RESUMEN
Alterations in the function of the p53 pathway are frequently described in chronic lymphocytic leukemia (CLL), mostly associated with deletion of 17p13 and/or mutations of the TP53 gene. In the present study, we investigated 103 CLLs for the impact of protein expression of full-length p53 and its isoforms ß and γ. A strong correlation between deletions of 17p13 and an accumulation of full-length p53 protein was found and was associated with a worse outcome compared to CLL with normal p53 (treatment-free survival p < 0.001, overall survival p = 0.04). Interestingly, the relative expression levels between full-length p53 protein and its isoforms ß and γ were significantly altered in CLL even without deletions of 17p13, compared to normal B-cells (p = 0.005). Furthermore, CLLs with higher p53 protein ratios showed worse clinical courses compared to CLLs with lower p53 protein ratios. Taken together, the differential expression of p53 isoforms could disrupt the p53 response and contribute to CLL pathogenesis.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 17/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Proteína p53 Supresora de Tumor/genética , Western Blotting , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Estimación de Kaplan-Meier , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Pronóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
To understand the influence of chromosomal alterations on gene expression in a genome-wide view, chromosomal imbalances detected by single nucleotide polymorphism (SNP) chips were compared with global gene expression in 16 cases of chronic lymphocytic leukemia (CLL). A strong concordance between chromosomal gain or loss and increased or reduced expression of genes in the affected regions was found, respectively. Regions of uniparental disomy (UPD) were rare and had usually no consistent influence on gene expression, but in one instance, a large UPD was associated with a downregulation of most genes in the affected chromosome. The frequently deleted miRNAs, MIRN15A and MIRN16-1, did not show a reduced expression in cases with monoallelic deletions. The BCL2 protein, considered to be downregulated by these miRNAs, was upregulated not only in CLL with biallelic deletion of MIRN15A and MIRN16-1, but also in cases with monoallelic deletion. This suggests a complex regulation of BCL2 levels in CLL cells. Taken together, in CLL, a global gene dosage effect exists for chromosomal gains and deletions and in some instances for UPDs. We did not confirm a consistent correlation between MIRN15A and MIRN16-1 expression levels and BCL2 protein levels, indicating a complex regulation of BCL2 expression.
Asunto(s)
Dosificación de Gen , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Polimorfismo de Nucleótido Simple , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Mapeo Cromosómico , ARN Helicasas DEAD-box/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , MicroARNs/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genética , Disomía Uniparental/genética , Regulación hacia ArribaRESUMEN
The transcription factor network in Hodgkin lymphoma (HL) represents a unique composition of proteins found in no other hematopoietic cell. Among these factors, an aberrant expression of the T-cell transcription factor GATA3 is observed in B cell-derived Hodgkin and Reed/Sternberg (HRS) tumor cells. Herein, we elucidate the regulation and function of this factor in HL. We demonstrate binding of NFκB and Notch-1, 2 factors with deregulated activity in HL to GATA3 promoter elements. Interference with NFκB and Notch-1 activity led to decreased GATA3 expression, indicating a dependency of deregulated GATA3 expression on these transcription factors. Down-regulation of GATA3 in HL cell lines demonstrated its role in the regulation of IL-5, IL-13, STAT4, and other genes. A correlation between GATA3 and IL-13 expression was confirmed for HRS cells in HL tissues. Thus, GATA3 shapes the cytokine expression and signaling that is typical of HL. Conclusively, aberrant GATA3 expression in HRS cells is stimulated by the deregulated constitutive activity of NFκB and Notch-1, indicating a complex network of deregulated transcription factors in these cells. GATA3 activity significantly contributes to the typical cytokine secretion of and signaling in HRS cells, which presumably plays an essential role in HL pathogenesis.
