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1.
Nat Commun ; 12(1): 5111, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433825

RESUMO

Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sistemas CRISPR-Cas/efeitos dos fármacos , Morfolinas/farmacologia , Mutagênese Insercional/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Tioxantenos/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Edição de Genes , Humanos , Deleção de Sequência/efeitos dos fármacos
2.
Neurology ; 94(21): e2270-e2282, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32139505

RESUMO

OBJECTIVE: To report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping. METHODS: Part 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry. RESULTS: Twelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%-4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry. CONCLUSION: Golodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization. CLINICALTRIALSGOV IDENTIFIER: NCT02310906. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.


Assuntos
Distrofina/biossíntese , Distrofia Muscular de Duchenne/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Administração Intravenosa , Adolescente , Criança , Relação Dose-Resposta a Droga , Método Duplo-Cego , Distrofina/genética , Imunofluorescência , Humanos , Masculino , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/genética , Deleção de Sequência/efeitos dos fármacos
3.
Mutagenesis ; 34(5-6): 421-429, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31676900

RESUMO

The tetrahydrofuran-type abasic site analog (THF) induces large deletion mutations in human cells. To compare the large deletions induced by THF on leading and lagging strand templates, plasmid DNAs bearing the analog at a specific position outside the supF gene were introduced into human U2OS cells. The replicated DNAs recovered from the transfected cells were electroporated into an Escherichia coli indicator strain. THF on the lagging strand template produced more supF mutants than THF on the leading strand template. This unequal mutagenicity was due to the higher frequencies of not only large deletions but also untargeted base substitutions induced in the gene. These results suggested that both types of mutations occur more frequently when abasic sites are formed on the lagging strand template.


Assuntos
Deleção de Sequência/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Humanos , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Mutagênicos/farmacologia , Plasmídeos/genética , Deleção de Sequência/efeitos dos fármacos , Transfecção/métodos
4.
FASEB J ; 33(7): 8634-8647, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31090455

RESUMO

Reduced expression of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) in humans and mice causes white matter inflammation and catatonic signs. These consequences are experimentally alleviated by microglia ablation via colony-stimulating factor 1 receptor (CSF1R) inhibition using PLX5622. Here we address for the first time preclinical topics crucial for translation, most importantly 1) the comparison of 2 long-term PLX5622 applications (prevention and treatment) vs. 1 treatment alone, 2) the correlation of catatonic signs and executive dysfunction, 3) the phenotype of leftover microglia evading depletion, and 4) the role of intercellular interactions for efficient CSF1R inhibition. Based on our Cnp-/- mouse model and in vitro time-lapse imaging, we report the unexpected discovery that microglia surviving under PLX5622 display a highly inflammatory phenotype including aggressive premortal phagocytosis of oligodendrocyte precursor cells. Interestingly, ablating microglia in vitro requires mixed glial cultures, whereas cultured pure microglia withstand PLX5622 application. Importantly, 2 extended rounds of CSF1R inhibition are not superior to 1 treatment regarding any readout investigated (magnetic resonance imaging and magnetic resonance spectroscopy, behavior, immunohistochemistry). Catatonia-related executive dysfunction and brain atrophy of Cnp-/- mice fail to improve under PLX5622. To conclude, even though microglia depletion is temporarily beneficial and worth pursuing, complementary treatment strategies are needed for full and lasting recovery.-Fernandez Garcia-Agudo, L., Janova, H., Sendler, L. E., Arinrad, S., Steixner, A. A., Hassouna, I., Balmuth, E., Ronnenberg, A., Schopf, N., van der Flier, F. J., Begemann, M., Martens, H., Weber, M. S., Boretius, S., Nave, K.-A., Ehrenreich, H. Genetically induced brain inflammation by Cnp deletion transiently benefits from microglia depletion.


