RESUMO
Aberrant DNA promoter methylation with associated gene silencing is a common epigenetic abnormality in acute lymphoblastic leukaemia (ALL) and is associated with poor survival. We have identified a family of transmembrane tyrosine phosphatase proteins as targets of hypermethylation in ALL and high-grade B cell lymphoma and demonstrated that this abnormal methylation correlates with transcript expression. PTPRG was methylated in 63% of ALL samples, PTPRK in 47%, PTPRM in 64% and PTPRO in 54% of cases, with most ALL samples containing methylation at multiple phosphatase loci. PTPRK promoter methylation was associated with a decreased overall survival in the cohort. Restoration of PTPRK transcript levels in leukaemia cells, where phosphatase transcript was silenced, reduced cell proliferation, inhibited colony formation and increased sensitivity to cytotoxic chemotherapy. These biological changes were associated with a reduction in levels of phosphorylated Erk1/2, Akt, STAT3 and STAT5 suggesting functional phosphatase activity after transcript re-expression. Methylation of the phosphatase promoters was reversible with decitabine and a histone deacetylase inhibitor, suggesting that PTPRK-mediated cell signalling pathways may be targeted with epigenetic therapies in lymphoid malignancy.
Assuntos
Metilação de DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Tirosina Fosfatases/genética , Adolescente , Adulto , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Ilhas de CpG , Humanos , Janus Quinase 1/genética , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismoRESUMO
We performed a genome-wide analysis of promoter associated CpG island methylation using methylated CpG island amplification (MCA) coupled to representational differential analysis (RDA) or a DNA promoter microarray in acute lymphoblastic leukemia (ALL). We identified 65 potential targets of methylation with the MCA/RDA approach, and 404 with the MCA/array. Thirty-six (77%) of the genes identified by MCA/RDA were shared by the MCA/array approach. Chromosomal location of these genes was evenly distributed in all autosomes. Functionally, 303 of these genes clustered in 18 molecular pathways. Of the 36 shared genes, 31 were validated and 26 were confirmed as being hypermethylated in leukemia cell lines. Expression analysis of eight of these genes was epigenetically modulated by hypomethylating agents and/or HDAC inhibitors in leukemia cell lines. Subsequently, DNA methylation of 15 of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) was validated in primary ALL samples. Patients with methylation of multiple CpG islands had a worse overall survival. This is the largest published list of potential methylation target genes in human leukemia offering the possibility of performing rational unbiased methylation studies in ALL.
Assuntos
Ilhas de CpG , Metilação de DNA , Genoma Humano , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Epigênese Genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Cromossomo Filadélfia , Regiões Promotoras GenéticasRESUMO
To investigate if the tumor suppressor properties of p57KIP2 are dependent on its DNA methylation status, we studied the impact of several stress stimuli in leukemic cell lines with different p57KIP2 promoter DNA methylation levels. p57KIP2 reactivation was observed after stimulation with transforming growth factor-beta, other cytokines, high-density culture or serum withdrawal in p57KIP2 promoter unmethylated cells but not in methylated cells. In these cells, p57KIP2 reactivation required the use of a hypomethylating agent or a histone deacetylase inhibitor. Overexpression of p57KIP2 in p57KIP2 promoter methylated leukemic cell lines resulted in cell growth arrest and the induction of apoptosis. In contrast, overexpression of p57KIP2 in partially methylated cells only resulted in a moderate inhibition of cell growth and had no impact on apoptosis. Transduction of unmethylated cells expressing high levels of p57KIP2 with p57KIP2 short hairpin RNA resulted in increased cell proliferation. These results suggest that the tumor suppressive properties of p57KIP2 in leukemia may depend on the intrinsic promoter DNA methylation status of the gene.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57/genética , Metilação de DNA , Genes Supressores de Tumor , Leucemia/genética , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta/farmacologia , Apoptose , Técnicas de Cultura de Células/métodos , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Células JurkatRESUMO
BACKGROUND: Aneurysms and dissections affecting the ascending aorta are associated primarily with degeneration of the aortic media, called medial necrosis. Families identified with dominant inheritance of thoracic aortic aneurysms and dissections (TAA/dissections) indicate that single gene mutations can cause medial necrosis in the absence of an associated syndrome. METHODS AND RESULTS: Fifteen families were identified with multiple members with TAAs/dissections. DNA from affected members from 2 of the families was used for a genome-wide search for the location of the defective gene by use of random polymorphic markers. The data were analyzed by the affected-pedigree-member method of linkage analysis. This analysis revealed 3 chromosomal loci with multiple markers demonstrating evidence of linkage to the phenotype. Linkage analysis using further markers in these regions and DNA from 15 families confirmed linkage of some of the families to 5q13-14. Genetic heterogeneity for the condition was confirmed by a heterogeneity test. Data from 9 families with the highest conditional probability of being linked to 5q were used to calculate the pairwise and multipoint logarithm of the odds (LOD) scores, with a maximum LOD of 4.74, with no recombination being obtained for the marker D5S2029. In 6 families, the phenotype was not linked to the 5q locus. CONCLUSIONS: A major locus for familial TAAs and dissections maps to 5q13-14, with the majority (9 of 15) of the families identified demonstrating evidence of linkage to this locus. The condition is genetically heterogeneous, with 6 families not demonstrating evidence of linkage to any loci previously associated with aneurysm formation.
