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1.
ACS Chem Biol ; 17(8): 2074-2087, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35830623

RESUMO

Impaired DNA repair activity has been shown to greatly increase rates of cancer clinically. It has been hypothesized that upregulating repair activity in susceptible individuals may be a useful strategy for inhibiting tumorigenesis. Here, we report that selected tyrosine kinase (TK) inhibitors including nilotinib, employed clinically in the treatment of chronic myeloid leukemia, are activators of the repair enzyme Human MutT Homolog 1 (MTH1). MTH1 cleanses the oxidatively damaged cellular nucleotide pool by hydrolyzing the oxidized nucleotide 8-oxo-2'-deoxyguanosine (8-oxo-dG)TP, which is a highly mutagenic lesion when incorporated into DNA. Structural optimization of analogues of TK inhibitors resulted in compounds such as SU0448, which induces 1000 ± 100% activation of MTH1 at 10 µM and 410 ± 60% at 5 µM. The compounds are found to increase the activity of the endogenous enzyme, and at least one (SU0448) decreases levels of 8-oxo-dG in cellular DNA. The results suggest the possibility of using MTH1 activators to decrease the frequency of mutagenic nucleotides entering DNA, which may be a promising strategy to suppress tumorigenesis in individuals with elevated cancer risks.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Neoplasias , Monoéster Fosfórico Hidrolases/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Carcinogênese , DNA , Dano ao DNA , Humanos , Nucleotídeos , Estresse Oxidativo
2.
Genes Cancer ; 3(2): 131-40, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23050045

RESUMO

The protein product of the xeroderma pigmentosum group C (XPC) gene is a DNA damage recognition factor that functions early in the process of global genomic nucleotide excision repair. Regulation of XPC expression is governed in part by p53 at the transcriptional level. To identify the regulatory elements involved in the p53-dependent control of XPC expression, we performed a quantitative PCR tiling experiment using multiple regularly spaced primer pairs over an 11-kb region centered around the XPC transcriptional start site. p53 chromatin immunoprecipitation was performed following ultraviolet irradiation, and DNA was analyzed for enrichment at each of 48 amplicons covering this region. A segment just upstream of the XPC translational initiation site was significantly enriched, whereas no enrichment of any other region was noted. In vitro promoter reporter assays and gel retardation assays were used to confirm the p53 responsiveness of this region and to define the minimal region with stimulating activity. We identified a p53 response element that has significant similarity to a consensus sequence, with 3 mismatches. This response element is unique in that part of the p53 binding site included the coding sequence for the first 2 amino acids in the XPC protein.

3.
Neoplasia ; 13(11): 1093-100, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22131884

RESUMO

Myc, a pleiotropic transcription factor that is deregulated and/or overexpressed in most human cancers, instructs multiple extracellular programs that are required to sustain the complex microenvironment needed for tumor maintenance, including remodeling of tumor stroma, angiogenesis, and inflammation. We previously showed in a model of pancreatic ß-cell tumorigenesis that acute Myc activation in vivo triggers rapid recruitment of mast cells to the tumor site and that this is absolutely required for angiogenesis and macroscopic tumor expansion. Moreover, systemic inhibition of mast cell degranulation with sodium cromoglycate induced death of tumor and endothelial cells in established tumors. Hence, mast cells are required both to establish and to maintain the tumors. Whereas this intimates that selective inhibition of mast cell function could be therapeutically efficacious, cromoglycate is not a practical drug for systemic delivery in humans, and no other systemic inhibitor of mast cell degranulation has hitherto been available. PCI-32765 is a novel inhibitor of Bruton tyrosine kinase (Btk) that blocks mast cell degranulation and is currently in clinical trial as a therapy for B-cell non-Hodgkin lymphoma. Here, we show that systemic treatment of insulinoma-bearing mice with PCI-32765 efficiently inhibits Btk, blocks mast cell degranulation, and triggers collapse of tumor vasculature and tumor regression. These data reinforce the notion that mast cell function is required for maintenance of certain tumor types and indicate that the Btk inhibitor PCI-32765 may be useful in treating such diseases.


