RESUMEN
The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML.
Asunto(s)
Perfilación de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , MicroARNs/genética , Piperazinas/farmacología , Pirimidinas/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzamidas , Análisis por Conglomerados , Resistencia a Antineoplásicos/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Whereas transcriptional silencing of genes due to epigenetic mechanisms is one of the most important alterations in acute lymphoblastic leukemia (ALL), some recent studies indicate that DNA methylation contributes to down-regulation of miRNAs during tumorigenesis. To explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL. Expression of miR-124a was down-regulated in ALL by hypermethylation of the promoter and histone modifications including decreased levels of 3mk4H3 and AcH3 and increased levels of 2mK9H3, 3mK9H3, and 3mK27H3. Epigenetic down-regulation of miR-124a induced an up-regulation of its target, CDK6, and phosphorylation of retinoblastoma (Rb) and contributed to the abnormal proliferation of ALL cells both in vitro and in vivo. Cyclin-dependent kinase 6 (CDK6) inhibition by sodium butyrate or PD-0332991 decreased ALL cell growth in vitro, whereas overexpression of pre-miR124a led to decreased tumorigenicity in a xenogeneic in vivo Rag2(-/-)gammac(-/-) mouse model. The clinical implications of these findings were analyzed in a group of 353 patients diagnosed with ALL. Methylation of hsa-miR-124a was observed in 59% of the patients, which correlated with down-regulation of miR-124a (P < 0.001). Furthermore, hypermethylation of hsa-miR-124a was associated with higher relapse rate (P = 0.001) and mortality rate (P < 0.001), being an independent prognostic factor for disease-free survival (P < 0.001) and overall survival (P = 0.005) in the multivariate analysis. These results provide the grounds for new therapeutic strategies in ALL either targeting the epigenetic regulation of microRNAs and/or directly targeting the CDK6-Rb pathway.
Asunto(s)
Quinasa 6 Dependiente de la Ciclina/genética , Epigénesis Genética/genética , Silenciador del Gen , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animales , División Celular , Línea Celular Tumoral , Cromatina/genética , Cromatina/fisiología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Supervivencia sin Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , ARN Mensajero/genética , Tasa de Supervivencia , Transcripción GenéticaRESUMEN
BACKGROUND: Expression of the pro-apoptotic BCL-2-interacting mediator (BIM) has recently been implicated in imatinib-induced apoptosis of BCR-ABL1(+) cells. However, the mechanisms involved in the regulation of BIM in CML and its role in the clinical setting have not been established. DESIGN AND METHODS: We analysed the mRNA expression of BIM in 100 newly diagnosed patients with CML in chronic phase by Q-RT-PCR and the protein levels by Western blot analysis. Methylation status was analysed by bisulphite genomic sequencing and MSP. CML cell lines were treated with imatinib and 5-aza-2'-deoxycytidine, and were transfected with two different siRNAs against BIM and cell proliferation and apoptosis were analysed. RESULTS: We demonstrated that down-regulation of BIM expression was present in 36% of the patients and was significantly associated with a lack of optimal response to imatinib as indicated by the decrease in cytogenetic and molecular responses at 6, 12 and 18 months in comparison with patients with normal BIM expression (p<0.05). Expression of BIM was mediated by promoter hypermethylation as demonstrated by restoration of BIM expression after treatment of CML cells with 5-aza-2'-deoxycytidine. Using CML cell lines with low and normal expression of BIM we further demonstrated that the expression of BIM is required for imatinib-induced CML apoptosis. CONCLUSION: Our data indicate that down-regulation of BIM is epigenetically controlled by methylation in a percentage of CML patients and has an unfavourable prognostic impact, and that the combination of imatinib with a de-methylating agent may result in improved responses in patients with decreased expression of BIM.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Regulación hacia Abajo/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas de la Membrana/biosíntesis , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/biosíntesis , Pirimidinas/farmacología , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Proteína 11 Similar a Bcl2 , Benzamidas , Proliferación Celular/efectos de los fármacos , Metilación de ADN , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Células Tumorales CultivadasRESUMEN
