RESUMEN

Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions. Despite the importance of histone methylation on gene regulation, characterization and quantitation of this modification have proven to be quite challenging. Great advances have been made in the analysis of histone modification using both bottom-up and top-down mass spectrometry (MS). However, MS-based analysis of histone posttranslational modifications (PTMs) is still problematic, due both to the basic nature of the histone N-terminal tails and to the combinatorial complexity of the histone PTMs. In this report, we describe a simplified MS-based platform for histone methylation analysis. The strategy uses chemical acetylation with d0-acetic anhydride to collapse all the differently acetylated histone forms into one form, greatly reducing the complexity of the peptide mixture and improving sensitivity for the detection of methylation via summation of all the differently acetylated forms. We have used this strategy for the robust identification and relative quantitation of H4R3 methylation, for which stoichiometry and symmetry status were determined, providing an antibody-independent evidence that H4R3 is a substrate for both Type I and Type II PRMTs. Additionally, this approach permitted the robust detection of H4K5 monomethylation, a very low stoichiometry methylation event (0.02% methylation). In an independent example, we developed an in vitro assay to profile H3K27 methylation and applied it to an EZH2 mutant xenograft model following small-molecule inhibition of the EZH2 methyltransferase. These specific examples highlight the utility of this simplified MS-based approach to quantify histone methylation profiles.

Asunto(s)

Histonas/metabolismo , Acetilación , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Humanos , Espectrometría de Masas , Metilación

RESUMEN

While c-MYC is well established as a proto-oncogene, its structure and function as a transcription factor have made c-MYC a difficult therapeutic target. To identify small-molecule inhibitors targeting c-MYC for anticancer therapy, we designed a high-throughput screening (HTS) strategy utilizing cellular assays. The novel approach for the HTS was based on the detection of cellular c-MYC protein, with active molecules defined as those that specifically decreased c-MYC protein levels in cancer cells. The assay was based on a dual antibody detection system using Förster/fluorescence resonance energy transfer (FRET) and was utilized to detect endogenous c-MYC protein in the MYC amplified cancer cell lines DMS273 and Colo320 HSR. The assays were miniaturized to 1536-well plate format and utilized to screen the GlaxoSmithKline small-molecule collection of approximately 2 million compounds. In addition to the HTS assay, follow-up assays were developed and used to triage and qualify compounds. Two cellular assays used to eliminate false-positive compounds from the initially selected HTS hits were (1) a cellular toxicity assay and (2) an unstable protein reporter assay. Three positive selection assays were subsequently used to qualify compounds: (1) 384-well cell cycle flow cytometry, (2) 384-well cell growth, and (3) c-MYC gene signature reverse transcription quantitative PCR (RT-qPCR). The HTS and follow-up assays successfully identified three compounds that specifically decreased c-MYC protein levels in cancer cells and phenocopied c-MYC siRNA in terms of cell growth inhibition and gene signatures. The HTS, triage, and three compounds identified are described.

Asunto(s)

Descubrimiento de Drogas/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes myc , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

In eukaryotes, post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumorigenesis and correlates with poor prognosis in several tumour types. Additionally, somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 occur in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. The Y641 residue is the most frequently mutated residue, with up to 22% of germinal centre B-cell DLBCL and follicular lymphoma harbouring mutations at this site. These lymphomas have increased H3K27 tri-methylation (H3K27me3) owing to altered substrate preferences of the mutant enzymes. However, it is unknown whether specific, direct inhibition of EZH2 methyltransferase activity will be effective in treating EZH2 mutant lymphomas. Here we demonstrate that GSK126, a potent, highly selective, S-adenosyl-methionine-competitive, small-molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and markedly inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.

Asunto(s)

Indoles/farmacología , Indoles/uso terapéutico , Linfoma Folicular/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Mutación/genética , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Piridonas/farmacología , Piridonas/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Linfoma Folicular/enzimología , Linfoma Folicular/genética , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Metilación/efectos de los fármacos , Ratones , Trasplante de Neoplasias , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Activación Transcripcional/efectos de los fármacos , Trasplante Heterólogo

RESUMEN

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

Asunto(s)

Alanina/genética , Proteínas de Unión al ADN/genética , Histonas/metabolismo , Linfoma de Células B/enzimología , Linfoma de Células B/genética , Lisina/metabolismo , Mutación/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Análisis Mutacional de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Neoplásica de la Expresión Génica , Glicina/genética , Heterocigoto , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Complejo Represivo Polycomb 2 , Especificidad por Sustrato , Factores de Transcripción/química , Factores de Transcripción/metabolismo

RESUMEN

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low-density lipoprotein receptor-related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF-luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF-luciferase activation. DKK-1 protein blocks upregulation of the TCF-luciferase reporter by the Wnt3-Fz1 chimera, suggesting involvement of LRP in Wnt3-Fz1 signaling. Expression of a Wnt3-Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3-Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt-Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases.