Asunto(s)
Citocinas/genética , Factor de Transcripción GATA3/genética , Enfermedad de Hodgkin/genética , Células de Reed-Sternberg/inmunología , Apoptosis , Línea Celular Tumoral , Citocinas/metabolismo , Regulación hacia Abajo/genética , Factor de Transcripción GATA3/antagonistas & inhibidores , Factor de Transcripción GATA3/metabolismo , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/patología , Humanos , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Lentivirus/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Interferente Pequeño/metabolismo , Receptor Notch1/metabolismo , Factor de Transcripción STAT4/genética , Factor de Transcripción STAT4/metabolismo , Análisis de SupervivenciaRESUMEN
Proliferation and survival of Hodgkin and Reed/Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), are dependent on constitutive activation of nuclear factor kappaB (NF-kappaB). NF-kappaB activation through various stimuli is negatively regulated by the zinc finger protein A20. To determine whether A20 contributes to the pathogenesis of cHL, we sequenced TNFAIP3, encoding A20, in HL cell lines and laser-microdissected HRS cells from cHL biopsies. We detected somatic mutations in 16 out of 36 cHLs (44%), including missense mutations in 2 out of 16 Epstein-Barr virus-positive (EBV(+)) cHLs and a missense mutation, nonsense mutations, and frameshift-causing insertions or deletions in 14 out of 20 EBV(-) cHLs. In most mutated cases, both TNFAIP3 alleles were inactivated, including frequent chromosomal deletions of TNFAIP3. Reconstitution of wild-type TNFAIP3 in A20-deficient cHL cell lines revealed a significant decrease in transcripts of selected NF-kappaB target genes and caused cytotoxicity. Extending the mutation analysis to primary mediastinal B cell lymphoma (PMBL), another lymphoma with constitutive NF-kappaB activity, revealed destructive mutations in 5 out of 14 PMBLs (36%). This report identifies TNFAIP3 (A20), a key regulator of NF-kappaB activity, as a novel tumor suppressor gene in cHL and PMBL. The significantly higher frequency of TNFAIP3 mutations in EBV(-) than EBV(+) cHL suggests complementing functions of TNFAIP3 inactivation and EBV infection in cHL pathogenesis.
Asunto(s)
Deleción Cromosómica , Genes Supresores de Tumor , Enfermedad de Hodgkin/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Linfoma de Células B/genética , Mutación , Proteínas Nucleares/genética , Línea Celular Tumoral , Elementos Transponibles de ADN , Proteínas de Unión al ADN , Infecciones por Virus de Epstein-Barr/genética , Mutación del Sistema de Lectura , Humanos , Mutación Missense , Polimorfismo de Nucleótido Simple , Transcripción Genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (HL) and lymphocytic and histiocytic (L&H) cells in nodular lymphocyte-predominant HL (NLPHL) are derived from germinal-center B cells. HRS cells have, however, largely lost their B cell phenotype and aberrantly express markers and transcriptional regulators of other hematolymphoid cell types. Deregulation of multiple signaling pathways and downstream transcription factors, including receptor tyrosine kinases, nuclear factor-kappa B (NF-kappaB), and Janus kinase/signal transducer and activator of transcription (JAK/STAT), is a further hallmark of HRS cells. These cells harbor genetic lesions that contribute to or cause increases in the activity of transcription factors of the NF-kappaB and STAT families. HRS cells are found within a mixed reactive cellular infiltrate and interact with these nonmalignant cells in a complex fashion that appears to be essential for HRS cell survival and proliferation. Less is known about the pathogenesis of L&H cells in NLPHL, but increases in the activity of receptor tyrosine kinases, NF-kappaB, and JAK/STAT have also been detected.
Asunto(s)
Histiocitos/patología , Enfermedad de Hodgkin/patología , Linfocitos/patología , Células de Reed-Sternberg/patología , Diferenciación Celular , Linaje de la Célula , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Histiocitos/inmunología , Histiocitos/metabolismo , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/metabolismo , Humanos , Quinasas Janus/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Linfoma de Células B/patología , Neoplasias del Mediastino/patología , FN-kappa B/metabolismo , Fenotipo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células de Reed-Sternberg/inmunología , Células de Reed-Sternberg/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genéticaRESUMEN
EBV-associated Hodgkin lymphoma (HL) and some post-transplant lymphoproliferative disease (PTLD) cases originate from pro-apoptotic germinal center (GC) B cells that have acquired destructive somatic Ig V gene mutations and were presumably rescued from apoptosis by EBV. To find out whether B cell receptor-crippled GC B cells acquire features of HL and/or PTLD cells upon EBV-infection and to reveal the impact of EBV on expression of B cell differentiation markers, we compared lymphoblastoid cell lines (LCLs) from GC B cells (including BCR-crippled GC-LCLs) to monoclonal LCLs from naïve B cells (N-LCLs). In addition, we analyzed the controversially discussed effect of EBV-infection on the GC B-cell-specific process of somatic hypermutation in vitro. Irrespective of their cellular origin, LCLs expressed CD20, CD30, CD38, AID, Pu.1, and with one exception Syk, but lacked expression of the GC B cell marker BCL-6. Interestingly, the T cell transcription factor GATA-3 that is aberrantly expressed in HL was induced in most GC-LCLs and the memory B cell marker CD27 was activated in N-LCLs. Remarkably, only 4 of 24 GC-LCLs showed significant somatic hypermutation activity, demonstrating that EBV usually silences hypermutation upon infection of GC B cells. Notably, one of three N-LCL showed a low level of intraclonal diversification. Thus, EBV-infection deregulates multiple differentiation factors and processes in B cells, leading to a largely homogenous phenotype of EBV-infected B cells in latency III.