PURPOSE: To identify microRNAs (miRNAs) epigenetically regulated in acute lymphoblastic leukemia (ALL). METHODS: We first examined ALL-derived cell lines for the presence of abnormal levels of two different histone modifications (trimethylation of H3 lysine 4 [K4H3me3] and dimethylation of H3 lysine 9 [K9H3me2]) in the 5'UTR regions around CpG islands of 78 miRNAs by chromatin immunoprecipitation (ChIP)-on-ChIP analysis. Methylation status (methylation-specific polymerase chain reaction [PCR]) and expression (quantitative PCR) of miRNAs showing a pattern of histone modifications linked to a closed chromatin structure were analyzed in a panel of six ALL cell lines and in 353 ALL patients. RESULTS: CpG islands around 13 miRNAs disclosed high levels of K9H3me2 and/or low levels of K4H3me3, a pattern of histone modifications underlying a closed chromatin structure associated with repressive gene expression. Complete consistency in the correlation between both histone marks, the presence of DNA methylation around these miRNAs, and their expression patterns was confirmed in the six ALL cell lines. Treatment with 5-Aza-2'-deoxycytidine upregulated the expression levels of these genes, suggesting that epigenetic mechanisms deregulate the expression of these miRNAs. A total of 65% of the ALL samples had at least one miRNA methylated (methylated group). Estimated disease-free survival (DFS) and overall survival (OS) at 14 years were 78% and 71% for nonmethylated patients and 24% and 28% for methylated patients (P = .00001 for both). Multivariate analysis demonstrated that methylation profile was an independent prognostic factor for predicting DFS (P = .0001) and OS (P = .0001). CONCLUSION: Aberrant miRNA methylation is a common phenomenon in ALL that affects the clinical outcome of these patients.
Asunto(s)
Epigénesis Genética , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Islas de CpG , Metilación de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , PronósticoRESUMEN
MicroRNAs (miRNA) are small noncoding, single-stranded RNAs that inhibit gene expression at a posttranscriptional level, whose abnormal expression has been described in different tumors. The aim of our study was to identify miRNAs potentially implicated in chronic myeloid leukemia (CML). We detected an abnormal miRNA expression profile in mononuclear and CD34(+) cells from patients with CML compared with healthy controls. Of 157 miRNAs tested, hsa-miR-10a, hsa-miR-150, and hsa-miR-151 were down-regulated, whereas hsa-miR-96 was up-regulated in CML cells. Down-regulation of hsa-miR-10a was not dependent on BCR-ABL1 activity and contributed to the increased cell growth of CML cells. We identified the upstream stimulatory factor 2 (USF2) as a potential target of hsa-miR-10a and showed that overexpression of USF2 also increases cell growth. The clinical relevance of these findings was shown in a group of 85 newly diagnosed patients with CML in which expression of hsa-miR-10a was down-regulated in 71% of the patients, whereas expression of USF2 was up-regulated in 60% of the CML patients, with overexpression of USF2 being significantly associated with decreased expression of hsa-miR-10a (P = 0.004). Our results indicate that down-regulation of hsa-miR-10a may increase USF2 and contribute to the increase in cell proliferation of CML implicating a miRNA in the abnormal behavior of CML.
Asunto(s)
Regulación Leucémica de la Expresión Génica/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/fisiopatología , MicroARNs/fisiología , Factores Estimuladores hacia 5'/genética , Antígenos CD34/metabolismo , División Celular/fisiología , Línea Celular Tumoral , Regulación hacia Abajo/fisiología , Perfilación de la Expresión Génica , Genes abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , MicroARNs/química , Conformación de Ácido Nucleico , Transfección , Regulación hacia Arriba/fisiologíaRESUMEN
The clinical significance of aberrant promoter methylation of the canonical Wnt pathway antagonist genes (sFRP1, sFRP2, sFRP4, sFRP5, Wif1, Dkk3, and Hdpr1) and also putative tumor-suppressor gene Wnt5a, belonging to the non-canonical Wnt signaling pathway, was investigated in a large series of 75 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia by methylation-specific polymerase chain reaction. At least one methylated gene was observed in cells from 66% (49/75) of patients (methylated group). Disease-free survival and overall survival at 9 years were 51 and 40%, respectively, for the unmethylated group and 3 and 2%, respectively, for the methylated group (both P < 0.0001). Multivariate analysis demonstrated that the Wnt methylation profile was an independent prognostic factor predicting disease-free survival (P = 0.007) and overall survival (P = 0.039). Abnormal DNA methylation of promoter-associated CpG islands in the Wnt signaling pathway is very common in Philadelphia chromosome-positive acute lymphoblastic leukemia and potentially defines subgroups with distinct clinical characteristics.
Asunto(s)
Metilación de ADN , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Wnt/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pronóstico , Factores de Riesgo , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.
