RESUMO
Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.
Assuntos
Hidrolases Anidrido Ácido/deficiência , Deficiência Intelectual/patologia , Microcefalia/patologia , Hipotonia Muscular/patologia , Mutação , Transtornos do Neurodesenvolvimento/patologia , Monoéster Fosfórico Hidrolases/genética , Alelos , Animais , Pré-Escolar , Feminino , Humanos , Lactente , Deficiência Intelectual/etiologia , Deficiência Intelectual/metabolismo , Masculino , Camundongos , Microcefalia/etiologia , Microcefalia/metabolismo , Hipotonia Muscular/etiologia , Hipotonia Muscular/metabolismo , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/metabolismo , Linhagem , FenótipoRESUMO
Desbuquois dysplasia (DD) type 1 is a rare skeletal dysplasia characterized by a short stature, round face, progressive scoliosis, and joint laxity. The causative gene has been identified as calcium-activated nucleotidase 1 (CANT1), which encodes a nucleotidase that preferentially hydrolyzes UDP to UMP and phosphate. In this study, we generated Cant1 KO mice using CRISPR/Cas9-mediated genome editing. All F0 mice possessing frameshift deletions at both Cant1 alleles exhibited a dwarf phenotype. Germline transmission of the edited allele was confirmed in an F0 heterozygous mouse, and KO mice were generated by crossing of the heterozygous breeding pairs. Cant1 KO mice exhibited skeletal defects, including short stature, thoracic kyphosis, and delta phalanx, all of which are observed in DD type 1 patients. The glycosaminoglycan (GAG) content and extracellular matrix space were reduced in the growth plate cartilage of mutants, and proliferating chondrocytes lost their typical flat shape and became round. Chondrocyte differentiation, especially terminal differentiation to hypertrophic chondrocytes, was impaired in Cant1 KO mice. These findings indicate that CANT1 is involved in the synthesis of GAG and regulation of chondrocyte differentiation in the cartilage and contribute to a better understanding of the pathogenesis of DD type 1.
Assuntos
Hidrolases Anidrido Ácido/deficiência , Anormalidades Craniofaciais/genética , Nanismo/genética , Lâmina de Crescimento/patologia , Instabilidade Articular/genética , Ossificação Heterotópica/genética , Polidactilia/genética , Hidrolases Anidrido Ácido/genética , Animais , Diferenciação Celular/genética , Condrócitos/patologia , Anormalidades Craniofaciais/diagnóstico , Anormalidades Craniofaciais/patologia , Modelos Animais de Doenças , Nanismo/diagnóstico , Nanismo/patologia , Feminino , Glicosaminoglicanos/biossíntese , Lâmina de Crescimento/citologia , Lâmina de Crescimento/crescimento & desenvolvimento , Humanos , Instabilidade Articular/diagnóstico , Instabilidade Articular/patologia , Masculino , Camundongos , Camundongos Knockout , Ossificação Heterotópica/diagnóstico , Ossificação Heterotópica/patologia , Polidactilia/diagnóstico , Polidactilia/patologia , Esqueleto/diagnóstico por imagem , Esqueleto/crescimento & desenvolvimentoRESUMO
Genome instability is an enabling characteristic of cancer that facilitates the acquisition of oncogenic mutations that drive tumorigenesis. Underlying much of the instability in cancer is DNA replication stress, which causes both chromosome structural changes and single base-pair mutations. Common fragile sites are some of the earliest and most frequently altered loci in tumors. Notably, the fragile locus, FRA3B, lies within the fragile histidine triad (FHIT) gene, and consequently deletions within FHIT are common in cancer. We review the evidence in support of FHIT as a DNA caretaker and discuss the mechanism by which FHIT promotes genome stability. FHIT increases thymidine kinase 1 (TK1) translation to balance the deoxyribonucleotide triphosphates (dNTPs) for efficient DNA replication. Consequently, FHIT-loss causes replication stress, DNA breaks, aneuploidy, copy-number changes (CNCs), small insertions and deletions, and point mutations. Moreover, FHIT-loss-induced replication stress and DNA breaks cooperate with APOBEC3B overexpression to catalyze DNA hypermutation in cancer, as APOBEC family enzymes prefer single-stranded DNA (ssDNA) as substrates and ssDNA is enriched at sites of both replication stress and DNA breaks. Consistent with the frequent loss of FHIT across a broad spectrum of cancer types, FHIT-deficiency is highly associated with the ubiquitous, clock-like mutation signature 5 occurring in all cancer types thus far examined. The ongoing destabilization of the genome caused by FHIT loss underlies recurrent inactivation of tumor suppressors and activation of oncogenes. Considering that more than 50% of cancers are FHIT-deficient, we propose that FRA3B/FHIT fragility shapes the mutational landscape of cancer genomes.