Asunto(s)

Receptores Frizzled/metabolismo , Modelos Biológicos , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Línea Celular , Receptores Frizzled/química , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Wnt/química , Proteína Wnt3

RESUMEN

In addition to a common polymorphism at codon 72, the p53 tumor suppressor gene also contains a rare single nucleotide polymorphism at amino acid 47. Wild type p53 encodes proline at this residue, but in <5% of African Americans, this amino acid is serine. Notably, phosphorylation of the adjacent serine 46 by the proline-directed kinase p38 MAPK is known to greatly enhance the ability of p53 to induce apoptosis. Here we showed that the serine 47 polymorphic variant, which replaces the proline residue necessary for recognition by proline-directed kinases, is a markedly poorer substrate for phosphorylation on serine 46 by p38 MAPK. Consistent with this finding, we showed that the serine 47 variant has up to 5-fold decreased ability to induce apoptosis compared with wild type p53. Mechanistically, we found that this variant has decreased ability to transactivate two p53 target genes, p53AIP1 and PUMA, but not other p53 response genes; this is the first time that phosphorylation of serine 46 has been implicated in transactivation of PUMA by p53. Down-regulation of PUMA in cells with wild type p53 using short interfering RNAs reduced apoptosis in these cells to a level comparable to that in cells containing the serine 47 variant. The combined data indicated that, like the codon 72 polymorphism, the codon 47 polymorphism of p53 is functionally significant and may play a role in cancer risk, progression, and the efficacy of therapy.

Asunto(s)

Codón , Polimorfismo Genético , Proteína p53 Supresora de Tumor/genética , Secuencia de Aminoácidos , Secuencia de Bases , Población Negra , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

There is substantial evidence in the literature that, in addition to functioning as an activator of transcription, the p53 tumor suppressor protein can also function as a sequence-specific transcriptional repressor of a separate set of genes. However, elucidation of the mechanism whereby p53 functions as a transcriptional repressor has been obscured by the use of artificial assays to measure this activity; these assays include transient transfection analyses, where both p53 and target promoters are overexpressed. This chapter describes alternative approaches for the definition of sequence elements that mediate transcriptional repression by p53. These include the McKay (immunobinding) assay, which measures the in vitro binding of large fragments of DNA, as well as chromatin immunoprecipitations (ChIPs), which measure in vivo binding. The use of such assays should better define the mechanism of transcriptional repression by p53 and should aid in the elucidation of the contribution of this activity to p53-dependent growth arrest and programmed cell death (apoptosis).

Asunto(s)

Regulación de la Expresión Génica , Pruebas de Precipitina/métodos , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Humanos , Unión Proteica , Proteínas Represoras/genética , Transcripción Genética

RESUMEN

The gene TP53, encoding p53, has a common sequence polymorphism that results in either proline or arginine at amino-acid position 72. This polymorphism occurs in the proline-rich domain of p53, which is necessary for the protein to fully induce apoptosis. We found that in cell lines containing inducible versions of alleles encoding the Pro72 and Arg72 variants, and in cells with endogenous p53, the Arg72 variant induces apoptosis markedly better than does the Pro72 variant. Our data indicate that at least one source of this enhanced apoptotic potential is the greater ability of the Arg72 variant to localize to the mitochondria; this localization is accompanied by release of cytochrome c into the cytosol. These data indicate that the two polymorphic variants of p53 are functionally distinct, and these differences may influence cancer risk or treatment.

Asunto(s)

Apoptosis/genética , Genes p53 , Polimorfismo Genético , Apoptosis/efectos de los fármacos , Arginina/genética , Línea Celular , Chaperonina 60/metabolismo , Codón/genética , Ácidos Grasos Insaturados/farmacología , Variación Genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Neoplasias/etiología , Neoplasias/genética , Prolina/genética , Supresión Genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/química