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1.
J Vis Exp ; (134)2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29683453

RESUMO

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas/genética , RNA/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/patologia , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo
2.
Oncotarget ; 7(45): 72395-72414, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27590350

RESUMO

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.


Assuntos
Regulação Neoplásica da Expressão Gênica , Genes Mitocondriais , Genes myc , Neoplasias/genética , Animais , Linhagem Celular Tumoral , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc , Espécies Reativas de Oxigênio/metabolismo , Transfecção
3.
Elife ; 3: e02200, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24867637

RESUMO

The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms.DOI: http://dx.doi.org/10.7554/eLife.02200.001.


Assuntos
Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Apoptose/genética , Sítios de Ligação , Linhagem Celular , Humanos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo
4.
Mol Cell Biol ; 31(24): 5037-45, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21986497

RESUMO

Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed "intrinsic tumor suppressor activity," and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.


Assuntos
Apoptose/genética , Neoplasias da Mama/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes myc , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Divisão Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Feminino , Loci Gênicos , Humanos , Camundongos , Camundongos Knockout , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética
5.
J Biol Chem ; 286(6): 4264-70, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21148320

RESUMO

In unstressed cells, the p53 tumor suppressor is highly unstable. DNA damage and other forms of cellular stress rapidly stabilize and activate p53. This process is regulated by a complex array of post-translational modifications that are dynamically deposited onto p53. Recent studies show that these modifications orchestrate p53-mediated processes such as cell cycle arrest and apoptosis. Cancer cells carry inherent genetic damage, but avoid arrest and apoptosis by inactivating p53. Defining the enzymatic machinery that regulates the stress-induced modification of p53 at single-residue resolution is critical to our understanding of the biochemical mechanisms that control this critical tumor suppressor. Specifically, acetylation of p53 at lysine 120, a DNA-binding domain residue mutated in human cancer, is essential for triggering apoptosis. Given the oncogenic properties of deacetylases and the success of deacetylase inhibitors as anticancer agents, we investigated the regulation of Lys(120) deacetylation using pharmacologic and genetic approaches. This analysis revealed that histone deacetylase 1 is predominantly responsible for the deacetylation of Lys(120). Furthermore, treatment with the clinical-grade histone deacetylase inhibitor entinostat enhances Lys(120) acetylation, an event that is mechanistically linked to its apoptotic effect. These data expand our understanding of the mechanisms controlling p53 function and suggest that regulation of p53 modification status at single-residue resolution by targeted therapeutics can selectively alter p53 pathway function. This knowledge may impact the rational application of deacetylase inhibitors in the treatment of human cancer.


Assuntos
Apoptose , Proteína Supressora de Tumor p53/metabolismo , Acetilação/efeitos dos fármacos , Benzamidas/farmacologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Mutação de Sentido Incorreto , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Estrutura Terciária de Proteína , Piridinas/farmacologia , Proteína Supressora de Tumor p53/genética
6.
Exp Cell Res ; 317(4): 488-95, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21111732

RESUMO

MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.


Assuntos
Diferenciação Celular/genética , Inibidores do Crescimento/análise , MicroRNAs/análise , Regulação para Cima , Apoptose , Butiratos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Inibidores do Crescimento/farmacologia , Humanos , Cloreto de Lítio/farmacologia , MicroRNAs/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacos
7.
Trends Biochem Sci ; 34(11): 571-8, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19819149

RESUMO

Protein phosphorylation is regulated dynamically in eukaryotic cells via modulation of the enzymatic activity of kinases and phosphatases. Like phosphorylation, acetylation has emerged as a critical regulatory protein modification that is altered dynamically in response to diverse cellular cues. Moreover, acetyltransferases and deacetylases are tightly linked to cellular signaling pathways. Recent studies provide clues about the mechanisms utilized to regulate acetyltransferases and deacetylases. The therapeutic value of deacetylase inhibitors suggests that understanding acetylation pathways will directly impact our ability to rationally target these enzymes in patients. Recently discovered mechanisms that directly regulate the catalytic activity of acetyltransferases and deacetylases provide exciting new insights about these enzymes.


Assuntos
Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Transdução de Sinais , Acetilação , Animais , Catálise , Histonas/metabolismo , Humanos , Modelos Biológicos , Proteína Supressora de Tumor p53/metabolismo
8.
Mol Cell ; 36(2): 174-5, 2009 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-19854127

RESUMO

Dynamic lysine acetylation regulates proteins involved in diverse cellular processes, with individual enzymes often acetylating multiple substrates. Here, Li et al. (2009) show that the substrate specificity of hMOF/MYST1/KAT8 is controlled by differential interaction with two mutually exclusive partners.


Assuntos
Histona Acetiltransferases/metabolismo , Acetilação , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Histonas/metabolismo , Humanos , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo
9.
Mol Cell ; 24(6): 841-51, 2006 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-17189187

RESUMO

The ability of p53 to induce apoptosis plays an important role in tumor suppression. Here, we describe a previously unknown posttranslational modification of the DNA-binding domain of p53. This modification, acetylation of lysine 120 (K120), occurs rapidly after DNA damage and is catalyzed by the MYST family acetyltransferases hMOF and TIP60. Mutation of K120 to arginine, as occurs in human cancer, debilitates K120 acetylation and diminishes p53-mediated apoptosis without affecting cell-cycle arrest. The K120R mutation selectively blocks the transcription of proapoptotic target genes such as BAX and PUMA while the nonapoptotic targets p21 and hMDM2 remain unaffected. Consistent with this, depletion of hMOF and/or TIP60 inhibits the ability of p53 to activate BAX and PUMA transcription. Furthermore, the acetyllysine 120 (acetyl-K120) form of p53 specifically accumulates at proapoptotic target genes. These data suggest that K120 acetylation may help distinguish the cell-cycle arrest and apoptotic functions of p53.


Assuntos
Apoptose , Histona Acetiltransferases/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação , Ciclo Celular , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Lisina Acetiltransferase 5 , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteína X Associada a bcl-2/genética
10.
Nature ; 432(7015): 406-11, 2004 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-15525939

RESUMO

The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.


Assuntos
Dano ao DNA , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Sequência Conservada , Reagentes de Ligações Cruzadas/química , DNA/química , DNA/genética , Histona-Lisina N-Metiltransferase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Metilação , Metiltransferases/deficiência , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosfoproteínas/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
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