Assuntos
Hidrolases Anidrido Ácido/genética , Instabilidade Genômica , Proteínas de Neoplasias/genética , Neoplasias/genética , Hidrolases Anidrido Ácido/deficiência , Animais , Sítios Frágeis do Cromossomo , Replicação do DNA , Humanos , Proteínas de Neoplasias/deficiênciaRESUMO
Expression of Fhit and Wwox protein is frequently lost or reduced in many human cancers. In this report, we provide data that further characterizes the molecular consequences of Fhit loss in the initiation of DNA double-strand breaks (DSBs), and of Wwox loss in altered repair of DSBs. We show that loss of Fhit initiates mild genome instability in early passage mouse kidney cells, confirming that DNA damage associated with Fhit-deficiency is not limited to cancer cells. We also demonstrate that the cause of Fhit-deficient DSBs: thymidine deficiency-induced replication stress, can be resolved with thymidine supplementation in early passage mouse kidney cells before extensive genome instability occurs. As for consequences of Wwox loss in cancer, we show in a small panel of breast cancer cells and mouse embryonic fibroblasts that Wwox expression predicts response to radiation and mitomycin C, all agents that cause DSBs. In addition, loss of Wwox significantly reduced progression free survival in a cohort of ovarian cancer patients treated with platin-based chemotherapies. Finally, stratification of a cohort of squamous lung cancers by Fhit expression reveals that Wwox expression is significantly reduced in the low Fhit-expressing group, suggesting that loss of Fhit is quickly succeeded by loss of Wwox. We propose that Fhit and Wwox loss work synergistically in cancer progression and that DNA damage caused by Fhit could be targeted early in cancer initiation for prevention, while DNA damage caused by Wwox loss could be targeted later in cancer progression, particularly in cancers that develop resistance to genotoxic therapies.
Assuntos
Hidrolases Anidrido Ácido/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/genética , Hidrolases Anidrido Ácido/deficiência , Animais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Camundongos , Proteínas de Neoplasias/deficiência , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Transdução de Sinais , Análise de Sobrevida , Proteínas Supressoras de Tumor/deficiência , Oxidorredutase com Domínios WW/deficiênciaRESUMO
Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.
Assuntos
Hidrolases Anidrido Ácido/genética , Antituberculosos/farmacologia , Farmacorresistência Bacteriana/genética , Isoniazida/farmacologia , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Hidrolases Anidrido Ácido/deficiência , Animais , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Ciclo do Ácido Cítrico/genética , Clofazimina/farmacologia , Modelos Animais de Doenças , Expressão Gênica , Glicerofosfatos/metabolismo , Isoenzimas/deficiência , Isoenzimas/genética , Meropeném , Camundongos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Naftoquinonas/farmacologia , Fosfotransferases (Aceptor do Grupo Fosfato)/deficiência , Polifosfatos/metabolismo , Tienamicinas/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/prevenção & controle , Vacinas de DNA/administração & dosagem , Xilose/análogos & derivados , Xilose/metabolismoRESUMO
The FHIT gene, encompassing an active common fragile site, FRA3B, is frequently silenced in preneoplasia and cancer, through gene rearrangement or methylation of regulatory sequences. Silencing of Fhit protein expression causes thymidine kinase 1 downregulation, resulting in dNTP imbalance, and spontaneous replication stress that leads to chromosomal aberrations, allele copy number variations, insertions/deletions, and single-base substitutions. Thus, Fhit, which is reduced in expression in the majority of human cancers, is a genome "caretaker" whose loss initiates genome instability in preneoplastic lesions. To follow the early genetic alterations and functional changes induced by Fhit loss that may recapitulate the neoplastic process in vitro, we established epithelial cell lines from kidney tissues of Fhit-/- and +/+ mouse pups early after weaning, and subjected cell cultures to nutritional and carcinogen stress, which +/+ cells did not survive. Through transcriptome profiling and protein expression analysis, we observed changes in the Trp53/p21 and survivin apoptotic pathways in -/- cells, and in expression of proteins involved in epithelial-mesenchymal transition. Some Fhit-deficient cell lines showed anchorage-independent colony formation and increased invasive capacity in vitro. Furthermore, cells of stressed Fhit-/- cell lines formed s.c. and metastatic tumors in nude mice. Collectively, we show that Fhit loss and subsequent thymidine kinase 1 inactivation, combined with selective pressures, leads to neoplasia-associated alterations in genes and gene expression patterns in vitro and in vivo.