Assuntos
Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/genética , Proteínas da Matriz Extracelular , Proteoglicanas , Dissecção Aórtica/patologia , Aneurisma da Aorta Torácica/patologia , Proteoglicanas de Sulfatos de Condroitina/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 5/genética , Saúde da Família , Feminino , Heterogeneidade Genética , Genoma Humano , Genótipo , Haplótipos , Humanos , Lectinas Tipo C , Escore Lod , Masculino , Repetições de Microssatélites , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Análise de Sequência de DNA , Trombospondinas/genética , VersicanasRESUMO
A large east Texas family with autosomal dominant inheritance of a novel bleeding disorder has been identified. The disorder is characterized clinically by easy bruising, life-threatening bleeding with trauma or surgery, and menorrhagia in affected women. Laboratory studies demonstrated prolongation of the prothrombin time and activated partial thromboplastin time in affected individuals. Paradoxically, assays of known coagulation factors are all within normal limits. To determine the molecular basis of this disease, a candidate gene linkage analysis in this kindred was done. Initially it was hypothesized that the cause of the disease in this family could be an antithrombin III (AT3) mutation that resulted in a constitutively active AT3 in the absence of heparin binding. Linkage studies using DNA from the family and an intragenic polymorphic marker within the AT3 gene showed that the disease mapped to this locus. The coding region and intron/exon junctions of AT3 were sequenced using the proband's DNA, but this analysis failed to identify a mutation. Additional family members were recruited for the study, and 16 polymorphic markers around the AT3 gene were analyzed. Using 2 recombinants, the critical interval for the defective gene was narrowed to approximately 1.5 Mb, centromeric to AT3. The factor V (FV) gene was mapped into the disease interval and sequenced; there were no mutations found. Elucidation of the genetic defect causing the bleeding disorder in this family may reveal a novel protein involved in the coagulation cascade.
Assuntos
Genes Dominantes/genética , Hemorragia/genética , Adulto , Antitrombina III/genética , Fatores de Coagulação Sanguínea , Testes de Coagulação Sanguínea , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 1/genética , Fator V/genética , Saúde da Família , Feminino , Ligação Genética , Marcadores Genéticos , Haplótipos , Hemorragia/epidemiologia , Hemorragia/etiologia , Humanos , Masculino , Mutação , Linhagem , Texas/epidemiologiaRESUMO
The TAL-1 gene is located on chromosome 1p32. In about 20% of T-cell acute lymphoblastic leukemias (T-ALL), this gene is disrupted in its 5' portion by a site-specific 100-kg deletion and is fused with the 5' part of the SIL gene, to form SIL-TAL-1 chimeric gene. In this study, we established a "nested" retrotranscriptase/polymerase chain reaction (RT/PCR) technique which allows detection of the SIL-TAL-1 transcriptional expression. A chimeric mRNA was observed in four of 17 T-ALL cases and has been shown to result from the fusion between the exon 1 of SIL and exon 3 of TAL. A sensitivity test showed that this RT/PCR procedure could detect one leukemic cell among 10(6) normal cells. A positive RT/PCR result was obtained in two cases during clinical remission, suggesting the presence of minimal residual disease (MRD). One patient developed clinical relapse 3 months after PCR positivity. Moreover, analysis of the Tald rearrangement by DNA-based PCR in four patients with SIL-TAL-1 fusion revealed the type A (Tald1) rearrangement in all cases. Sequence analysis demonstrated the presence of N region and non-random "P" nucleotide, as well as base deletions at the genomic SIL-TAL-1 joining site. These data indicate that detection of TAL-1 gene abnormality is important for diagnosis and monitoring of MRD in a subset of T-ALL.