Assuntos
2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/genética , Encéfalo/patologia , Encefalite/genética , Microglia/patologia , Deleção de Sequência/genética , Adulto , Animais , Encéfalo/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Fenótipo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Deleção de Sequência/efeitos dos fármacos
5.
Appl Microbiol Biotechnol ; 103(3): 1465-1474, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30607491

RESUMO

Markerless genetic engineering of bacterial genomes is commonly performed by two-step homologous recombination methods using vectors carrying flanking regions of the target gene for site-specific vector integration and counterselection markers to provide positive selection pressure on the second recombination step resulting in vector excision. Here, we provide the proof-of-principle of a novel counterselection method that exploits antagonistic activities between bactericidal and bacteriostatic antibiotics and which can provide selection pressure on the second recombination step by selective killing of bacteria retaining the antibiotic selection marker. This method was optimized for the bacterial pathogens Listeria monocytogenes and Neisseria meningitidis by screening for antagonistic activities between the bactericidal aminoglycosides kanamycin, streptomycin, and gentamicin in combination with the bacteriostatic antibiotics chloramphenicol and erythromycin. The largest difference in selective killing of both L. monocytogenes and N. meningitidis containing an antibiotic selection marker versus wild-type bacteria was observed for the combination of erythromycin, gentamicin, and ermC as antibiotic selection marker. Therefore, this combination was used to generate two markerless deletion mutants for both L. monocytogenes and N. meningitidis. After applying the dual-antimicrobial selection pressure on cultures during the second recombination step, surviving colonies were replica plated on agar with and without erythromycin. On average, 12-13% of the randomly selected bacterial colonies had lost the selection marker due to a second recombination event and approximately half of these colonies were the desired markerless in-frame deletion mutants. Therefore, this method proved to be easy and fast and should be applicable to a wide variety of bacterial species.


Assuntos
Antibacterianos/farmacologia , Sequência de Bases/genética , Engenharia Genética/métodos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/genética , Deleção de Sequência/efeitos dos fármacos , Cloranfenicol/farmacologia , Combinação de Medicamentos , Eritromicina/farmacologia , Genoma Bacteriano/genética , Gentamicinas/farmacologia , Canamicina/farmacologia , Metiltransferases/genética , Deleção de Sequência/genética , Estreptomicina/farmacologia
6.
J Steroid Biochem Mol Biol ; 185: 200-211, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30194976

RESUMO

Bisphenol A (BPA), an endocrine-disrupting chemical, is capable of producing reproductive toxicity. BPA results in mitochondrial DNA (mtDNA) deletion and mitochondrial dysfunction; however, the effect of BPA on the mitochondria of ovarian granulosa cells is not clear. Further, 1,25-dihydroxyvitamin D3 (1,25D3) may play a role in reproduction, because its receptor, VDR, contributes to the inhibition of oxidative stress and predominantly exists in the nuclei of granulosa cells. Hence, the role of 1,25D3 in BPA-mediated effects on mitochondrial function was examined in this study. Primary rat granulosa cells treated with BPA, 1,25D3, or both were subjected to molecular/biochemical assays to measure cell survival, mtDNA content, mtDNA deletion, superoxide dismutase activity, levels of proteins related to mitochondrial biogenesis, and mitochondrial function. We found that cell viability was dose-dependently reduced and reactive oxygen species (ROS) levels were increased by BPA treatment. BPA administration elevated Mn-superoxide dismutase (MnSOD) expression but negatively regulated total SOD activity. 1,25D3 treatment alone increased 17ß-estradiol secretion, ATP production, and cellular oxygen consumption. In cells treated with both agents, 1,25D3 enhanced BPA-induced MnSOD protein upregulation and blocked the BPA-mediated decline in total SOD activity. Furthermore, 1,25D3 attenuated BPA-mediated mtDNA deletion but showed no effect on BPA-induced increases in mtDNA content. Although BPA had no influence on the levels of peroxisome proliferator-activated receptor-γ coactivator-1 α, nuclear respiratory factor-1, mitochondrial transcription factor A, or cytochrome c oxidase subunit IV, 1,25D3 plus BPA markedly increased mitochondrial biogenesis-related protein expression via the PI3K-Akt pathway. Moreover, BPA-mediated negative regulation of cytochrome c oxidase subunit I levels and 17ß-estradiol secretion was attenuated by 1,25D3 pre-treatment. Our results suggest that 1,25D3 attenuates BPA-induced decreases in 17ß-estradiol and that treatment with 1,25D3 plus BPA regulates granulosa cell mitochondria by elevating mitochondrial biogenesis-related protein levels.