Assuntos
Hidrolases Anidrido Ácido/deficiência , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Progressão da Doença , Proteínas de Neoplasias/deficiência , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Apoptose/genética , Movimento Celular/genética , Células Cultivadas , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/genética , Timidina Quinase/genética , Fatores de Tempo , Transcrição GênicaRESUMO
Inadequate dietary Zn consumption increases susceptibility to esophageal and other cancers in humans and model organisms. Since Zn supplementation can prevent cancers in rodent squamous cell carcinoma (SCC) models, we were interested in determining if it could have a preventive effect in a rodent skin cancer model, as a preclinical basis for considering a role for Zn in prevention of human nonmelanoma skin cancers, the most frequent cancers in humans. We used the 7,12-dimethyl benzanthracene carcinogen/phorbol myristate acetate tumor promoter treatment method to induce skin tumors in Zn-sufficient wild-type and Fhit (human or mouse protein) knockout mice. Fhit protein expression is lost in >50% of human cancers, including skin SCCs, and Fhit-deficient mice show increased sensitivity to carcinogen induction of tumors. We hypothesized that: (1) the skin cancer burdens would be reduced by Zn supplementation; (2) Fhit(-/-) (Fhit, murine fragile histidine triad gene) mice would show increased susceptibility to skin tumor induction versus wild-type mice. 30 weeks after initiating treatment, the tumor burden was increased ~2-fold in Fhit(-/-) versus wild-type mice (16.2 versus 7.6 tumors, P < 0.001); Zn supplementation significantly reduced tumor burdens in Fhit(-/-) mice (males and females combined, 16.2 unsupplemented versus 10.3 supplemented, P = 0.001). Most importantly, the SCC burden was reduced after Zn supplementation in both strains and genders of mice, most significantly in the wild-type males (P = 0.035). Although the mechanism(s) of action of Zn supplementation in skin tumor prevention is not known in detail, the Zn-supplemented tumors showed evidence of reduced DNA damage and some cohorts showed reduced inflammation scores. The results suggest that mild Zn supplementation should be tested for prevention of skin cancer in high-risk human cohorts.
Assuntos
Carcinoma de Células Escamosas/patologia , Suplementos Nutricionais , Neoplasias Cutâneas/patologia , Pele/efeitos dos fármacos , Pele/patologia , Zinco/farmacologia , Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Animais , Carcinoma de Células Escamosas/etiologia , Dano ao DNA , Modelos Animais de Doenças , Feminino , Inflamação/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Neoplasias Cutâneas/etiologia , Carga TumoralRESUMO
In this work, the impact of space weather on the staining and the motility of volutin granules ("dancing bodies") in vacuoles of yeast Saccharoniyces cerevisiae with normal and changed phosphoric metabolism was studied. It was showed that the installed ear- lier relation between metachromatic reaction of volutin granules and galactic cosmic rays was confirmed by DAPI-staining of inorganic polyphosphates in cells S. cerevisiae UCM Y-517. The inverse correlation of efficiency of DAPI-staining of volutin granules with some solar activity indexes was observed. During the period of study, the rhythmicity of motile volutin granules in the studied cultures (S. cerevisiae UCM Y-517, S. cerevisiae CRY, S. cerevisiae CNX) was marked. The strains S. cerevisiae UCM Y-517 and CRY, both with unmodified phosphoric metabolism, showed the same rhythmicity of mobile volutin granules. However, this rhythmicity in the strain CNX, which cannot synthesize exopolyphosphatases PPX1 and PPN1 (CF 3.6.1.11), was changed. The volutin granule motion coincided with the rhythmicity of galactic cosmic rays within the period of 9 days. It can be suggested that "dancing bodies" are synchronized with rhythms of galactic cosmic rays. Observed differences between mutant and parental strains may indirectly indicate participation of polyphosphates in the reaction of cells on the changes of space weather pa- rameters. Thus, correlating variability of staining and motion of polyphosphate-containing volutin granules under action of cosmophysical factors may be an evidence of possible key role of phosphoric metabolism in sensitivity of yeast cells to space weather changes.
