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BACKGROUND: Association studies have implicated the glycosaminoglycan hyaluronan (hyaluronic acid, HA) and its degrading enzymes the hyaluronidases in tumour progression and metastasis. Oligosaccharides of degraded HA have been ascribed a number of biological functions that are not exerted by high-molecular-weight HA (HMW-HA). However, whether these small HA oligosaccharides (sHA) have a role in tumour progression currently remains uncertain due to an inability to analyse their concentration in tumours. METHODS: We report a novel method to determine the concentration of sHA ranging from 6 to 25 disaccharides in tumour interstitial fluid (TIF). Levels of sHA were measured in TIF from experimental rat tumours and human colorectal tumours. RESULTS: While the majority of HA in TIF is HMW-HA, concentrations of sHA up to 6 µg ml(-1) were detected in a subset of tumours, but not in interstitial fluid from healthy tissues. In a cohort of 72 colorectal cancer patients we found that increased sHA concentrations in TIF are associated with lymphatic vessel invasion by tumour cells and the formation of lymph node metastasis. CONCLUSIONS: These data document for the first time the pathophysiological concentration of sHA in tumours, and provide evidence of a role for sHA in tumour progression.
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Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Líquido Extracelular/metabolismo , Ácido Hialurónico/metabolismo , Adenocarcinoma/secundario , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Humanos , Metástasis Linfática , Invasividad Neoplásica , Trasplante de Neoplasias , RatasRESUMEN
BACKGROUND AND OBJECTIVES: Despite its dwindling occurrence, gastric cancer remains a leading cause of cancer related mortality worldwide. Molecular determinants of prognosis that impact survival are being sought out as a means to facilitate rational clinical decision-making and enhance patient management. In this study, we evaluated three molecules implicated in gastric carcinogenesis and demonstrated that the differential expression of cyclooxygenase-2 (COX-2) and the viral oncogene homolog Src proteins could explain the differences in survival observed in patients older and younger than 50 years of age. METHODS: We evaluated 5-year survival in a cohort of 423 gastric cancer patients using chronological age as a variable. Additionally, we assessed the protein expression of three molecules (COX-2, TFF1, Src) implicated in the pathogenesis of gastric cancer using immunohistochemistry. RESULTS: We found that patients younger than 50 years of age had a better 5-year survival rate in all tumor stages. We found that the expression of COX-2 and Src correlated significantly with survival in this group without any significant impact attributable to TFF1. CONCLUSIONS: Our study demonstrates that young gastric cancer patients have a better prognostic outlook that could in part be explained by the differential expression of COX-2 and Src.
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Biomarcadores de Tumor/análisis , Carcinoma/química , Carcinoma/mortalidad , Ciclooxigenasa 2/análisis , Proteínas Proto-Oncogénicas pp60(c-src)/análisis , Neoplasias Gástricas/química , Neoplasias Gástricas/mortalidad , Proteínas Supresoras de Tumor/análisis , Adulto , Factores de Edad , Carcinoma/patología , Carcinoma/cirugía , Unión Esofagogástrica , Femenino , Alemania , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Estudios Retrospectivos , Neoplasias Gástricas/patología , Neoplasias Gástricas/cirugía , Análisis de Matrices Tisulares , Factor Trefoil-1RESUMEN
BACKGROUND AND AIMS: Transanal real-time elastography (RTE) was demonstrated to yield valuable information regarding elastic properties of the anal sphincter in patients with fecal incontinence. We studied the role of RTE findings as a risk factor for the outcome of patients with fecal incontinence following anorectal surgery in irradiated and non-irradiated individuals and compared these observations with conventional B-mode/color Doppler EUS.â PATIENTS AND METHODS: 90 patients with postsurgical fecal incontinence were included in this prospective monocentric study. Baseline and follow-up (after 3 weeks and 1 year) assessment included an incontinence severity score questionnaire, rectal manometry, B-mode/color Doppler EUS and RTE with quantitation of the sphincter elastograms. RESULTS: 81 patients could be finally assessed, in 24 patients (29.6%) a pathological elastogram with predominantly hard elements was found; logistic regression analysis revealed no significant association with the short- and long-term clinical outcome nor were any differences seen between irradiated and non-irradiated patients. Defined sphincter defects as seen with conventional EUS were significanntly associated with a worse short- and long-term outcome: odds ratio ORshort-term: 1.414 (1.107 - 1.807, p = 0.0101); ORlong-term: 1.675 (95% CI: 1.133 - 2.477; p = 0.0294). Submucosal thickening and hypervascularization were found more frequently in the irradiated group (p < 0.01). CONCLUSION: RTE with quantitation of sphincter elastic properties yields no further diagnostic and prognostic information compared to conventional EUS in irradiated and non-irradiated patients and, therefore, cannot be regarded as a new tool in the assessment of those patients. Our data further confirm the view that defined sphincter defects may be a major risk factor for an unfavorable outcome.
