/métodos , Trasplante de Riñón , Riñón/fisiopatología , MicroARNs/orina , Insuficiencia Renal Crónica/diagnóstico , Adulto , Anciano , Atrofia , Biopsia , Estudios de Casos y Controles , Femenino , Fibrosis , Marcadores Genéticos , Humanos , Riñón/patología , Trasplante de Riñón/efectos adversos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Proteinuria/diagnóstico , Proteinuria/genética , Proteinuria/orina , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/orina , Reproducibilidad de los Resultados , Factores de Tiempo , Resultado del Tratamiento , Urinálisis , Adulto JovenRESUMEN
BACKGROUND: Ischemia-reperfusion (I/R) injury is a multifactorial phenomenon that occurs during the transplant event and frequently compromises early graft function after liver transplantation (LT). Current comprehension of molecular mechanisms and regulation processes of I/R injury lacks clarity. MicroRNA (miRNA) regulation results critical in several biological processes. METHODS: This study evaluated gene expression and miRNA expression profiles using microarrays in 34 graft biopsies collected at preimplantation (L1) and at 90 min postreperfusion (L2) from consecutives deceased-donor LT recipients. miRNA profiles were first analyzed. Data integration analysis (gene expression/miRNA expression) aimed to identify potential target genes for each identified miRNA from the L1/L2 differential gene expression profile. RESULTS: Pairwise comparison analyses identified 40 miRNAs and 3168 significantly differentially expressed genes at postreperfusion time compared with preimplantation time. Pathway analysis of miRNAs associated these profiles with antiapoptosis, inhibition of cellular proliferation, and proinflammatory processes. Target analysis identified an miRNA-associated molecular profile of 2172 genes involved in cellular growth and proliferation modulation by cell cycle regulation, cell death and survival, and proinflammatory and anti-inflammatory processes. miRNA-independent genes involved proinflammatory molecules. CONCLUSION: We identified a miRNA profile involved in posttranscriptional regulatory mechanisms in I/R injury post-LT. A better understanding of these molecular processes involved in I/R may contribute to develop new strategies to minimize graft injury.
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Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Rechazo de Injerto/genética , Trasplante de Hígado , MicroARNs/genética , Daño por Reperfusión/genética , Adulto , Anciano , Biopsia , Femenino , Estudios de Seguimiento , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Humanos , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad , Periodo Posoperatorio , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transcripción Genética , Adulto JovenRESUMEN
Liver transplantation represents the treatment of choice for acute hepatic failure or chronically induced end stage of liver disease. Molecular characterization of hepatic injury and recovery processes encloses the key for biomarker discovery in the liver transplantation field. Several pathological hepatic processes were demonstrated to be associated with deregulated miRNA profiles. Moreover, abnormal concentration levels of circulating cell-free miRNAs correlate with specific hepatic injury. The high molecular stability and emerging rapid assessment techniques invite further consideration of miRNAs as feasible monitoring and outcome predictive biomarkers for liver disease and liver transplantation. The present review aims to provide an overview of recent achievements in research on the potential applicability of miRNAs as biomarkers in liver disease and liver transplantation.