Assuntos
Radiação Cósmica , Grânulos Citoplasmáticos/efeitos da radiação , Polifosfatos/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Atividade Solar , Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Análise de Variância , Grânulos Citoplasmáticos/metabolismo , Expressão Gênica , Periodicidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
UNLABELLED: Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here that M. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identify M. tuberculosis Rv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found that M. tuberculosis poly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of the ppx2 knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role in M. tuberculosis growth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability. IMPORTANCE: The stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediate Mycobacterium tuberculosis persistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in the ppx2 gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Parede Celular/fisiologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Permeabilidade , Fosfatos/metabolismo , Polifosfatos/metabolismo , Hidrolases Anidrido Ácido/deficiência , Parede Celular/ultraestrutura , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Viabilidade Microbiana , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/ultraestrutura , Análise de Sequência de DNARESUMO
Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome 'caretaker' whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit (-/-) MEFs escape senescence to become immortal more rapidly than Fhit (+/+) MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a 'mutator' phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.
Assuntos
Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/metabolismo , Instabilidade Genômica/fisiologia , Rim/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/metabolismo , Hidrolases Anidrido Ácido/genética , Animais , Caspase 3/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Instabilidade Genômica/genética , Perda de Heterozigosidade , Camundongos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas de Neoplasias/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Hidrolases Anidrido Ácido/deficiência , Animais , Antibacterianos/farmacologia , Elementos de DNA Transponíveis/genética , Feminino , Teste de Complementação Genética , Cobaias , Humanos , Hidrólise/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Modelos Moleculares , Mutação/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Polifosfatos/metabolismo , Recombinação Genética/genética , Homologia Estrutural de Proteína , Virulência/efeitos dos fármacosRESUMO
Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest k(cat)/K(m) values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Quebras de DNA de Cadeia Simples , Pirofosfatases/metabolismo , Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Sequência de Aminoácidos , Núcleo Celular/química , Proliferação de Células , Técnicas de Silenciamento de Genes , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Nucleotídeos de Inosina/metabolismo , Inosina Trifosfato/metabolismo , Dados de Sequência Molecular , Pirofosfatases/deficiência , Pirofosfatases/genética , Ribonucleotídeos/metabolismoRESUMO
OBJECTIVES: Aberrant or increased expression of cyclooxygenase-2 (COX-2) has been implicated in the pathogenesis of many diseases, including cancer. However, the exact mechanism by which COX-2 may influence tumorigenesis has yet to be described. To investigate the chemopreventive role of a COX-2 inhibitor, rofecoxib, in the development of urinary bladder cancer, we studied the effect of this drug in heterozygous and nullizygous fragile histidine triad (FHIT) gene-deficient mice in a chemically induced carcinogenesis model. MATERIALS AND METHODS: Two-hundred eight mice consisting of 50 FHIT +/+, 63 FHIT +/- and 95 FHIT -/-, were divided into five treatment groups and followed up for 15 weeks. Mice were treated with freshly prepared solution of 0.1% or 0.01% N-butyl-N-(-4-hydroxybutyl)-nitrosamine (BBN) in their drinking water and rofecoxib was administered in mouse chow at 150 parts per million concentration. Mice were sacrificed, and accurate histological analysis of the bladder was performed. RESULTS: Rofecoxib treatment significantly reduced the incidence of preneoplastic lesions/bladder tumors (P = 0.016). Comparing the incidence of neoplastic lesions in mice treated with rofecoxib and BBN (22/56, 39.3%) and mice treated only with BBN (32/57, 56.1%), a protective role of rofecoxib on the BBN tumor induction has been observed (P = 0.024). A similar result (P = 0.002) has been reached observing the incidence of mild and moderate dysplasia in mice treated with a lower concentration of BBN (8/16, 50.0% vs. 20/24, 83.3%).Moreover, as previously observed, a significant increase in neoplastic lesions in the FHIT +/- and FHIT -/- vs. FHIT +/+ mice after BBN treatment has been observed (P = 0.003). CONCLUSIONS: These findings suggest that rofecoxib provides a therapeutic defense against bladder carcinogenesis in our model and confirmed that the FHIT knock-out mouse is a suitable system to study in vivo bladder carcinogenesis.