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Canal Anal/diagnóstico por imagen , Canal Anal/cirugía , Procedimientos Quirúrgicos del Sistema Digestivo/efectos adversos , Diagnóstico por Imagen de Elasticidad/métodos , Endosonografía/métodos , Incontinencia Fecal/diagnóstico por imagen , Incontinencia Fecal/radioterapia , Canal Anal/efectos de la radiación , Sistemas de Computación , Incontinencia Fecal/etiología , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Resultado del Tratamiento , Ultrasonografía Doppler en Color/métodosRESUMEN
Chemoprotection of haematopoietic stem cells (HSCs) by gene therapeutic transfer of drug-resistance genes represents the encouraging approach to prevent myelosuppression, which is one of the most severe side effects in tumor therapy. Thus, we cloned and evaluated six different bicistronic lentiviral SIN vectors encoding two transgenes, MGMT(P140K) (an O(6)-benzylguanine-resistant mutant of methylguanine-DNA methyltransferase) and MDR1 (multidrug resistance 1), using various linker sequences (IRESEMCV, IRESFMDV and 2A-element of FMDV (F2A)). Expression of both transgenes in HL-60 and in K562 cells was assayed by quantitative real-time PCR. Combination therapy with ACNU plus paclitaxel in HL-60 cells and with carmustin (BCNU) plus doxorubicin in K562 cells resulted in the most significant survival advantage of cells transduced with the lentiviral vector HR'SIN-MGMT(P140K)-F2A-MDR1 compared with untransduced cells. In human HSCs, overexpression of both transgenes by this vector also caused significantly increased survival and enrichment of transduced cells after treatment with BCNU plus doxorubicin or temozolomide plus paclitaxel. In summary, we could show significant chemoprotection by overexpression of MDR1 and MGMT(P140K) with a lentiviral vector using the F2A linker element in two different haematopoietic cell lines and in human primary HSCs with various combination regimens. Consequently, we are convinced that these in vitro investigations will help to improve combination chemotherapy regimens by reducing myelotoxic side effects and increasing the therapeutic efficiency.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Vectores Genéticos/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Lentivirus/metabolismo , Péptidos/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Fitogénicos/farmacología , Carmustina/farmacología , Supervivencia Celular , Clonación Molecular , Citoprotección , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Doxorrubicina/farmacología , Vectores Genéticos/genética , Células HL-60 , Células Madre Hematopoyéticas/metabolismo , Humanos , Células K562 , Lentivirus/genética , Nimustina/farmacología , Paclitaxel/farmacología , Péptidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Temozolomida , Transgenes , Proteínas Supresoras de Tumor/genéticaRESUMEN
PURPOSE: Histology still forms the backbone for the diagnosis of the myxoid and round cell subtypes of liposarcoma but the molecular identification of the different transcript variants remains a challenge, due, in part, to the complexity of multiple overlapping exons that they share between them. This study was conducted to develop and evaluate a more sensitive platform than existing semi-quantitative approaches for detecting FUS-CHOP transcripts. MATERIALS AND METHODS: In the present investigation we describe a novel approach using real-time PCR to identify and differentiate the fusion transcripts formed in the t(12; 16)(q13; p11) chromosomal translocation. This method is founded on the basis of transcript individualized primers and probes, which were designed to detect specifically the different variants in both frozen and FFPE tissues. RESULTS: Our results show that the method is highly specific, sensitive, and superior to the widely used nested PCR approach, and is accurately able to differentiate the most common variants, as well as quantify copy numbers. Primer amplification and probe detection of FUS-CHOP from genomic DNA of human, mouse, cocker spaniel and chicken sources all resulted in completely negative results indicating this technique is specific for human RNA derived transcripts. CONCLUSION: This new method offers an additional tool in the investigation of liposarcoma that may impact considerably on missed diagnosis and it's accompanying clinical ramifications.