Assuntos
Compostos Benzidrílicos/toxicidade , Calcitriol/farmacologia , Disruptores Endócrinos/toxicidade , Estradiol/metabolismo , Células da Granulosa/metabolismo , Mitocôndrias/patologia , Fenóis/toxicidade , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores de Calcitriol/metabolismo , Deleção de Sequência/efeitos dos fármacos , Deleção de Sequência/genética , Superóxido Dismutase/metabolismo
7.
G3 (Bethesda) ; 9(1): 61-71, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30389796

RESUMO

In at least some unicellular organisms, mutation rates are temporarily raised upon exposure to environmental stress, potentially contributing to the evolutionary response to stress. Whether this is true for multicellular organisms, however, has received little attention. This study investigated the effects of chronic mild stress, in the form of low-level copper and nickel exposure, on mutational processes in Daphnia pulex using a combination of mutation accumulation, whole genome sequencing and life-history assays. After over 100 generations of mutation accumulation, we found no effects of metal exposure on the rates of single nucleotide mutations and of loss of heterozygosity events, the two mutation classes that occurred in sufficient numbers to allow statistical analysis. Similarly, rates of decline in fitness, as measured by intrinsic rate of population increase and of body size at first reproduction, were negligibly affected by metal exposure. We can reject the possibility that Daphnia were insufficiently stressed to invoke genetic responses as we have previously shown rates of large-scale deletions and duplications are elevated under metal exposure in this experiment. Overall, the mutation accumulation lines did not significantly depart from initial values for phenotypic traits measured, indicating the lineage used was broadly mutationally robust. Taken together, these results indicate that the mutagenic effects of chronic low-level exposure to these metals are restricted to certain mutation classes and that fitness consequences are likely minor and therefore unlikely to be relevant in determining the evolutionary responses of populations exposed to these stressors.


Assuntos
Daphnia/genética , Aptidão Genética/genética , Genoma/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Animais , Cobre/toxicidade , Daphnia/efeitos dos fármacos , Aptidão Genética/efeitos dos fármacos , Mutação/efeitos dos fármacos , Acúmulo de Mutações , Taxa de Mutação , Níquel/toxicidade , Reprodução/genética , Deleção de Sequência/efeitos dos fármacos
8.
Am J Med Sci ; 356(5): 492-498, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30177262

RESUMO

Thrombotic microangiopathy (TMA) may result from a variety of clinical conditions, including thrombotic thrombocytopenic purpura, Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome and complement-mediated hemolytic uremic syndrome. Thrombocytopenic purpura is diagnosed when ADAMTS13 is <10%, while a diagnosis of Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome is made with the evidence of infection by Shiga toxin-producing Escherichia coli. Diagnosis of complement-mediated hemolytic uremic syndrome is not dependent on a specific laboratory test and is a diagnosis of exclusion. TMA is a rare disease and finding individuals that have more than 1 concurrent etiology leading to TMA is even more rare. Here we describe the presentation and management of an individual with CFHR1 deletion-associated TMA also found to have a positive stool Shiga toxin. We discuss the significance of Shiga toxin in serving as a trigger for development of TMA in an individual predisposed to development of TMA due to presence of a homozygous deletion in CFHR1.


Assuntos
Sequência de Bases/efeitos dos fármacos , Proteínas Inativadoras do Complemento C3b/genética , Deleção de Sequência/efeitos dos fármacos , Toxina Shiga/efeitos adversos , Microangiopatias Trombóticas/genética , Adulto , Proteínas Inativadoras do Complemento C3b/metabolismo , Feminino , Homozigoto , Humanos , Microangiopatias Trombóticas/microbiologia
9.
Cancer Res ; 78(15): 4386-4395, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29844120