Assuntos
Antineoplásicos/uso terapêutico , Lactonas/uso terapêutico , Sulfonas/uso terapêutico , Neoplasias da Bexiga Urinária/prevenção & controle , Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Animais , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Lesões Pré-Cancerosas/prevenção & controleRESUMO
Loss of heterozygosity at the FHIT locus is coincident with activation of DNA damage response checkpoint proteins; thus damage at fragile loci may trigger checkpoint activation. We examined preneoplastic lesions adjacent to non-small cell lung carcinomas for alterations to expression of Fhit and activated checkpoint proteins. Expression scores were analyzed for pair-wise associations and correlations among proteins and type of lesion. Hyperplastic and dysplastic lesions were positive for nuclear gammaH2AX expression; 12/20 dysplastic lesions were negative for Fhit expression. Fhit positive lesions showed expression of most checkpoint proteins examined, while Fhit negative lesions showed absence of expression of Chk1 and phosphoChk1. The results show that loss of expression of Fhit is significantly directly correlated with absence of activated Chk1 in dysplasia, and suggest a connection between loss of Fhit and modulation of checkpoint activity.
Assuntos
Hidrolases Anidrido Ácido/deficiência , Sítios Frágeis do Cromossomo/genética , Dano ao DNA/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/deficiência , Lesões Pré-Cancerosas/genética , Hidrolases Anidrido Ácido/genética , Quinase 1 do Ponto de Checagem , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Humanos , Hiperplasia , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteínas de Neoplasias/genética , Proteínas Quinases/genéticaRESUMO
The fragile histidine triad gene (human FHIT, mouse Fhit) has been shown to act as a tumor suppressor gene. Nit1 and Fhit form a fusion protein, encoded by the NitFhit gene in flies and worms, suggesting that mammalian Nit1 and Fhit proteins, which are encoded by genes on different chromosomes in mammals, may function in the same signal pathway(s). A previous study showed that Nit1 deficiency in knockout mice confers a cancer prone phenotype, as does Fhit deficiency. We have now assessed the tumor susceptibility of Fhit(-/-)Nit1(-/-) mice and observed that double knockout mice develop more spontaneous and carcinogen-induced tumors than Fhit(-/-) mice, suggesting that the extent of tumor susceptibility due to Nit1 and Fhit deficiency is additive, and that Nit1 and Fhit affect distinct signal pathways in mammals. Nit1, like Fhit, is present in cytoplasm and mitochondria but not nuclei. Because Fhit deficiency affects responses to replicative and oxidative stress, we sought evidence for Nit1 function in response to such stresses in tissues and cultured cells: when treated with hydroxyurea, the normal kidney-derived double-deficient cells appear not to activate the pChk2 pathway and when treated with H(2)O(2), show little evidence of DNA damage, compared with wild type and Fhit(-/-) cells. The relevance of Nit1 deficiency to human cancers was examined in human esophageal cancer tissues, and loss of Nit1 expression was observed in 48% of esophageal adenocarcinomas.