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Liposarcoma/genética , Proteína FUS de Unión a ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Factor de Transcripción CHOP/genética , Humanos , Técnicas In Vitro , Proteínas de Fusión Oncogénica/genética , Reacción en Cadena de la PolimerasaRESUMEN
INTRODUCTION: Epidemiological studies have found an increased risk for colon cancer and faster disease progression in patients with type 2 diabetes mellitus (T2DM). We aimed to determine whether patients with T2DM are diagnosed with more advanced stages of colorectal cancer, i. e., metastasized disease (UICC III and IV), at the time of diagnosis, since such a finding may have an impact on future guidelines for patients with T2DM. MATERIALS AND METHODS: A cross-sectional analysis of colorectal cancer patients was performed. Stages at diagnosis in patients with (18.0%) or without (82%) T2DM were compared using logistic regression analysis to correct for confounders. RESULTS: Patients with T2DM were older, more obese, and more often male (each p<0.05). Unexpectedly, patients with T2DM had a lower risk for metastasized disease at diagnosis (p=0.023). Correction for age, gender, BMI, smoking and aspirin intake in a multiple logistic regression analysis did not change the result (OR=0.57, p=0.037). When looking at individual cancer stages rather than collapsed categories, there was a trend for less advanced stages in patients with T2DM (p=0.093). Excluding stage I because of potential screening bias due to the introduction of (insurance-covered) colonoscopy screening improved model fit, and confirmed less advanced cancer stages (p=0.0246). CONCLUSIONS: Possibly because of earlier detection, patients with T2DM may be at lower risk for advanced stages of colon cancer at diagnosis. Further studies are warranted to confirm our results and to investigate the impact of closer medical surveillance in patients with type 2 diabetes mellitus.
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Carcinoma/complicaciones , Carcinoma/patología , Neoplasias del Colon/complicaciones , Neoplasias del Colon/patología , Diabetes Mellitus Tipo 2/complicaciones , Anciano , Carcinoma/epidemiología , Neoplasias del Colon/epidemiología , Comorbilidad , Complicaciones de la Diabetes/epidemiología , Diabetes Mellitus Tipo 2/epidemiología , Progresión de la Enfermedad , Femenino , Humanos , Hallazgos Incidentales , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Oportunidad RelativaRESUMEN
MicroRNAs (miRNAs) as modulators of gene expression have been described to display both tumor-promoting and tumor-suppressive functions. Although their role has been studied in different tumor types, little is known about how they regulate nuclear factor κB (NF-κB) signaling in breast cancer. Here, we performed an unbiased whole genome miRNA (miRome) screen to identify novel modulators of NF-κB pathway in breast cancer. The screen identified 13 miRNA families whose members induced consistent effects on NF-κB activity. Among those, the miR-520/373 family inhibited NF-κB signaling through direct targeting of RELA and thus strongly reduced expression and secretion of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8. With a combination of in vitro and in vivo approaches, we propose a metastasis-suppressive role of miR-520/373 family. miR-520c and miR-373 abrogated both in vitro cell invasion and in vivo intravasation of highly invasive MDA-MB-231 cells. However, knockdown of RELA did not affect their metastatic ability. mRNA profiling of MDA-MB-231 cells on overexpression of miR-520/373 members revealed a strong downregulation of transforming growth factor-ß (TGF-ß) signaling. Mechanistically, the metastasis-suppressive role of miR-520/373 can be attributed to direct suppression of TGFBR2, as the silencing of TGFBR2 phenocopied the effects of miR-520/373 overexpression on suppression of Smad-dependent expression of the metastasis-promoting genes parathyroid hormone-related protein, plasminogen activator inhibitor-1 and angiopoietin-like 4 as well as tumor cell invasion, in vitro and in vivo. A negative correlation between miR-520c and TGFBR2 expression was observed in estrogen receptor negative (ER(-)) breast cancer patients but not in the ER positive (ER(+)) subtype. Remarkably, decreased expression of miR-520c correlated with lymph node metastasis specifically in ER(-) tumors. Taken together, our findings reveal that miR-520/373 family has a tumor-suppressive role in ER(-) breast cancer by acting as a link between the NF-κB and TGF-ß pathways and may thus contribute to the interplay of tumor progression, metastasis and inflammation.