RESUMO

Homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene is a frequent event in a wide variety of human cancers and is a possible molecular target for therapy. One potential therapeutic strategy to target MTAP-deleted tumors involves combining toxic purine analogues such as 6'-thioguanine (6TG) or 2'-fluoroadenine (2FA) with the MTAP substrate 5'-deoxy-5'-methylthioadenosine (MTA). The rationale is that excess MTA will protect normal MTAP+ cells from purine analogue toxicity because MTAP catalyzes the conversion of MTA to adenine, which then inhibits the conversion of purine base analogues into nucleotides. However, in MTAP- tumor cells, no protection takes place because adenine is not formed. Here, we examine the effects of 6TG and 2FA in combination with MTA in vitro and in vivoIn vitro, MTA protected against both 6TG and 2FA toxicity in an MTAP-dependent manner, shifting the IC50 concentration by one to three orders of magnitude. However, in mice, MTA protected against toxicity from 2FA but failed to protect against 6TG. Addition of 100 mg/kg MTA to 20 mg/kg 2FA entirely reversed the toxicity of 2FA in a variety of tissues and the treatment was well tolerated by mice. The 2FA+MTA combination inhibited tumor growth of four different MTAP- human tumor cell lines in mouse xenograft models. Our results suggest that 2FA+MTA may be a promising combination for treating MTAP-deleted tumors.Significance: Loss of MTAP occurs in about 15% of all human cancers; the MTAP protection strategy presented in this study could be very effective in treating these cancers. Cancer Res; 78(15); 4386-95. ©2018 AACR.


Assuntos
Adenina/análogos & derivados , Desoxiadenosinas/farmacologia , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Tionucleosídeos/farmacologia , Adenina/metabolismo , Adenina/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Homozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células NIH 3T3 , Purina-Núcleosídeo Fosforilase/metabolismo , Deleção de Sequência/efeitos dos fármacos , Tioguanina/farmacologia
10.
Cell Mol Life Sci ; 74(1): 57-66, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27722768

RESUMO

Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Deleção de Sequência , Animais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/análise , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Descoberta de Drogas , Humanos , Fosforilação/efeitos dos fármacos , Mutação Puntual/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Deleção de Sequência/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
11.
PLoS Genet ; 12(10): e1006368, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27755535

RESUMO

For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs). This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3' overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles.


Assuntos
Caenorhabditis elegans/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Animais , Caenorhabditis elegans/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/biossíntese , Metanossulfonato de Etila/toxicidade , Estudos de Associação Genética , Genoma/efeitos dos fármacos , Mutagênese , Mutagênicos/toxicidade , Deleção de Sequência/efeitos dos fármacos , DNA Polimerase teta
12.
Metallomics ; 8(2): 228-35, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26688044

RESUMO

Arsenic is omnipresent in soil, air, food and water. Chronic exposure to arsenic is a serious problem to human health. In-depth understanding of this metalloid's toxicity is a fundamental step towards development of arsenic-free foods and measures for bioremediation. By screening the complete set of gene deletion mutants (4873) of Saccharomyces cerevisiae, this study uncovered 75 sensitive and 39 resistant mutants against arsenite [As(III)]. Functional analysis of the corresponding genes revealed the molecular details for its uptake, toxicity and detoxification. On the basis of the hypersensitivity of yap3Δ, the transcription factor, Yap3p, is for the first time linked to the cell's detoxification against As(III). Apart from confirming the previously described role of the mitogen-activated protein kinase (MAPK) Hog1 pathway in combating arsenic toxicity, the results show that the regulatory subunits (Ckb1p and Ckb2p) of protein kinase CK2 are also involved in the process, suggesting possible crosstalk between the two key protein kinases. The sensitivity to As(III) conferred by deletion of the genes involved in protein degradation and chromatin remodelling demonstrates protein damage is the key mode of toxicity for the metalloid. Furthermore, the resistant phenotype of fps1Δ, snf3Δ and pho81Δ against As(III) links arsenic uptake with the corresponding plasma membrane-bound transporters-aquaglyceroporin (Fps1p), hexose (Snf3p) and phosphate transporters. The molecular details obtained in this screen for As(III) uptake, detoxification and toxicity provide the basis for future investigations into arsenic-related problems in the environment, agriculture and human health.