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Biomarcadores/sangre , Enfermedad Hepática en Estado Terminal/sangre , Enfermedad Hepática en Estado Terminal/cirugía , Trasplante de Hígado , MicroARNs/sangre , Animales , Progresión de la Enfermedad , Enfermedad Hepática en Estado Terminal/genética , Enfermedad Hepática en Estado Terminal/fisiopatología , Humanos , Hígado/patología , Hepatopatías Alcohólicas/fisiopatología , Regeneración Hepática/fisiología , MicroARNs/análisis , MicroARNs/fisiología , Soluciones Preservantes de Órganos/química , Daño por Reperfusión/fisiopatología , Trasplante HomólogoRESUMEN
BACKGROUND: Early hepatocellular carcinoma (HCC) detection is difficult because low accuracy of surveillance tests. Genome-wide analyses were performed using HCV-cirrhosis with HCC to identify predictive signatures. METHODOLOGY/PRINCIPAL FINDINGS: Cirrhotic liver tissue was collected from 107 HCV-infected patients with diagnosis of HCC at pre-transplantation and confirmed in explanted livers. Study groups included: 1) microarray hybridization set (nâ=â80) including patients without (woHCCâ=â45) and with (wHCCâ=â24) HCC, and with incidental HCC (iHCCâ=â11); 2) independent validation set (nâ=â27; woHCCâ=â16, wHCCâ=â11). Pairwise comparisons were performed using moderated t-test. FDR<1% was considered significant. L(1)-penalized logistic regression model was fit for woHCC and wHCC microarrays, and tested against iHCC. Prediction model genes were validated in independent set by qPCR. The genomic profile was associated with genetic disorders and cancer focused on gene expression, cell cycle and cell death. Molecular profile analysis revealed cell cycle progression and arrest at G2/M, but progressing to mitosis; unregulated DNA damage check-points, and apoptosis. The prediction model included 17 molecules demonstrated 98.6% of accuracy and correctly classified 6 out of 11 undiagnosed iHCC cases. The best model performed even better in the additional independent set. CONCLUSIONS/SIGNIFICANCES: The molecular analysis of HCV-cirrhotic tissue conducted to a prediction model with good performance and high potential for HCC surveillance.
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Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Hepacivirus , Hepatitis C Crónica/metabolismo , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Adulto , Anciano , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/virología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Detección Precoz del Cáncer , Femenino , Regulación Neoplásica de la Expresión Génica , Hepatitis C Crónica/virología , Humanos , Hígado/metabolismo , Hígado/patología , Hígado/virología , Cirrosis Hepática/virología , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , TranscriptomaRESUMEN
The mammalian Krüppel-like factor 6 (KLF6) is involved in critical roles such as growth-related signal transduction, cell proliferation and differentiation, development, apoptosis and angiogenesis. Also, KLF6 appears to be an emerging key factor during cancer development and progression. Its expression is thoroughly regulated by several cell-damaging stimuli. DNA damaging agents at lethal concentrations induce a p53-independent down-regulation of the klf6 gene. To investigate the impact of external stimuli on human klf6 gene expression, its mRNA level was analyzed using a cancer cell line profiling array system, consisting in an assortment of immobilized cDNAs from multiple cell lines treated with several cell-damaging agents at growth inhibitory concentrations (IC(50)). Cell-damaging agents affected the klf6 expression in 62% of the cDNA samples, though the expression pattern was not dependent on the cell origin type. Interestingly, significant differences (p<0.0001) in KLF6 mRNA levels were observed depending on the cellular p53 status upon cell damage. KLF6 expression was significantly increased in 63% of p53-deficient cells (122/195). Conversely, KLF6 mRNA level decreased nearly 4 fold in more than 70% of p53+/+ cells. In addition, klf6 gene promoter activity was down-regulated by DNA damaging agents in cells expressing the functional p53 protein whereas it was moderately increased in the absence of functional p53. Consistent results were obtained for the endogenous KLF6 protein level. Results indicate that human klf6 gene expression is responsive to external cell damage mediated by IC(50) concentrations of physical and chemical stimuli in a p53-dependent manner. Most of these agents are frequently used in cancer therapy. Induction of klf6 expression in the absence of functional p53 directly correlates with cell death triggered by these compounds, whereas it is down-regulated in p53+/+ cells. Hence, klf6 expression level could represent a valuable marker for the efficiency of cell death upon cancer treatment.