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Neoplasias de la Mama/metabolismo , MicroARNs/metabolismo , FN-kappa B/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/biosíntesis , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Metástasis de la Neoplasia/genética , Proteína Relacionada con la Hormona Paratiroidea/biosíntesis , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Estrógenos/deficiencia , Receptores de Estrógenos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
Axl is a receptor that induces proliferation, migration and invasion in cancer. In this study, we show that specific microRNAs (miRNAs) target the 3'-UTR of Axl. Luciferase-reporter assays with wild-type and deleted miR-34 and miR-199a/b seed sequences of Axl 3'-UTR confirmed the specificity of targeting. An inverse correlation between Axl protein and miR-34a expression in a panel of non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and breast cancer (BRC) cell lines was observed, while miR-199a/b expression was completely suppressed. Pre-miR transfection inhibited in vitro migration and invasion and, in vivo, reduced the number of distant lung- or liver-metastases in a chorion-allantoic-membrane (CAM) assay. Moreover, methylation-specific PCR on bisulfite-converted DNA obtained from the cell lines showed that the miR-34a promoter methylation status was inversely correlated with its expression, and that miR-199a/b promoter regions were methylated in all cells tested. In a panel of NSCLC tissues (n=44), miR-34a and miR-199a/b were found to be downregulated and significantly co-expressed. A lower expression of all three miRs was significantly associated with squamous histotypes, and, in a preliminary series, NSCLC patients with miR-34a upregulation showed a positive association towards a longer survival. These results indicate that Axl receptor expression can be regulated by miR-34a and miR-199a/b, which are suppressed by promoter methylation in solid cancer cells.
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MicroARNs/fisiología , Neoplasias/enzimología , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Línea Celular Tumoral , Metilación de ADN , Humanos , MicroARNs/genética , Datos de Secuencia Molecular , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Neoplasias/genética , Neoplasias/patología , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico , Tirosina Quinasa del Receptor AxlRESUMEN
BACKGROUND: Enzastaurin (Enz) is a serine/threonine kinase inhibitor blocking protein kinase C (PKC)beta/AKT pathway. However, an ability of this compound to inhibit cancer invasion and metastasis is not yet clearly elucidated. METHODS: The ability of Enz to inhibit invasion and metastasis, and to target molecules was investigated in non-small cell lung cancer (NSCLC) by RT-PCR validated microarray, Matrigel, and in vivo chorionallantoic membrane (CAM) assays. RESULTS: Enzastaurin significantly reduced migration, invasion, and in vivo metastasis to lungs and liver (CAM assay) of diverse NSCLC cell lines. Genes promoting cancer progression (u-PAR, VEGFC, and HIF1alpha) and tumour suppression (VHL, RASSF1, and FHIT) of NSCLC were significantly (P<0.05) down- or upregulated after Enz treatment in H460, A549, and H1299 cells, respectively. Luciferase/chromatin immunoprecipitation analysis showed that Enz transcriptionally controls urokinase-type plasminogen activator receptor (u-PAR) expression by promoter inhibition through Sp1, Sp3, and c-Jun(AP-1). Moreover, siRNA knockdown of u-PAR re-sensitised Enz-resistant cells and induced apoptosis, suggesting u-PAR as a marker of Enz resistance. CONCLUSION: This study shows that Enz inhibits migration, invasion, and in vivo metastasis by targeting u-PAR, besides further targeting progression-related and tumour-suppressor genes in NSCLC. Together with u-PAR being a novel putative marker of Enz response, these data encourage molecularly tailored clinical studies on Enz in NSCLC therapy.