Asunto(s)
Proteínas del Choque Térmico HSC70/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Antígenos CD34/metabolismo , Línea Celular Tumoral/metabolismo , Supervivencia Celular , Ciclina D , Ciclinas/metabolismo , Perfilación de la Expresión Génica , Proteínas del Choque Térmico HSC70/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Análisis por Micromatrices , Transducción de SeñalRESUMEN
The aim of our study was to determine the potential mechanism(s) implicated in Imatinib resistance in patients with Ph+ ALL. Resistance of Ph+ ALL cells to Imatinib-induced apoptosis was associated with lack of inhibition of Akt phosphorylation. Addition of the PI3K inhibitor LY294002 to Imatinib significantly increased apoptosis of Ph+ ALL cells. Interestingly, expression of PTEN was reduced in Ph+ ALL cells which was due to PTEN promoter hypermethylation. Treatment of Ph+ ALL cells with 5-Aza-2'-deoxycytidine was associated with an increased expression of PTEN and an increase in cell apoptosis. These results suggest that Imatinib resistance in patients with ALL may be dependent at least in part to PTEN down-regulation due to the abnormal promoter hypermethylation and support the potential role of de-methylating agents for the treatment of patients with Ph+ ALL.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Metilación de ADN , Fosfohidrolasa PTEN/genética , Piperazinas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Regiones Promotoras Genéticas , Pirimidinas/farmacología , Benzamidas , Línea Celular Tumoral , Regulación hacia Abajo , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/fisiología , Humanos , Mesilato de Imatinib , Fosfatidilinositol 3-Quinasas/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Proto-Oncogénicas c-akt/fisiologíaRESUMEN
In the search for new derivatives with anticancer activity that are able to induce a selective pro-apoptotic mechanism in cancer cells, we have designed, synthesized, and evaluated a series of new 2-(alkylsulfanyl)-N-alkylpyrido[2,3-d]pyrimidine-4-amine derivatives as cytotoxic and apoptosis inducers. The potential antitumor activity of the compounds was evaluated in vitro by examining their cytotoxic effects against human breast, colon, and bladder cancer-cell lines. The IC(50)values of the compounds that showed cytotoxic activity were calculated. The cytotoxic compounds were then tested for their ability to induce caspase-3 activation and nuclear-chromatin degradation. Some compounds, such as 6c, 6d, 6e, 6j, 6o, and 6p, show significant in-vitro cytotoxicity in at least two of the three tested cell lines, induced apoptosis, and also produced a rapid dose-dependent increase in the caspase-3 level in some of the cell lines tested. In order to test the selectivity of the compounds, two non-tumoral human cell lines were used. Several compounds of the did not show cytotoxicity in these cell lines.
Asunto(s)
Antineoplásicos/síntesis química , Apoptosis , Piridinas/síntesis química , Pirimidinas/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática , Humanos , Modelos Moleculares , Piridinas/química , Piridinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
Repetitive elements are heavily methylated in normal tissues, but hypomethylated in malignant tissues, driving the global genomic hypomethylation found in cancer. This hypomethylation results in chromosomal instability, a well-characterized feature of the advanced phases of chronic myeloid leukemia (CML). We investigated methylation changes of DNA repetitive elements (LINE1, Alu, Satellite-alpha and Satellite-2) during the progression of CML from chronic phase (CP) to blast crisis (BC). CP-CML samples were significantly more hypomethylated for all repetitive sequences compared with normal samples. Furthermore, a more profound level of hypomethylation was observed among BC samples compared with CP samples. Our data suggest that repetitive DNA hypomethylation are closely associated with CML progression.