Assuntos
Hidrolases Anidrido Ácido/fisiologia , Aminoidrolases/fisiologia , Neoplasias Esofágicas/etiologia , Proteínas de Neoplasias/fisiologia , Hidrolases Anidrido Ácido/deficiência , Aminoidrolases/deficiência , Animais , Células Cultivadas , Quinase do Ponto de Checagem 2 , Dano ao DNA , Suscetibilidade a Doenças , Sinergismo Farmacológico , Neoplasias Esofágicas/patologia , Peróxido de Hidrogênio/farmacologia , Hidroxiureia/farmacologia , Rim/citologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Proteínas Serina-Treonina Quinases , Células Tumorais CultivadasRESUMO
AIM: To investigate the expression of fragile histidine triad (FHIT) protein in 64 patients with ulcerative colitis (UC) and Crohn's disease (CD), and its relation with clinicopathological data. METHODS: Rabbit-anti-FHIT antibody was used to detect FHIT protein expression in 64 formalin-fixed, paraffin-embedded tissue specimens of inflammatory bowel disease (IBD) by citrate-microwave-streptavidin (SP)-HRP immunohistochemical method. RESULTS: The positive FHIT protein expression was 22.79% +/- 16.16%, 42.14% +/- 16.82% in active and remittent phases of UC, 36.07% +/- 19.23% in CD, and 57.05% +/- 8.86% in normal colon mucosa. Statistically significant differences in FHIT protein expression were observed between the active and remittent phases of UC, between the active phase of UC and normal colon mucosa, as well as between the remittent phase of UC and normal colon mucosa, and between CD and normal colon mucosa. CONCLUSION: Our results show that FHIT protein expression is completely absent or reduced in IBD, suggesting that the FHIT gene might be associated with the oncogenesis and progression of IBD, an early event from inflammatory conditions to carcinoma in IBD.
Assuntos
Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Doenças Inflamatórias Intestinais/genética , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Adulto , Idoso , Biópsia , China , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colonoscopia , Doença de Crohn/genética , Doença de Crohn/patologia , Feminino , Humanos , Inflamação , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , SigmoidoscopiaRESUMO
Loss of fragile histidine triad (Fhit) expression is often associated with human malignancies, and Fhit functions as a tumor suppressor in controlling cell growth and apoptosis, although specific signal pathways are still undefined. We have used a proteomic approach to define proteins in the Fhit-mediated tumor suppression pathway. Because substitution of Tyr(114) (Y114) with phenylalanine (Y114F) diminishes Fhit functions, we did protein expression profiling to identify proteins differentially expressed in Fhit-negative H1299 lung cancer cells infected with wild-type (Ad-FHIT-wt) and Y114 mutant FHIT-expressing (Ad-FHIT-Y114F) adenoviruses. Among 12 distinct proteins that exhibited 4-fold differences in expression on comparison of the two infected cell lysates, cyclophilin A, the intracellular reporter of the immunosuppressive drug cyclosporine A, showed a remarkably decreased protein level in cells infected with Ad-FHIT-wt versus Ad-FHIT-Y114F. Conversely, loss of Fhit expression resulted in increased cyclophilin A expression in mouse tissues and cell lines. Restoration of Fhit expression led to down-regulated cyclophilin A protein expression and subsequently prevented cyclophilin A-induced up-regulation of cyclin D1, Cdk4, and resultant cell cycle progression (G(1)-S transition), which was independent of Ca(2+)/calmodulin-dependent kinase inhibitor, KN-93. Interestingly, Fhit down-modulation of phosphatase activity of calcineurin, which controls cyclin D1/Cdk4 activation, was reversed by cyclophilin A treatment in a concentration-dependent manner, a reversal that was inhibited by additional cyclosporine A treatment. Thus, cyclophilin A is a downstream target in Fhit-mediated cessation of cell cycle progression at late G(1) phase. Elucidation of the protein effectors of Fhit signaling may lead to identification of targets for lung cancer therapy.