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Indoles/farmacología , Invasividad Neoplásica/prevención & control , Metástasis de la Neoplasia/prevención & control , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/patología , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Abstract Hematotoxicity is a major and frequently dose-limiting side effect of chemotherapy. Retroviral methylguanine-DNA-methyltransferase (MGMT; EC 2.1.1.63) gene transfer to primitive hematopoietic progenitor cells (CD34(+) cells) might allow the application of high-dose alkylator chemotherapy with almost mild to absent myelosuppression. Because gammaretroviral vector integration was found in association with malignant or increased proliferation, novel lentiviral vectors with self-inactivating (SIN) capacity might display a safer option for future gene transfer studies. We assessed the influence of chemoselection on integration patterns in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-treated and untreated human CD34(+) cells transduced with an SIN lentiviral vector carrying the MGMT(P140K) transgene, using ligation-mediated PCR (LM-PCR) and next-generation sequencing. In addition, for the first time, the local influence of the lentiviral provirus on the expression of hit and flanking genes in human CD34(+) cells was analyzed at a clonal level. For each colony, the integration site was detected (LM-PCR) and analyzed (QuickMap), and the expression of hit and flanking genes was measured (quantitative RT-PCR). Analyses of both treated and untreated CD34(+) cells revealed preferential integration into genes. Integration patterns in BCNU-treated cells showed mild, but not significant, differences compared with those found in untreated CD34(+) cells. Most importantly, when analyzing the local influence of the provirus, we saw no significant deregulation of the integration-flanking genes. These findings demonstrate that SIN vector-mediated gene transfer might display a feasible and possibly safe option for MGMT(P140K)-mediated chemoprotection of CD34(+) cells.
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Antineoplásicos Alquilantes/uso terapéutico , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Lentivirus/genética , Secuencias Repetidas Terminales , Antígenos CD34/genética , Antígenos CD34/metabolismo , Células Sanguíneas/química , Células Sanguíneas/metabolismo , Terapia Genética , Vectores Genéticos , Humanos , Lentivirus/metabolismo , Reacción en Cadena de la Polimerasa , Transducción Genética , TransgenesRESUMEN
We reported earlier that knockdown of tumor suppressor Pdcd4 (programed cell death 4) downregulates E-cadherin expression and activates beta-catenin/Tcf (T-cell factor)-dependent transcription in colon tumor cells. However, the underlying mechanism of these observations remains unknown. In this study, we showed that knockdown of Pdcd4 downregulates E-cadherin expression through elevated protein level of Snail. Over-expression of Pdcd4 upregulates E-cadherin expression and inhibits beta-catenin/Tcf-dependent transcription. We then showed that knockdown of E-cadherin activates beta-catenin/Tcf-dependent transcription. Conversely, over-expression of E-cadherin in Pdcd4 knockdown cells inhibits beta-catenin/Tcf-dependent transcription. In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells. Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells. Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown. Taken together, our data showed that elevated Snail expression by Pdcd4 knockdown leads to downregulation of E-cadherin resulting in activating beta-catenin/Tcf-dependent transcription and stimulating the expression of c-Myc and u-PAR, thus providing molecular explanation of how Pdcd4 suppresses tumor invasion.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Cadherinas/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción TCF/metabolismo , beta Catenina/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Western Blotting , Cadherinas/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HT29 , Humanos , Mutación , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Factores de Transcripción TCF/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , beta Catenina/genéticaRESUMEN
OBJECTIVE: In fecal incontinence the role of elastography has not yet been evaluated. We performed a trial to further characterize the internal and external anal sphincter in patients with fecal incontinence and compared a visual assessment scale with a computerized program for quantifying elastic properties of the anal sphincter. MATERIAL AND METHODS: Fifty consecutive patients with fecal incontinence were studied (n = 31 following lower anterior resection, n = 8 with Crohn's disease, n = 9 following colon surgery, n = 2 others). Elastogram color distribution within the sphincter representing elastic properties was quantified using a visual analog scale and an off-line computerized area calculation program. RESULTS: The main finding was that the inner anal sphincter (IAS) differed significantly from the external anal sphincter (EAS) with regard to elastogram color distribution. There were no significant correlations with clinical and functional parameters. There was, however, a non-significant increase in the percentage of blue (hard) areas in the IAS in patients neoadjuvantly irradiated for rectal or cervical cancer compared to non-irradiated patients, which was accompanied by a significant decrease in the resting sphincter pressure (p < 0.009). CONCLUSIONS: The IAS, a smooth muscle, and the EAS, a striated muscle, have different elastogram color distributions, probably reflecting their different elastic properties. The absence of significant correlations with the major clinical and functional parameters suggests that in routine clinical practice ultrasound real-time elastography may not yield additional information in patients with fecal incontinence. There may be exceptions, particularly in irradiated patients.