Assuntos
Arsênio/toxicidade , Poluentes Ambientais/toxicidade , Genoma Fúngico/efeitos dos fármacos , Saccharomyces cerevisiae , Deleção de Sequência/efeitos dos fármacos , Deleção de Sequência/genética , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
13.
Anticancer Res ; 35(7): 3885-91, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26124334

RESUMO

AIM: Non-small cell lung cancer (NSCLC) with minor mutations in the epidermal growth factor receptor (EGFR) gene, except for the common 15 base-pair deletions in exon 19 and the L858R mutation in exon 21, is rare, and only few data exist on this patient population. The aim of the present study was to describe the clinical characteristics and to clarify the efficacy of EGFR-tyrosine kinase inhibitors (TKIs) in patients with NSCLC harboring minor mutations of the EGFR gene. PATIENTS AND METHODS: This was a multicenter, retrospective study that analyzed specimens from patients with NSCLC who had minor EGFR gene mutations and were treated with EGFR-TKIs between June 2002 and March 2012. RESULTS: Out of 56 patients with minor mutations of the EGFR gene, 44 were treated with either gefitinib or erlotinib. Mutation sites were G719X in exon 18 (n=35), L861Q in exon 21 (n=11), and G874S in exon 21 (n=1). Three patients had both the G719S and the L861Q mutation. The response rate to TKI treatment was 29.5%, and the disease control rate was 63.6%. The median progression-free survival (PFS) was 6.7 months [95% confidence interval (CI)=2.06-8.66 months]. The median PFS was 7.2 months (95% CI=4.23-12.3 months) in 32 patients who received first- or second-line treatment with EGFR-TKIs, whereas the median PFS was 1.57 months (95% CI=0.73-3.8 months) in 12 patients treated with EGFR-TKIs as a third-line or later treatment. In multivariate Cox analysis, erlotinib therapy was associated with a longer PFS than gefitinib (p=0.025). CONCLUSION: Patients with NSCLC harboring minor mutations of the EGFR gene exhibited a modest response to EGFR-TKI treatment. Treatment with first-generation EGFR-TKIs, in particular erlotinib, may be considered a first- or second-line option for patients with NSCLC with minor EGFR mutations.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Povo Asiático/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Intervalo Livre de Doença , Cloridrato de Erlotinib , Éxons/efeitos dos fármacos , Éxons/genética , Feminino , Gefitinibe , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Mutação/genética , Quinazolinas/uso terapêutico , Estudos Retrospectivos , Deleção de Sequência/efeitos dos fármacos , Deleção de Sequência/genética
14.
J Cancer Res Ther ; 11(2): 397-402, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26148607

RESUMO

AIM: BIM deletion polymorphism was deemed to be associated with downregulation of BIM, resulting in a decreased apoptosis induced by epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutation-positive non-small cell lung cancer (NSCLC). However, accumulating evidences concerning the association between BIM deletion polymorphism and efficacy of EGFR-TKI and survival in EGFR-mutation-driven NSCLC patient reported contradictory results. MATERIALS AND METHODS: A meta-analysis was conducted by combing six original eligible studies including 871 NSCLC patients. RESULTS: Our study showed that BIM deletion polymorphism was significantly associated with poor response to EGFR-TKI therapy in mutant EGFRNSCLC patients (P(h) = 0.309, P(z) = 0.001, OR = 0.39, 95% confidence interval (CI) = 0.23-0.67). Disease control rate (DCR) in mutant EGFRNSCLC patient with treatment of EGFR-TKI was significantly decreased in patients with BIM deletion polymorphism comparing to patients harbored BIM wild variant (P(h) = 0.583, P(Z) = 0.007, OR = 0.46, 95%CI = 0.25-0.85). EGFR mutation-derived NSCLC patient carrying BIM deletion polymorphism had a shorter progression-free survival (PFS; P(h) < 0.001, P(z) < 0.001, hazard ratio (HR) = 1.37, 95%CI = 1.09-1.71) and overall survival (OS; P(h) = 0.90, P(z) = 0.003, HR = 1.25, 95%CI = 1.08-1.45), than those harbored BIM wild variant. CONCLUSION: These results suggested that BIM deletion polymorphism might be a cause that contributes to primary EGFR-TKI resistance, and it could be used as a genetic predictor for EGFR-TKI outcome and an independent prognostic factor of EGFR mutation-driven NSCLC patient.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Polimorfismo Genético/genética , Inibidores de Proteínas Quinases/uso terapêutico , Antineoplásicos/uso terapêutico , Intervalo Livre de Doença , Humanos , Polimorfismo Genético/efeitos dos fármacos , Deleção de Sequência/efeitos dos fármacos , Deleção de Sequência/genética
15.
J Photochem Photobiol B ; 149: 164-71, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26083904