Assuntos
Degranulação Celular/efeitos dos fármacos , Insulinoma/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Degranulação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Genes myc , Insulinoma/genética , Insulinoma/patologia , Mastócitos/metabolismo , Mastócitos/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Teóricos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Piperidinas , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Células Tumorais Cultivadas
4.
Proc Natl Acad Sci U S A ; 104(49): 19482-7, 2007 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-18042714

RESUMO

Histone deacetylase (HDAC) inhibitors such as the phenyl hydroxamic acid PCI-24781 have emerged recently as a class of therapeutic agents for the treatment of cancer. Recent data showing synergy of HDAC inhibitors with ionizing radiation and other DNA-damaging agents have suggested that HDAC inhibitors may act, in part, by inhibiting DNA repair. Here we present evidence that HDAC enzymes are important for homologous recombinational repair of DNA double-strand breaks. Combination studies of PCI-24781 with the poly(ADP-ribose) polymerase inhibitor PJ34, an agent thought to produce lesions repaired by homologous recombination (HR), resulted in a synergistic effect on apoptosis. Immunofluorescence analysis demonstrated that HDAC inhibition caused a complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigations revealed that inhibition of HDAC enzymes by PCI-24781 led to a significant reduction in the transcription of genes specifically associated with HR, including RAD51. RAD51 protein levels were significantly decreased after 24 h of drug exposure both in vitro and in vivo. Consistent with inhibition of HR, treatment with PCI-24781 resulted in a decreased ability to perform homology directed repair of I-SceI-induced chromosome breaks in transfected CHO cells. In addition, an enhancement of cell killing was observed in Ku mutant cells lacking functional nonhomologous end joining compared with WT cells. Together these results demonstrate that HDAC enzymes are critically important to enable functional HR by controlling the expression of HR-related genes and promoting the proper assembly of HR-directed subnuclear foci.


Assuntos
Benzofuranos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Rad51 Recombinase/metabolismo , Radiossensibilizantes/farmacologia , Animais , Apoptose , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fenantrenos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Rad51 Recombinase/genética , Tolerância a Radiação/efeitos dos fármacos , Recombinação Genética/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima
5.
J Biol Chem ; 280(41): 34473-80, 2005 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-16091363

RESUMO

The p53 pathway is a central mediator of the apoptotic response. ASPP2/(53BP2L) (apoptosis-stimulating protein of p53 2, also known as 53BP2L) enhances apoptosis through selective stimulation of p53 transactivation of proapoptotic target genes. Although the Rb/E2F pathway regulates ASPP2/(53BP2L) transcription, the complex mechanisms controlling ASPP2/(53BP2L) levels and function remain unknown. We now report that proteasomal degradation modulates ASPP2/(53BP2L) protein levels and apoptotic function. Treatment of cells with proteasomal inhibitors, including the clinically utilized proteasomal inhibitor bortezomib, increases ASPP2/(53BP2L) protein but not RNA levels. Likewise, anthracycline-based chemotherapy, which has multiple mechanisms of action, including proteasomal inhibition, increases ASPP2/(53BP2L) protein but not RNA levels. Proteasomal inhibition or anthracycline treatment increases ASPP2/(53BP2L) protein stability and half-life. Furthermore, the central region of the ASPP2/(53BP2L) protein is ubiquitinated as would be expected for a proteasomal substrate. More importantly, small interfering RNA knockdown of ASPP2/(53BP2L) levels attenuated bortezomib-induced apoptosis, and this effect was greater in wild-type p53 cells. Because elevated levels of ASPP2/(53BP2L) are proapoptotic, these results described an important new molecular mechanism that modulates the p53-ASPP2/(53BP2L) apoptotic pathway.