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Antineoplásicos/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Inhibidores de Crecimiento/farmacología , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias/genética , Estrés Oxidativo , Proteínas Proto-Oncogénicas/genética , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Genes Supresores de Tumor , Genes p53 , Células Hep G2 , Humanos , Factor 6 Similar a Kruppel , Mutágenos/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
An essential role for the Krüppel-like transcription factor family has been determined in the regulation of remarkable processes including cell proliferation, differentiation, signal transduction, oncogenesis, and cell death. A member of this group, Krüppel-like factor 6 (KLF6), identified on the basis of its ability to regulate a group of genes belonging to the carcinoembryonic antigen gene family, has been involved in human carcinogenesis. Early studies proposed a tumor suppressor function for KLF6 because of its ability to reduce cell proliferation through several biochemical mechanisms including regulation of cell cycle components, oncogene products, and apoptosis. Mutations within the klf6 gene, decreased expression and/or loss-of-heterozygosity were associated with the development of different human malignancies, and, hence, further supporting the tumor suppressor function of KLF6. This view has been challenged by other studies in distinct types of human cancers describing infrequent genetic alterations of klf6 gene or even enhanced expression in some tumors. The scenario about KLF6 function became still more complex as the description of oncogenic KLF6 splice variant 1 (SV1) with dominant negative activity against the wild type KLF6 (wtKLF6) protein. Additionally, increased evidence is suggesting that KLF6 is a bonafide target of several signaling cascades, which ultimate regulatory effect on this protein could drive decisions of cell life and death, facing the dilemma about how wtKLF6 could be involved in both processes. These apparently conflicting situations, emerged by apparently opposite effects mediated by wtKLF6, may be related, at least in part, to the biological cross-talk with the c-Jun oncoprotein. Depending on the stimulus received by the cell, wtKLF6 interaction with c-Jun determines different cell outcomes such as proliferation control or apoptosis. Thus, KLF6 responsiveness represents a kind of cell warning signal on receiving different stimuli, including oncogenic activation and microbial infections, orchestrating the implementation of proliferation and apoptotic programs.
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Apoptosis , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel , Proteínas Proto-Oncogénicas , Transducción de Señal , Animales , Apoptosis/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Genes Supresores de Tumor/fisiología , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Pérdida de Heterocigocidad , Ratones , Ratones Noqueados , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Transducción de Señal/genéticaRESUMEN
BACKGROUND: B cells and antibodies are involved not only in controlling the spread of blood circulating Trypanosoma cruzi, but also in the autoreactive manifestations observed in Chagas disease. Acute infection results in polyclonal B cell activation associated with hypergammaglobulinemia, delayed specific humoral immunity and high levels of non-parasite specific antibodies. Since TNF superfamily B lymphocyte Stimulator (BAFF) mediates polyclonal B cell response in vitro triggered by T. cruzi antigens, and BAFF-Tg mice show similar signs to T. cruzi infected mice, we hypothesized that BAFF can mediate polyclonal B cell response in experimental Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: BAFF is produced early and persists throughout the infection. To analyze BAFF role in experimental Chagas disease, Balb/c infected mice were injected with BR3:Fc, a soluble receptor of BAFF, to block BAFF activity. By BAFF blockade we observed that this cytokine mediates the mature B cell response and the production of non-parasite specific IgM and IgG. BAFF also influences the development of antinuclear IgG and parasite-specific IgM response, not affecting T. cruzi-specific IgG and parasitemia. Interestingly, BAFF inhibition favors the parasitism in heart. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate, for the first time, an active role for BAFF in shaping the mature B cell repertoire in a parasite infection.