RESUMO

Inactivation of Escherichia coli K-12 was conducted by applying a continuous supplying of commercial H2O2 to mimic the H2O2 production in a photocatalytic system, and the contribution of H2O2 in photocatalytic inactivation was investigated using a modified "partition system" and five E. coli mutants. The concentration of exogenous H2O2 required for complete inactivation of bacterial cells was much higher than that produced in-situ in common photocatalytic system, indicating that H2O2 alone plays a minor role in photocatalytic inactivation. However, the concentration of exogenously produced H2O2 required for effective inactivation of E. coli K-12 was much lower when the light irradiation was applied. To further investigate the possible physiological changes, inactivation of E. coli BW25113 (the parental strain), and its corresponding isogenic single-gene deletion mutants with light pretreatment was compared. The results indicate that light irradiation increases the bacterial intracellular Fe(2+) level and favors hydroxyl radical (OH) production via the catalytic reaction of Fe(2+), leading to increase in DNA damage. Moreover, the results indicate that the properties of light source, such as intensity and major emission wavelength, may alter the physiology of bacterial cells and affect the susceptibility to in-situ resultant H2O2 in the photocatalytic inactivation processes, leading to significant influence on the photocatalytic inactivation efficiencies of E. coli K-12.


Assuntos
Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/efeitos da radiação , Peróxido de Hidrogênio/farmacologia , Luz , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Catálise , Meio Ambiente , Escherichia coli K12/genética , Deleção de Sequência/efeitos dos fármacos , Deleção de Sequência/efeitos da radiação , Especificidade da Espécie , Fatores de Tempo
17.
Lung Cancer ; 88(2): 181-6, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25726043

RESUMO

INTRODUCTION: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment is the standard therapy for non-small cell lung cancer (NSCLC) harbouring EGFR-activating mutations. The NEJ002 phase 3 clinical trial demonstrated the efficacy of EGFR-TKI; gefitinib was significantly superior in both progression-free survival (PFS) and objective response rate (ORR) than carboplatin plus paclitaxel. However, several cases showed no response. In this study, we performed further analysis of the characteristics of these non-responders. METHODS: Available data from NEJ002 on maximum changes in tumour size were obtained from 103 cases (90.4%) and 110 cases (96.5%) in the carboplatin-paclitaxel and gefitinib groups, respectively. Waterfall plots of maximum tumour size changes were created for non-responders. RESULTS: Five (4.9%) and 9 (8.2%) cases in the carboplatin-paclitaxel and gefitinib groups were non-responders, respectively. The mean pack years of the non-responders in the carboplatin-paclitaxel and gefitinib groups were 0.33 and 31.7, respectively. The ORR of total smokers (61.5%) and heavy smokers (over 40 pack years, 52.6%) in the gefitinib group were significantly lower compared to people who have never smoked (80.0%) (P=0.044 and P=0.020, respectively). Smoker cases also showed a tendency towards lower PFS and overall survival (OS). In addition, the EGFR common mutation types did not affect PFS and OS in gefitinib-treated cases in NEJ002. However, in this study, the ORR and waterfall plots showed that gefitinib-treated non-responders who had a deletion in exon 19 in the EGFR gene exhibited a tendency towards a higher response compared to those with a L858R mutation. CONCLUSIONS: NSCLC patients with a smoking history or the EGFR L858R mutation may demonstrate a poorer response to gefitinib treatment.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Fumar/efeitos adversos , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/genética , Ensaios Clínicos Fase III como Assunto , Intervalo Livre de Doença , Receptores ErbB/antagonistas & inibidores , Éxons/efeitos dos fármacos , Éxons/genética , Feminino , Gefitinibe , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Mutação/genética , Paclitaxel/administração & dosagem , Estudos Retrospectivos , Deleção de Sequência/efeitos dos fármacos , Deleção de Sequência/genética , Resultado do Tratamento
18.
Am J Physiol Renal Physiol ; 306(5): F561-7, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24402098