Assuntos
Apoptose , Proteínas de Transporte/biossíntese , Proteínas de Transporte/fisiologia , Regulação Neoplásica da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Antraciclinas/farmacologia , Proteínas Reguladoras de Apoptose , Northern Blotting , Western Blotting , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fatores de Transcrição E2F/metabolismo , Humanos , Pirazinas/farmacologia , RNA/metabolismo , Interferência de RNA , Processamento Pós-Transcricional do RNA , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Tempo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo
6.
Circulation ; 108(24): 3042-7, 2003 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-14638548

RESUMO

BACKGROUND: NO is a major regulator of cardiovascular physiology that reduces vascular and cardiac contractility. Accumulating evidence indicates that endogenous inhibitors may regulate NOS. The NOS inhibitors asymmetric dimethylarginine (ADMA) and N-monomethylarginine are metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). This study was designed to determine if increased expression of DDAH could reduce tissue and plasma levels of the NOS inhibitors and thereby increase NO synthesis. METHODS AND RESULTS: We used gene transfer and transgenic approaches to overexpress human DDAH I in vitro and in vivo. The overexpression of DDAH in cultured endothelial cells in vitro induced a 2-fold increase in NOS activity and NO production. In the hDDAH-1 transgenic mice, we observed approximately 2-fold increases in tissue NOS activity and urinary nitrogen oxides, associated with a 2-fold reduction in plasma ADMA. The systolic blood pressure of transgenic mice was 13 mm Hg lower than that of wild-type controls (P<0.05). The systemic vascular resistance and cardiac contractility were decreased in response to the increase in NO production. CONCLUSIONS: DDAH I overexpression increases NOS activity in vitro and in vivo. The hDDAH-1 transgenic animal exhibits a reduced systolic blood pressure, systemic vascular resistance, and cardiac stroke volume. This study provides compelling evidence that the elaboration and metabolism of endogenous ADMA plays an important role in regulation of NOS activity.


Assuntos
Amidoidrolases/metabolismo , Arginina/análogos & derivados , Óxido Nítrico/biossíntese , Amidoidrolases/genética , Animais , Arginina/sangue , Células Cultivadas , Endotélio/metabolismo , Hemodinâmica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase/metabolismo , Transfecção
7.
DNA Repair (Amst) ; 2(9): 947-54, 2003 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-12967652

RESUMO

In response to a variety of types of DNA damage, the p53 tumor suppressor gene product is activated and regulates a number of downstream cellular processes such as cell cycle arrest, apoptosis and DNA repair. Recent discoveries concerning the regulation of DNA repair processes by p53, such as nucleotide excision repair (NER) and base excision repair (BER) have paved the way for studies to understand the mechanisms governing p53-dependent DNA repair. Although several theories have been proposed, accumulating evidence points to a transcriptional regulatory role for p53 in NER, mediating expression of the global genomic repair (GGR)-specific damage recognition genes, DDB2 and XPC. In BER, a more direct role for p53 has been proposed, potentially acting through protein-protein interactions with BER specific factors. These advances have greatly enhanced our understanding of the role of p53 in DNA repair and this review comprehensively summarizes current opinions on the mechanisms of p53-dependent DNA repair.


Assuntos
Dano ao DNA , Reparo do DNA/genética , Proteína Supressora de Tumor p53/fisiologia , Proteína BRCA1/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo
8.
DNA Repair (Amst) ; 2(7): 819-26, 2003 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-12826282