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Anticuerpos Antiprotozoarios/biosíntesis , Factor Activador de Células B/metabolismo , Linfocitos B/inmunología , Enfermedad de Chagas/inmunología , Bazo/inmunología , Trypanosoma cruzi/inmunología , Animales , Factor Activador de Células B/antagonistas & inhibidores , Modelos Animales de Enfermedad , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Krüppel-like factor 6 (KLF6) is an evolutionarily conserved and ubiquitously expressed protein that belongs to the mammalian Sp1/KLF family of transcriptional regulators. Though KLF6 is a transcription factor and harbors a nuclear localization signal it is not systematically located in the nucleus but it was detected in the cytoplasm of several tissues and cell lines. Hence, it is still not fully settled whether the tumor suppressor function of KLF6 is directly associated with its ability to regulate target genes. METHODOLOGY/PRINCIPAL FINDINGS: In this study we analyzed KLF6 expression and sub-cellular distribution by immunohistochemistry in several normal and tumor tissues in a microarray format representing fifteen human organs. Results indicate that while both nuclear and cytoplasmic distribution of KLF6 is detected in normal breast tissues, breast carcinomas express KLF6 mainly detected in the cytoplasm. Expression of KLF6 was further analyzed in breast cancer tissues overexpressing ERBB2 oncoprotein, which is associated with poor disease prognosis and patient's survival. The analysis of 48 ductal carcinomas revealed a significant population expressing KLF6 predominantly in the nuclear compartment (X(2)p = 0.005; Fisher p = 0.003). Moreover, this expression pattern correlates directly with early stage and small ductal breast tumors and linked to metastatic events in lymph nodes. CONCLUSIONS/SIGNIFICANCE: Data are consistent with a preferential localization of KLF6 in the nuclear compartment of early stage and small HER2-ERBB2 overexpressing ductal breast tumor cells, also presenting lymph node metastatic events. Thus, KLF6 tumor suppressor could represent a new molecular marker candidate for tumor prognosis and/or a potential target for therapy strategies.
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Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Núcleo Celular/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Inmunohistoquímica , Factor 6 Similar a Kruppel , Fracciones Subcelulares/metabolismoRESUMEN
The Krüppel-like transcription Factor 6 (KLF6) is regulated during cell proliferation and differentiation events like mammalian development and tissue regeneration, while its aberrant expression is associated with tumor formation. To investigate KLF6 transcriptional control, the genomic organization of human KLF6 together with its cis-regulatory region was analyzed. A high sequence homology of KLF6 regulatory regions was found in mammals, which in turn predicts a high degree of evolutionary conserved transcriptional mechanisms. A transcription start site was identified at the first nucleotide downstream of a potential initiator element. Also, the role of KLF6 regulatory regions was determined by transfection experiments. A minimal promoter region lacking a TATA-box yet containing an Initiator was identified and found to be active in all cells analyzed. In addition, two strong activating sequences were located between positions -407/-344 and -307/-207, where the latter contained Sp1 and CAAT-box sites. Furthermore, ectopic expression of Sp1 increased the transcriptional activity of the KLF6 promoter. In conclusion, our data revealed that KLF6 gene transcription is under control of a TATA-box independent initiation mechanism together with an evolutionary conserved array of positive cis-acting elements.
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Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores/genética , Transactivadores/fisiología , Animales , Secuencia de Bases , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Mamíferos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , TATA Box , Transcripción Genética , TransfecciónRESUMEN
The aim of this work was to evaluate by immunohistochemistry (IHC) the expression of both LRP-1 and urokinase-type plasminogen activator receptor (uPAR) at different developmental stages of rat prostate disease by using a prostate cancer model previously developed in our laboratory. We found that LRP-1 was weakly expressed in normal prostates and in rats with hyperplastic glands. The expression of this receptor increased and correlated with the degree of premalignant lesions (PIN I, II, and III). The IHC for uPAR in normal prostates and in premalignant lesions showed a score of immunostaining that correlated with the expression of LRP-1. On the other hand, in prostates with adenocarcinomas and undifferentiated carcinomas, LRP-1 was undetectable or weakly detected, whereas uPAR showed a significantly higher level of expression. Based on the IHC results in rat prostates with premalignant and malignant lesions and considering that LRP-1, by mediating the internalization of uPAR, is involved in the regulation of extracellular matrix remodeling and cell migration, we conclude that a decreased expression of LRP-1 could be involved with the increasing activation of plasminogen activators shown in cancers.