RESUMO

Epithelial Na(+) channel (ENaC) subunits (α, ß, and γ) found in functional complexes are translated from mature mRNAs that are similarly processed by the inclusion of 13 canonical exons. We examined whether individual exons 3-12, encoding the large extracellular domain, are required for functional channel expression. Human ENaCs with an in-frame deletion of a single α-subunit exon were expressed in Xenopus oocytes, and their functional properties were examined by two-electrode voltage clamp. With the exception of exon 11, deletion of an individual exon eliminated channel activity. Channels lacking α-subunit exon 11 were hyperactive. Oocytes expressing this mutant exhibited fourfold greater amiloride-sensitive whole cell currents than cells expressing wild-type channels. A parallel fivefold increase in channel open probability was observed with channels lacking α-subunit exon 11. These mutant channels also exhibited a lost of Na(+) self-inhibition, whereas we found similar levels of surface expression of mutant and wild-type channels. In contrast, in-frame deletions of exon 11 from either the ß- or γ-subunit led to a significant loss of channel activity, in association with a marked decrease in surface expression. Our results suggest that exon 11 within the three human ENaC genes encodes structurally homologous yet functionally diverse domains and that exon 11 in the α-subunit encodes a module that regulates channel gating.


Assuntos
Canais Epiteliais de Sódio/genética , Deleção de Sequência , Xenopus laevis/metabolismo , Amilorida/farmacologia , Animais , Células Cultivadas , Canais Epiteliais de Sódio/metabolismo , Éxons , Humanos , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Mutação Puntual/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Deleção de Sequência/efeitos dos fármacos , Xenopus laevis/genética
19.
Mutagenesis ; 29(1): 27-36, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24243707

RESUMO

Ochratoxin A (OTA) is a carcinogen targeting proximal tubules at the renal outer medulla (ROM) in rodents. We previously reported that OTA increased mutant frequencies of the red/gam gene (Spi(-)), primarily deletion mutations. In the present study, Spi(-) assays and mutation spectrum analyses in the Spi(-) mutants were performed using additional samples collected in our previous study. Spi(-) assay results were similar to those in our previous study, revealing large (>1kb) deletion mutations in the red/gam gene. To clarify the molecular progression from DNA damage to gene mutations, in vivo comet assays and analysis of DNA damage/repair-related mRNA and/or protein expression was performed using the ROM of gpt delta rats treated with OTA at 70, 210 or 630 µg/kg/day by gavage for 4 weeks. Western blotting and immunohistochemical staining demonstrated that OTA increased γ-H2AX expression specifically at the carcinogenic target site. In view of the results of comet assays, we suspected that OTA was capable of inducing double-strand breaks (DSBs) at the target sites. mRNA and/or protein expression levels of homologous recombination (HR) repair-related genes (Rad51, Rad18 and Brip1), but not nonhomologous end joining-related genes, were increased in response to OTA in a dose-dependent manner. Moreover, dramatic increases in the expression of genes involved in G2/M arrest (Chek1 and Wee1) and S/G2 phase (Ccna2 and Cdk1) were observed, suggesting that DSBs induced by OTA were repaired predominantly by HR repair, possibly due to OTA-specific cell cycle regulation, consequently producing large deletion mutations at the carcinogenic target site.


Assuntos
Carcinógenos/toxicidade , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Ocratoxinas/toxicidade , Deleção de Sequência/efeitos dos fármacos , Animais , Sequência de Bases , Peso Corporal/efeitos dos fármacos , Carcinógenos/administração & dosagem , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Ensaio Cometa , Proteínas de Escherichia coli/genética , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Testes de Mutagenicidade/métodos , Ocratoxinas/administração & dosagem , Tamanho do Órgão , Pentosiltransferases/genética , Ratos , Ratos Transgênicos
20.
Chem Biol ; 20(7): 943-55, 2013 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-23890012

RESUMO

Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.


Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Deleção de Sequência/efeitos dos fármacos , Temperatura , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Sinergismo Farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Cinética , Modelos Moleculares , Nucleotídeos/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína
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