Assuntos
Hidrolases Anidrido Ácido/fisiologia , Ciclofilina A/fisiologia , Genes Supressores de Tumor/fisiologia , Proteínas de Neoplasias/fisiologia , Análise Serial de Proteínas/métodos , Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Calcineurina/metabolismo , Ciclo Celular , Fase G1/genética , Humanos , Modelos Biológicos , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Mutação Puntual , Fase S/genética , Transdução Genética , Transgenes , Células Tumorais CultivadasRESUMO
The Fhit gene, encompassing the most active common human chromosomal fragile region, FRA3B, has been shown to act as a tumor suppressor. Several studies have shown significant Fhit alterations or Fhit protein loss in lung cancers from smokers compared with lung cancers from nonsmokers. To evaluate the role of Fhit under controlled experimental conditions, we exposed rodents to environmental cigarette smoke (ECS) and evaluated Fhit expression or Fhit protein in the respiratory tract. After 14 days of exposure to ECS, loss of Fhit protein in the bronchial/bronchiolar epithelium affected half of the tested B6-129(F(1)) mice, either wild type or Fhit(+/-). After 28 days, it affected the vast majority of the tested SKH-1 hairless mice and of A/J mice and all (UL53-3 x A/J)F(1) mice, either wild type or P53(+/-). In Sprague-Dawley rats, exposure to ECS for up to 30 days caused a time-dependent loss of Fhit in pulmonary alveolar macrophages. Moreover, ECS down-regulated Fhit expression and significantly decreased Fhit protein in the rat bronchial epithelium. The oral administration of N-acetylcysteine attenuated the ECS-related loss of Fhit, whereas oltipraz, 5,6-benzoflavone, phenethyl isothiocyanate, and indole 3-carbinol, and their combinations had no significant effect. Parallel studies evaluated a variety of molecular, biochemical, and cytogenetic alterations in the respiratory tract of the same animals. In conclusion, there is unequivocal evidence that Fhit is an early, critical target in smoke-related lung carcinogenesis in rodents, and that certain chemopreventive agents can attenuate the occurrence of this gene alteration.
Assuntos
Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Anticarcinógenos/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/enzimologia , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Poluição por Fumaça de Tabaco , Acetilcisteína/farmacologia , Hidrolases Anidrido Ácido/biossíntese , Animais , Western Blotting , Brônquios/patologia , Regulação para Baixo , Imuno-Histoquímica , Camundongos , Proteínas de Neoplasias/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulindaco/farmacologiaRESUMO
Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in approximately 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit-/- cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage-induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI-induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation-induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation-induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.
Assuntos
Hidrolases Anidrido Ácido/genética , Reparo do DNA/genética , Proteínas de Neoplasias/genética , Proteínas Quinases/genética , Hidrolases Anidrido Ácido/deficiência , Animais , Linhagem Celular , Quinase 1 do Ponto de Checagem , DNA/genética , DNA/efeitos da radiação , Dano ao DNA , Camundongos , Proteínas de Neoplasias/deficiência , RNA Interferente Pequeno/genética , Recombinação Genética/genéticaRESUMO
The FHIT gene plays important roles in cancer development, including lung cancers, in which the Fhit protein is frequently lost. To determine if Fhit-deficient mice exhibit increased susceptibility to carcinogen-induced lung cancer, mice were treated with the pulmonary carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone. Wild-type and Fhit-deficient animals did not exhibit significantly different frequencies of lung lesions, but Fhit-/- mice showed significantly increased average tumor volume (1.62 mm3) and multiplicity in tumor-bearing mice, compared with wild-type mice (0.70 mm3). Tumors of Fhit-/- mice were all carcinomas, whereas Fhit+/+ mice did not develop carcinomas. To determine if Fhit absence, in combination with deficiency of an additional 3p tumor suppressor, would affect the frequency of tumor induction, we examined the spontaneous and dimethylnitrosamine-induced tumor phenotype of Fhit-/-Vhl+/- mice. Whereas no spontaneous lung tumors were observed in Fhit-/- or Vhl+/- mice, 44% of Fhit-/-Vhl+/- mice developed adenocarcinomas by 2 years of age. Dimethylnitrosamine (6 mg/kg body weight) induced lung tumors (adenomas and carcinomas) in 100% of Fhit-/-Vhl+/- mice and adenomas in 40% of Fhit-/- mice by 20 months of age. Thus, double deficiency in murine homologues of 3p suppressor genes, including haploinsufficiency of Vhl, predisposes to spontaneous and induced lung cancers, showing that Fhit-deficient mice will be useful, in combination with other 3p tumor suppressors, in recapitulating a pattern of lung cancer development similar to the human pattern; such double- or triple-deficient mice will be excellent lung cancer prevention and therapy models.