Asunto(s)
Metilación de ADN , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Secuencias Repetitivas de Ácidos Nucleicos , Elementos Alu , Crisis Blástica/genética , ADN Satélite , Humanos , Leucemia Mieloide de Fase Crónica/genéticaRESUMEN
Wnt5a is a member of the Wnt family of proteins that signals through the non-canonical Wnt/Ca(2+) pathway to suppress cyclin D1 expression and negatively regulate B cell proliferation suggesting that it acts as an tumour suppressor for lymphoid leukemogenesis. Although canonical Wnt pathway is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Wnt5a abnormalities has never been evaluated in this clinical setting. The methylation status of the WNT5A promoter was analysed by methylation-specific PCR (MSP) and sequencing in six ALL-derived cell lines (TOM-1, NALM-20, MY, LOUCY, JURKAT and TANOUE) and in 307 ALL patients. WNT5A and CYCLIN D1 expressions were assessed by quantitative RT-PCR. We observed WNT5A hypermethylation in all cell lines and in cells from 43% (132/307) of ALL patients. WNT5A methylation was associated with decreased WNT5A mRNA expression (P<0.001) and this expression was restored after exposure to the demethylating agent 5-Aza-2'-deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P=0.002). Disease-free survival (DFS) and overall survival (OS) at 13 and 14 years, respectively, were 59% and 53% for unmethylated patients and 28% and 31% for hypermethylated patients (P=0.0003 and P=0.003). Multivariate analysis demonstrated that WNT5A methylation was an independent prognostic factor predicting DFS (P=0.003) and OS (P=0.04). We have demonstrated that WNT5A, a putative tumour suppressor gene in ALL, is silenced by methylation in this disease and that this epigenetic event is associated with upregulation of CYCLIN D1 expression and confers poor prognosis in this group of patients.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Wnt/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pronóstico , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMEN
Tumor associated antigens (TAA) provide attractive targets for cancer-specific immunotherapy. PRAME is a TAA gene up-regulated in advanced phases of chronic myeloid leukemia (CML). To date, molecular mechanisms for the expression of PRAME have never been studied. We found that some Ph'-positive cell lines did not express PRAME. The expression of PRAME was restored in these cell lines by treatment with 5'-aza-2'-deoxycytidine, suggesting that the expression of PRAME is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the CpG sites of the PRAME exon 2 in these cancer cell lines revealed a close relationship between the methylation status of the PRAME gene and its expression. A methylation-specific PCR analysis demonstrated that hypomethylation of PRAME was significantly more frequent in CML blast crisis (70%) than in chronic phase (36%) (P=0.01) and was correlated with high expression levels of PRAME transcripts (P<0.0001). These results suggest that hypomethylation of PRAME up-regulates its expression in CML and might play a significant role in the progression of the disease.
Asunto(s)
Antígenos de Neoplasias/genética , Epigénesis Genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Secuencia de Bases , Metilación de ADN , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS: In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.
Asunto(s)
Antígenos de Neoplasias/sangre , Islas de CpG , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/fisiología , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Regiones Promotoras Genéticas , Antineoplásicos/farmacología , Benzamidas , Línea Celular Tumoral , Humanos , Mesilato de Imatinib , Interferones/uso terapéutico , Masculino , Piperazinas/farmacología , Pronóstico , Pirimidinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/metabolismoRESUMEN
In order to obtain less toxic antitumoral compounds we have looked for novel compounds with anticancer activity based on proapoptotic mechanisms. The compounds studied in this work are derivatives of bicyclic aromatic systems like pyrido[2,3-d]pyrimidines. The potential antitumoral activity of the compounds was evaluated in vitro by examining their cytotoxic effects against human breast, colon, and bladder cancer lines (MD-MBA-231, HT-29, and T-24). The data indicate that HC-6 is a potent anticancer drug showing dose-dependent cytostatic and proapoptotic effects through activation of two different signaling pathways namely a pathway leading to cell cycle arrest and a transcription-independent route leading to rapid apoptosis.
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Antineoplásicos/química , Apoptosis/efectos de los fármacos , Pirimidinas/química , Pirimidinas/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Humanos , Transducción de Señal/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Activation of the Wnt/beta-catenin signaling pathway is a hallmark of a number of solid tumors. We analyzed the regulation of the Wnt/beta-catenin pathway in acute lymphoblastic leukemia (ALL) and its role in the pathogenesis of the disease. We found that expression of the Wnt inhibitors sFRP1, sFRP2, sFRP4, sFRP5, WIF1, Dkk3, and Hdpr1 was down-regulated due to abnormal promoter methylation in ALL cell lines and samples from patients with ALL. Methylation of Wnt inhibitors was associated with activation of the Wnt-signaling pathway as demonstrated by the up-regulation of the Wnt target genes WNT16, FZ3, TCF1, LEF1, and cyclin D1 in cell lines and samples and the nuclear localization of beta-catenin in cell lines. Treatment of ALL cells with the Wnt inhibitor quercetin or with the demethylating agent 5-aza-2'-deoxycytidine induced an inactivation of the Wnt pathway and induced apoptosis of ALL cells. Finally, in a group of 261 patients with newly diagnosed ALL, abnormal methylation of Wnt inhibitors was associated with decreased 10-year disease-free survival (25% versus 66% respectively, P < .001) and overall survival (28% versus 61% respectively, P = .001). Our results indicate a role of abnormal Wnt signaling in ALL and establish a group of patients with a significantly worse prognosis (methylated group).