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Canal Anal/diagnóstico por imagen , Endosonografía , Incontinencia Fecal/diagnóstico por imagen , Adulto , Diagnóstico por Imagen de Elasticidad , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cell's genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/- 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations ('random set'). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with 'safe' insertion profiles.
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Mapeo Cromosómico/métodos , Procesamiento Automatizado de Datos/métodos , Vectores Genéticos/uso terapéutico , Mutagénesis Insercional/genética , Retroviridae/genética , Programas Informáticos , Acceso a la Información , Animales , Secuencia de Bases/genética , Sitios de Unión , Sitios Frágiles del Cromosoma , Biología Computacional/métodos , Islas de CpG , Bases de Datos Genéticas , Terapia Genética/métodos , Terapia Genética/normas , Vectores Genéticos/efectos adversos , Genoma Humano/genética , Humanos , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetitivas de Ácidos Nucleicos , Seguridad , Análisis de Secuencia de ADN/métodosRESUMEN
Unclear perianal bleeding may cause diagnostic and therapeutic difficulty, particularly when the bleeding source cannot be detected. In this case record we report on a 64-year-old woman with systemic sclerosis and incomplete CRES(T) syndrome diagnosed more than 10 years ago with no detectable teleangiectasia/angiodysplasia at that time. During the course of the disease the initially incomplete CRES(T) syndrome developed into a complete CREST syndrome with repeated bleeding from teleangiectatic/angiodysplastic lesions of the rectum, stomach/duodenum. In addition, chronic diarrhoea with malabsorption, bacterial overgrowth and severe anal incontinence were present which all were seen as intestinal manifestations of the existing underlying disease. A complete motoric and sensoric insufficiency of the anal sphincter was found manometrically, anal endosonography with elastography revealed changes compatible with sclerosis. In the absence of a causal therapy symptomatic treatment strategies are described and discussed on the basis of existing pathophysiologic knowledge.
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Angiodisplasia/diagnóstico , Síndrome CREST/diagnóstico , Diarrea/etiología , Incontinencia Fecal/etiología , Enfermedades Gastrointestinales/diagnóstico , Hemorragia Gastrointestinal/etiología , Esclerodermia Sistémica/diagnóstico , Telangiectasia Hemorrágica Hereditaria/diagnóstico , Colonoscopía , Diagnóstico Diferencial , Diagnóstico por Imagen de Elasticidad , Endosonografía , Humanos , Mucosa Intestinal/patología , Persona de Mediana EdadRESUMEN
The rapidly advancing elucidation of molecular targets in human cancers during the last decade has provided an excellent basis for the development of novel therapeutics. A huge variety of potential target structures have been identified, many of which are already being exploited for therapeutic purposes. This review introduces the reader into the concept of molecular targeted therapies, and provides some prototypic examples.
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Antineoplásicos/uso terapéutico , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/terapia , Animales , HumanosRESUMEN
Tumor-suppressor Pdcd4 inhibits transformation and invasion and is downregulated in cancers. So far, it has not been studied as to whether miRNAs, suppressing target expression by binding to the 3'-UTR, regulate Pdcd4 or invasion. The present study was conducted to investigate the regulation of Pdcd4, and invasion/intra-vasation, by miRNAs. A bioinformatics search revealed a conserved target-site for miR-21 within the Pdcd4-3'-UTR at 228-249 nt. In 10 colorectal cell lines, an inverse correlation of miR-21 and Pdcd4-protein was observed. Transfection of Colo206f-cells with miR-21 significantly suppressed a luciferase-reporter containing the Pdcd4-3'-UTR, whereas transfection of RKO with anti-miR-21 increased activity of this construct. This was abolished when a construct mutated at the miR-21/nt228-249 target site was used instead. Anti-miR-21-transfected RKO cells showed an increase of Pdcd4-protein and reduced invasion. Moreover, these cells showed reduced intra-vasation and lung metastasis in a chicken-embryo-metastasis assay. In contrast, overexpression of miR-21 in Colo206f significantly reduced Pdcd4-protein amounts and increased invasion, while Pdcd4-mRNA was unaltered. Resected normal/tumor tissues of 22 colorectal cancer patients demonstrated an inverse correlation between miR-21 and Pdcd4-protein. This is the first study to show that Pdcd4 is negatively regulated by miR-21. Furthermore, it is the first report to demonstrate that miR-21 induces invasion/intravasation/metastasis.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/patología , Proteínas Reguladoras de la Apoptosis/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , MicroARNs/fisiología , Interferencia de ARN , Proteínas de Unión al ARN/genética , Animales , Secuencia de Bases , Células CACO-2 , Embrión de Pollo , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Células HCT116 , Células HT29 , Humanos , Datos de Secuencia Molecular , Invasividad Neoplásica , Metástasis de la Neoplasia , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.