Asunto(s)
Linfocitos B/citología , Linfocitos B/virología , Diferenciación Celular , Transformación Celular Viral/fisiología , Regulación de la Expresión Génica , Herpesvirus Humano 4/fisiología , Apoptosis , Linfocitos B/metabolismo , Biomarcadores , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/virología , Humanos , Fenotipo , Hipermutación Somática de Inmunoglobulina/genética , Proteínas de la Matriz Viral/metabolismo , Latencia del VirusRESUMEN
Bcl-2 plays a key role in the regulation of apoptosis. We investigated the role of a novel regulatory single-nucleotide polymorphism (-938C>A) in the inhibitory P2 BCL2 promoter in B-cell chronic lymphocytic leukemia (B-CLL). The -938C allele displayed significantly increased BCL2 promoter activity and binding of nuclear proteins compared with the A allele. Concomitantly, Bcl-2 protein expression in B cells from CLL patients carrying the -938 AA genotype was significantly increased compared with CC genotypes. Genotype distribution between 123 CLL patients (42 AA, 55 AC, 26 CC) and 120 genotyped healthy controls (36 AA, 63 AC, 21 CC) was not significantly different, suggesting that genotypes of this polymorphism do not increase the susceptibility for B-CLL. However, median time from first diagnosis to initiation of chemotherapy and median overall survival were significantly shorter in patients with -938AA genotype (38 and 199 months, respectively) compared with AC/CC genotypes (120 and 321 months, respectively; P = .008 and P = .003, respectively). Multivariable Cox regression identified the BCL2-938AA genotype as an independent prognostic factor for the time to first treatment (hazard ratio [HR] 1.9; P = .034) together with disease stage at diagnosis (HR 2.5; P = .004) and ZAP-70 status (HR 3.0; P = .001). The BCL2-938AA genotype is associated with increased Bcl-2 expression and a novel unfavorable genetic marker in patients with B-CLL.
Asunto(s)
Biomarcadores de Tumor/genética , Genes bcl-2 , Leucemia Linfocítica Crónica de Células B/genética , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Anciano , Alelos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular , Aberraciones Cromosómicas , Cromosomas Humanos/genética , Cromosomas Humanos/ultraestructura , Progresión de la Enfermedad , Femenino , Genotipo , Humanos , Estimación de Kaplan-Meier , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Análisis de Supervivencia , Transcripción Genética , Transfección , Proteína Tirosina Quinasa ZAP-70/análisisRESUMEN
The cellular transcription factor E2F1 is part of an anti-tumor safeguard mechanism: it engages cell-death pathways either alone or in cooperation with p53 to protect organisms from the development of tumors. E2F1 activates downstream factors, which in turn produce secondary changes in gene expression that trigger apoptosis. Although the mechanisms are incompletely understood, several studies have demonstrated that E2F1 is involved in many different aspects of programmed cell death depending on the cellular background. Here, these findings are highlighted in the context of the most recent follow-up studies that have used apoptotic E2F1 genes as new therapeutics or drug targets, thereby providing insight into the basic mechanisms of E2F1-induced apoptosis and its possible clinical implications.