Asunto(s)
Epigénesis Genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antimetabolitos Antineoplásicos/administración & dosificación , Azacitidina/administración & dosificación , Azacitidina/análogos & derivados , Línea Celular Tumoral , Niño , Preescolar , Metilación de ADN/efectos de los fármacos , Decitabina , Supervivencia sin Enfermedad , Epigénesis Genética/efectos de los fármacos , Femenino , Estudios de Seguimiento , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/genética , Humanos , Lactante , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Quercetina/administración & dosificación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/genética , beta Catenina/genéticaRESUMEN
PURPOSE: To examine cancer genes undergoing epigenetic inactivation in a set of ETV6/RUNX1-positive acute lymphoblastic leukemias in order to define the CpG island methylator phenotype (CIMP) in the disease and evaluate its relationship with clinical features and outcome. EXPERIMENTAL DESIGN: Methylation-specific PCR was used to analyze the methylation status of 38 genes involved in cell immortalization and transformation in 54 ETV6/RUNX1-positive samples in comparison with 190 ETV6/RUNX1-negative samples. RESULTS: ETV6/RUNX1-positive samples had at least one gene methylated in 89% of the cases. According to the number of methylated genes observed in each individual sample, 20 patients (37%) were included in the CIMP- group (0-2 methylated genes) and 34 (67%) in the CIMP+ group (>2 methylated genes). Remission rate did not differ significantly among either group of patients. Estimated disease-free survival and overall survival at 9 years were 92% and 100% for the CIMP- group and 33% and 73% for the CIMP+ group (P = 0.002 and P = 0.04, respectively). Multivariate analysis showed that methylation profile was an independent prognostic factor in predicting disease-free survival (P = 0.01) and overall survival (P = 0.05). A group of four genes (DKK3, sFRP2, PTEN, and P73) showed specificity for ETV6/RUNX1-positive subset of samples. CONCLUSION: Our results suggest that methylation profile may be a potential new biomarker of risk prediction in ETV6/RUNX1-positive acute lymphoblastic leukemias.
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Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 21/genética , Metilación de ADN , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocación Genética , Adolescente , Niño , Preescolar , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Islas de CpG , Femenino , Humanos , Masculino , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Proteína ETS de Variante de Translocación 6RESUMEN
The DBC1 gene is a potential tumour suppressor gene that is commonly hypermethylated in epithelial cancers. We studied the role of promoter hypermethylation in the regulation of DBC1 in acute lymphoblastic leukaemia (ALL) cell lines and 170 ALL patients at diagnosis. Abnormal methylation of DBC1 was observed in all ALL cell lines and in 17% of ALL patients. Moreover, DBC1 methylation was associated with decreased DBC1 expression, while treatment of ALL cells with 5-Aza-2'-deoxycytidine resulted in demethylation of the promoter and upregulation of DBC1 expression. Fluorescence in situ hybridisation identified the deletion of one allele of DBC1 in some ALL cell lines, which indicated that the lack of DBC1 expression was due to deletion of one allele and methylation of the other. In conclusion, these results demonstrate, for the first time, that the expression of DBC1 is downregulated in a percentage of patients with ALL due to the hypermethylation of its promoter and/or gene deletion.