Asunto(s)
Amplificación de Genes , Terapia Genética , Retroviridae/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Ratones , Ratones SCID , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
The prognosis of pancreatic adenocarcinoma is affected by early metastases and local tumour invasion beyond surgical margins. Gene expression profiling in pancreatic cancer tissue is complicated due to the high amount of RNAses being present in human tissue and that of suitable models. In order to demonstrate early metastases, the models should take into account the anatomical environment of the tumour. Using the orthotopic transplantation of pancreatic tumour cells in SCID (severe combined immunodeficiency) mice, these interactions are taken into consideration. In order to identify genes associated with local tumour invasion and metastases in ductal pancreatic cancer, we investigated a human pancreatic tumour cell line derived from an orthopic pancreatic tumour model in SCID mice. Differential gene expression was performed on the basis of microarray technique. The human MiaPaca-2 cell line was implanted orthotopically in SCID mice. Transcriptional profiling was performed on fresh frozen tissue derived from the primary tumour, the tumour invasion front and the liver metastases. Differentially expressed genes were identified using statistical analyses, and were validated with external databases and with immunohistochemistry. A total of 1066 of 14 500 genes were significantly differentially expressed. Comparing the primary tumour with the tumour invasion front, there were 614 statistically significant up- and 348 downregulated genes. Twenty-five statistically significant up- and 181 downregulated genes were identified comparing the liver metastases with the primary tumour. Eight genes (PAI-1, BNIP3l, VEGF, NSE, RGS4, HSP27, GADD45A, PTPN14) were chosen and validated in a semi-quantitative immunohistochemical analysis, which revealed a positive correlation to the array data. Overrepresentation analyses revealed a total of 66 significantly regulated pathways associated with cell proliferation, cell stress, cell communication metabolic and cytokine function. In conclusion, model marker genes for local invasion and liver metastases can be identified using transcriptional profiling in the SCID mouse. Overrepresentation analysis secures a good and fast overview about the significantly regulated genes and can assign genes to certain pathways. These marker genes can be related to the apoptotic cascade, angiogenesis and cell interaction.
Asunto(s)
Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Neoplasias Pancreáticas/genética , Animales , Línea Celular Tumoral , Bases de Datos Factuales , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones SCID , Invasividad Neoplásica , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
Underweight as a consequence of chronic diarrhoea may lead to fatigue, tiredness and impaired physical performance, especially when the underlying cause has not been evaluated. In spite of algorithms as a help in the differential diagnosis, an individual approach with critical consideration of diet history, laboratory data and imaging procedures is necessary. Additional difficulties may arise when the history of food intolerance is inconsistent and technical findings including endoscopy are inconclusive. We report on a 57-year-old female patient with underweight, chronic intermittent diarrhoea and cramp-like abdominal pain for more than 10 years following pelvic irradiation due to Hodgkin's disease of the ovary. A systematic diagnostic approach was not undertaken until very recently due to the deterioration of her clinical conditions pointing to jejunal malabsorption. In spite of the absence of a specific history of milk/milk product intolerance a lactose H (2)-breath test was performed showing lactase deficiency with lactose intolerance. The rapid improvement of all her symptoms after a lactose-poor diet had been started supported this diagnosis. Possible reasons for the long time period which had elapsed until the diagnosis was established and the discrepancy of the H (2)-breath test results with the absence of a clear-cut history for milk/milk product intolerance are discussed in terms of the importance of a structured history-taking with regard to nutrition and diet habits. In addition, potential explanations for radiation-induced functional damage in the absence of morphological abnormalities are provided. Based on the experience of this case and considerations regarding the consequences of radiation-induced jejunal damage, we recommend that a lactose-H (2) breath test be routinely included in the diagnostic work-up of patients with unclear chronic diarrhoea even if there is no defined history of milk/milk product intolerance.