Asunto(s)
Apoptosis , Factores de Transcripción E2F/fisiología , Animales , Proteínas de Ciclo Celular/fisiología , Factores de Transcripción E2F/genética , Terapia Genética/métodos , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/terapia , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
OBJECTIVE: The transcription factor E2F1 serves as a major regulator of the cell-cycle by controlling G1-S phase transition. However, apart from its proliferative function high levels of deregulated E2F1 are capable of inducing apoptosis depending on the cellular context. In particular the tumor suppressor p53 and its homologue p73 are implicated in this proapoptotic function. METHODS: Here, we investigated the mechanistic basis for E2F1-mediated apoptosis in vascular smooth muscle cells (VSMCs) which have previously been shown to be E2F1-responsive. RESULTS: Interestingly, E2F1-expression in these cells induced clear signs of apoptosis in the absence of any proliferative activity. Although cell-cycle regulated genes such as CCNE1 and CDC25A were activated, BrdU-staining revealed no S-phase entry. Instead, a rapid loss of cell viability by induction of apoptosis was observed. Using a transactivation-defective E2F1-mutant, we show that apoptosis induction is independent of the transactivation function and therefore independent of ARF and p73. However, this mutant retains its ability to stabilize and phosphorylate p53, suggesting that p53 is sufficient for the effect of E2F1. CONCLUSION: VSMCs therefore represent a cellular system in which the transactivation-independent, proapoptotic activity of E2F1 is the primary cellular function. Ectopic expression of E2F1 might therefore be a suitable therapy to prevent VSMC hyperproliferation.
Asunto(s)
Apoptosis , Proteínas de Unión al ADN , Músculo Liso Vascular/patología , Factores de Transcripción/farmacología , Adenoviridae/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Fragmentación del ADN , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Citometría de Flujo , Expresión Génica , Vectores Genéticos/administración & dosificación , Humanos , Músculo Liso Vascular/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The p53 family includes three members that share significant sequence homology, yet exhibit fundamentally different functions in tumorigenesis. Whereas p53 displays all characteristics of a classical tumor suppressor, its homologues p63 and p73 do not. We have previously shown, that NH(2)-terminally truncated isoforms of p73 (Delta TA-p73), which act as dominant-negative inhibitors of p53 are frequently overexpressed in cancer cells. Here we provide evidence that Delta TA-p73 isoforms also affect the retinoblastoma protein (RB) tumor suppressor pathway independent of p53. Delta TA-p73 isoforms inactivate RB by increased phosphorylation, resulting in enhanced E2F activity and proliferation of fibroblasts. By inactivating the two major tumor suppressor pathways in human cells they act functionally analogous to several viral oncoproteins. These findings provide an explanation for the fundamentally different functions of p53 and p73 in tumorigenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína de Retinoblastoma/metabolismo , Transactivadores/metabolismo , Adenoviridae/genética , Western Blotting , División Celular , Medio de Cultivo Libre de Suero/farmacología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteínas de Unión al ADN/química , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Genes Dominantes , Genes Supresores de Tumor , Humanos , Luciferasas/metabolismo , Microscopía Fluorescente , Neoplasias/metabolismo , Proteínas Nucleares/química , Fenotipo , Fosfoproteínas/química , Fosforilación , Plásmidos/metabolismo , Isoformas de Proteínas , Estructura Terciaria de Proteína , Transactivadores/química , Factores de Transcripción , Transfección , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteínas Supresoras de TumorRESUMEN
The p16/RB/E2F regulatory pathway, which controls transit through the G1 restriction point of the cell cycle, is one of the most frequent targets of genetic alterations in human cancer. Any of these alterations results in the deregulated expression of the transcription factor E2F, one of the key mediators of cell cycle progression. Under these conditions, E2F1 also participates in the induction of apoptosis by a p53-dependent pathway, and independently of p53. Recently, we identified the p53-homolog p73 as a first direct target of p53-independent apoptosis. Here, we used a cDNA microarray to screen an inducible E2F1-expressing Saos-2 cell line for E2F1 target genes. Expression analysis by cDNA microarray and RT-PCR revealed novel E2F1 target genes involved in E2F1-regulated cellular functions such as cell cycle control, DNA replication and apoptosis. In addition, the identification of novel E2F1 target genes participating in the processes of angiogenesis, invasion and metastasis supports the view that E2F1 plays a central role in many aspects of cancer development. These results provide new insight into the role of E2F1 in tumorigenesis as a basis for the development of novel anti-cancer therapeutics.