RESUMO

The tumor suppressor protein p53 functions in many cellular responses to UV-induced DNA damage, including activating the global nucleotide excision repair (NER) pathway. A potential mechanism for the effect on NER is through the ability of p53 to transcriptionally regulate genes that are directly involved in NER. DDB2 is one such gene that is regulated by p53 at both the basal and UV inducible levels. In order to further understand p53's role in NER, we transfected and selected clones that stably overexpress DDB2 in a human p53 deficient cell line. Global genomic repair (GGR) of cyclobutane pyrimidine dimers was significantly increased in the DDB2 expressing cells in comparison to controls, demonstrating that p53 wt protein itself is not directly required for efficient GGR. The protein product of DDB2, p48, is also post-translationally regulated by proteasomal degradation in response to UV irradiation. The regulation of p48 at both the transcriptional level by p53, and post-translationally by the proteasome suggests that p48 may be a rate limiting component of NER.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genes p53/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Humanos , Immunoblotting , Dímeros de Pirimidina/metabolismo , Raios Ultravioleta
9.
Proc Natl Acad Sci U S A ; 99(20): 12985-90, 2002 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-12242345

RESUMO

The p53 tumor suppressor gene product is a transcription factor involved in cell-cycle regulation, apoptosis, and DNA repair. We and others have shown that p53 is required for efficient nucleotide excision repair (NER) of UV-induced DNA lesions. p53-deficient cells are defective in the repair of UV photoproducts in genomic DNA but proficient for transcription-coupled repair. Therefore, we examined whether p53 regulates the expression of genes required for global genomic repair. In this study, we demonstrate that the mRNA and protein products of the xeroderma pigmentosum group C (XPC) gene are UV-inducible in a time- and dose-dependent manner in human WI38 fibroblasts and HCT116 colorectal cancer cells wild type for p53. However, no significant induction of XPC was observed in p53-deficient counterparts to these cells. Furthermore, regulated expression of wild-type p53 in p53 null Li-Fraumeni syndrome human fibroblasts significantly augmented the expression of XPC protein. Analysis of the human XPC gene sequence revealed a putative p53 response element in the XPC promoter that was capable of mediating sequence-specific DNA binding to p53 in vitro. These results provide strong evidence that the NER gene XPC is a DNA damage-inducible and p53-regulated gene and likely plays a role in the p53-dependent NER pathway.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/genética , Genes p53 , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Western Blotting , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica , Células Tumorais Cultivadas , Raios Ultravioleta
10.
Circulation ; 106(8): 987-92, 2002 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-12186805

RESUMO

BACKGROUND: An endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), is elevated in patients with type 2 diabetes mellitus (DM). This study explored the mechanisms by which ADMA becomes elevated in DM. METHODS AND RESULTS: Male Sprague-Dawley rats were fed normal chow or high-fat diet (n=5 in each) with moderate streptozotocin injection to induce type 2 DM. Plasma ADMA was elevated in diabetic rats (1.33+/-0.31 versus 0.48+/-0.08 micromol/L; P<0.05). The activity, but not the expression, of dimethylarginine dimethylaminohydrolase (DDAH) was reduced in diabetic rats and negatively correlated with their plasma ADMA levels (P<0.05). DDAH activity was significantly reduced in vascular smooth muscle cells and human endothelial cells (HMEC-1) exposed to high glucose (25.5 mmol/L). The impairment of DDAH activity in vascular cells was associated with an accumulation of ADMA and a reduction in generation of cGMP. In human endothelial cells, coincubation with the antioxidant polyethylene glycol-conjugated superoxide dismutase (22 U/mL) reversed the effects of the high-glucose condition on DDAH activity, ADMA accumulation, and cGMP synthesis. CONCLUSIONS: A glucose-induced impairment of DDAH causes ADMA accumulation and may contribute to endothelial vasodilator dysfunction in DM.


Assuntos
Amidoidrolases/fisiologia , Arginina/análogos & derivados , Arginina/fisiologia , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Aorta/enzimologia , Arginina/sangue , Arginina/metabolismo , Linhagem Celular , Células Cultivadas , GMP Cíclico/biossíntese , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/sangue , Glucose/farmacologia , Humanos , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Estresse Oxidativo , Polietilenoglicóis/farmacologia , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/farmacologia
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