Asunto(s)
Metilación de ADN , Regulación hacia Abajo , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Regiones Promotoras Genéticas/genética , Proteínas Supresoras de Tumor/sangre , Adolescente , Adulto , Proteínas de Ciclo Celular , Femenino , Eliminación de Gen , Humanos , Hibridación Fluorescente in Situ , Masculino , Proteínas del Tejido Nervioso , Reacción en Cadena de la Polimerasa/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genéticaRESUMEN
In the search of new symmetrical derivatives with anticancer activity, we have looked for novel compounds able to induce a selective proapoptotic mechanism in cancer cells. The potential antitumoral activity of several quinazoline derivatives was evaluated in vitro examining their cytotoxic effects against human breast, colon and bladder cancer cell lines. The IC(50) value of the compounds that showed cytotoxic activity was calculated. These compounds were tested for their ability to induce caspase-3 activation and nuclear chromatin degradation. Non-tumoral human cell lines were used to test the selectivity of the cytotoxic compounds against cancer cells. Several compounds showed no cytotoxicity in these cell lines. Finally, JRF12 (2,4-dibenzylaminoquinazoline) was chosen as the best candidate and its mechanism of action was studied in more detail. A time dependent evaluation of apoptosis was performed in the three cancer cell lines, followed by an evaluation of the cell cycle regulation involvement that showed a decrease of cells in G(1) phase and increase of cells in G(2) phase before cell death. 2,4-dibenzylaminoquinazoline treatment produces few changes in the expression of genes as evaluated by using oligonucleotide microarrays and Q-RT-PCR assays. In conclusion, 2,4-dibenzylaminoquinazoline is a promising anticancer drug showing cytostatic and apoptotic effects mainly in a transcription independent manner.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Quinazolinas/uso terapéutico , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis , Caspasa 3/metabolismo , Inhibidores de Caspasas , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/farmacología , Evaluación Preclínica de Medicamentos , Genes Relacionados con las Neoplasias/efectos de los fármacos , Humanos , Neoplasias/enzimología , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Quinazolinas/química , Quinazolinas/farmacología , ARN Neoplásico/análisisRESUMEN
The work described here involved the synthesis and biological evaluation of new heteroaryldiamides and heteroaryldiamines. A new general model in which the structures can be adjusted has been applied in this study. Three different structural units can be distinguished: a central nucleus and two symmetric terminal units. The central element is either an aliphatic chain of varying length and flexibility, piperazine, or a polyamine nucleus. However, the terminal units are pyridine, quinoline, indole, benzene or pyrido[2,3-d]pyrimidine with different substituents. The antitumoural activities of the compounds were evaluated in vitro by examining their cytotoxic effects against human breast, colon, and bladder cancer cell lines. Compounds that showed cytotoxic activity were subjected to both apoptosis and caspase-3 assays. With regard to selectivity, the cytotoxicity was also determined in cell cultures of two nontumoural lines. The most promising compounds are 4c, 5c and 7, which are amino-pyridinium, quinolyl-N-oxide, and pyridyl derivatives, respectively, and these reveal a significant in vitro cytotoxicity in at least two of the three cell lines tested. These compounds induced apoptosis and also produced a rapid dose-dependent increase in the caspase-3 level in HT-29 cells. Other encouraging profiles were found, such as those presented by 1k and 8d, which are cytotoxic and apoptotic but do not provoke an increase in the level of caspase-3, or those presented by 2f, 3c and 4a, which are slightly cytotoxic but do not show any other significant activity. The different types of behaviour of each compound are not necessarily parallel in the three cell lines tested.
Asunto(s)
Amidas/farmacología , Antineoplásicos/farmacología , Apoptosis , Caspasas/metabolismo , Diaminas/farmacología , Amidas/síntesis química , Antineoplásicos/síntesis química , Caspasa 3 , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Diaminas/síntesis química , Relación Dosis-Respuesta a Droga , Activación Enzimática , Células HT29 , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The PARK2 gene, previously identified as a mutated target in patients with autosomal recessive juvenile parkinsonism (ARJP), has recently been found to be a candidate tumor suppressor gene in ovarian, breast, lung and hepatocellular carcinoma that maps to the third common fragile site (CFS) FRA6E. PARK2 is linked to a novel described PACRG gene by a bidirectional promoter containing a defined CpG island in its common promoter region. We have studied the role of promoter hypermethylation in the regulation of PARK2 and PACRG expression in different tumor cell lines and primary patient samples. Abnormal methylation of the common promoter of PARK2 and PACRG was observed in 26% of patients with acute lymphoblastic leukemia and 20% of patients with chronic myelogenous leukemia (CML) in lymphoid blast crisis, but not in ovarian, breast, lung, neuroblastoma, astrocytoma or colon cancer cells. Abnormal methylation resulted in downregulation of PARK2 and PACRG gene expression, while demethylation of ALL cells resulted in demethylation of the promoter and upregulation of PARK2 and PACRG expression. By FISH, we demonstrated that a lack of PARK2 and PACRG expression was due to biallelic hypermethylation and not to deletion of either PARK2 or PACRG in ALL. In conclusion, our results demonstrate for the first time that the candidate tumor suppressor genes PARK2 and PACRG are epigenetically regulated in human leukemia, suggesting that abnormal methylation and regulation of PARK2 and PACRG may play a role in the pathogenesis and development of this hematological neoplasm.