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1.
Mutat Res ; 808: 48-52, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29518635

RESUMO

Recent research shows that extra-nuclear cell-free chromatin (cfCh) released from dying cells can freely enter into healthy cells and integrate into their genomes. Genomic integration of cfCh leads to dsDNA breaks and activation of inflammatory cytokines both of which occur concurrently with similar kinetics and that induction of inflammation can be abrogated by preventing DNA breaks with the use of cfCh inactivating agents. The proposal is put forward that inflammatory cytokines are a new family of DDR proteins that are activated following dsDNA breaks inflicted by genomic integration of cfCh.


Assuntos
Quebras de DNA de Cadeia Dupla , Inflamação/etiologia , Humanos
2.
BMC Mol Biol ; 18(1): 21, 2017 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-28793862

RESUMO

BACKGROUND: We have recently reported that cell-free DNA (cfDNA) fragments derived from dying cells that circulate in blood are biologically active molecules and can readily enter into healthy cells to activate DNA damage and apoptotic responses in the recipients. However, DNA is not conventionally known to spontaneously enter into cells or to have any intrinsic biological activity. We hypothesized that cellular entry and acquisition of biological properties are functions of the size of DNA. RESULTS: To test this hypothesis, we generated small DNA fragments by sonicating high molecular weight DNA (HMW DNA) to mimic circulating cfDNA. Sonication of HMW DNA isolated from cancerous and non-cancerous human cells, bacteria and plant generated fragments 300-3000 bp in size which are similar to that reported for circulating cfDNA. We show here that while HMW DNAs were incapable of entering into cells, sonicated DNA (sDNA) from different sources could do so indiscriminately without heed to species or kingdom boundaries. Thus, sDNA from human cells and those from bacteria and plant could enter into nuclei of mouse cells and sDNA from human, bacterial and plant sources could spontaneously enter into bacteria. The intracellular sDNA associated themselves with host cell chromosomes and integrated into their genomes. Furthermore, sDNA, but not HMW DNA, from all four sources could phosphorylate H2AX and activate the pro-inflammatory transcription factor NFκB in mouse cells, indicating that sDNAs had acquired biological activities. CONCLUSIONS: Our results show that small fragments of DNA from different sources can indiscriminately enter into other cells across species and kingdom boundaries to integrate into their genomes and activate biological processes. This raises the possibility that fragmented DNA that are generated following organismal cell-death may have evolutionary implications by acting as mobile genetic elements that are involved in horizontal gene transfer.


Assuntos
DNA/genética , Transferência Genética Horizontal , Animais , Linhagem Celular , Ácidos Nucleicos Livres/química , Ácidos Nucleicos Livres/genética , Cromossomos/química , Cromossomos/genética , Cromossomos/metabolismo , DNA/química , DNA/metabolismo , Imunofluorescência , Histonas/metabolismo , Humanos , Camundongos , Microscopia de Fluorescência , Peso Molecular , NF-kappa B/metabolismo , Especificidade da Espécie
3.
Int J Occup Med Environ Health ; 28(6): 921-39, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26294196

RESUMO

December 2014 marked the 30th year anniversary of Bhopal gas tragedy. This sudden and accidental leakage of deadly poisonous methyl isocyanate (MIC) gas instigated research efforts to understand the nature, severity of health damage and sufferings of 570 000 ailing survivors of this tragedy. In a decade-long period, our systematic laboratory investigations coupled with long-term molecular surveillance studies have comprehensively demonstrated that the risk of developing an environmental associated aberrant disease phenotype, including cancer, involves complex interplay of genomic and epigenetic reprogramming. These findings poised us to translate this knowledge into an investigative framework of "molecular biodosimetry" in a strictly selected cohort of MIC exposed individuals. A pragmatic cancer risk-assessment strategy pursued in concert with a large-scale epidemiological study might unfold molecular underpinnings of host-susceptibility and exposureresponse relationship. The challenges are enormous, but we postulate that the study will be necessary to establish a direct initiation-promotion paradigm of environmental carcinogenesis. Given that mitochondrial retrograde signaling-induced epigenetic reprogramming is apparently linked to neoplasticity, a cutting-edge tailored approach by an expert pool of biomedical researchers will be fundamental to drive these strategies from planning to execution. Validating the epigenomic signatures will hopefully result in the development of biomarkers to better protect human lives in an overburdened ecosystem, such as India, which is continuously challenged to meet population demands. Besides, delineating the mechanistic links between MIC exposure and cancer morbidity, our investigative strategy might help to formulate suitable regulatory policies and measures to reduce the overall burden of occupational and environmental carcinogenesis.


Assuntos
Vazamento Acidental em Bhopal , Carcinógenos , Desastres , Isocianatos/intoxicação , Neoplasias , Medição de Risco , Sobreviventes/estatística & dados numéricos , Antidrepanocíticos/intoxicação , Humanos , Incidência , Índia/epidemiologia , Neoplasias/induzido quimicamente , Neoplasias/diagnóstico , Neoplasias/epidemiologia , Radiometria , Taxa de Sobrevida/tendências
4.
J Biosci ; 40(1): 91-111, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25740145

RESUMO

Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.


Assuntos
Cromatina/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA/genética , Ácidos Nucleicos/sangue , Células 3T3 , Animais , Apoptose/genética , Sequência de Bases , Caspase 3/metabolismo , Linhagem Celular Tumoral , Desoxirribonuclease I/farmacologia , Células HeLa , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/sangue , Neoplasias/genética , Ácidos Nucleicos/genética , Análise de Sequência de DNA
5.
Int J Toxicol ; 33(2): 116-29, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24563415

RESUMO

Emerging studies have linked prooxidative carbamate compound exposures with various human pathologies including pancreatic cancer. In these studies, our aim was to examine mitochondrial oxidative stress-mediated aberrant chromatin responses in human pancreatic ductal epithelial cells. Posttranslational histone modifications, promoter DNA methylation, and micro-RNA (miRNA) expression patterns were evaluated following induction of mitochondrial oxidative stress by N-succinimidyl N-methylcarbamate exposure. In treated cells, perturbation in mitochondrial machinery led to hypermethylation of p16 and smad4 gene promoters and downregulation of respective gene products. Posttranslational histone modifications that include hypoacetylation of acetylated histone (AcH) 3 and AcH4, hypermethylation of monomethylated histone 3 at lysine 9 and trimethylated histone 4 at lysine 20 ubiquitinated histone (uH) 2A/uH2B, and increased phosphorylation of H2AX and H3 were observed in the treated cells. Altered expression of miRNAs denoted possible location of corresponding genes at oxidatively damaged fragile sites. Collectively, our results provide a direct role of mitochondrial oxidative stress-mediated epigenetic imbalance to perturbed genomic integrity in oxygen radical-induced pancreatic injury. Further, identification and characterization of molecular switches that affect these epigenomic signatures and targets thereof will be imperative to understand the complex role of redox-regulatory network in pancreatic milieu.


Assuntos
Epigênese Genética/fisiologia , Células Epiteliais/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Pâncreas/metabolismo , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína Smad4/metabolismo
6.
Indian J Med Res ; 140 Suppl: S120-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25673532

RESUMO

Stress induced premature senescence (SIPS) is a relative extension to the concept of exogenous cellular insult. Besides persistent double strand (ds) DNA breaks and increased ß-galactosidase activity, biological significance of telomeric attrition in conjunction with senescence associated secretory phenotype (SASP) has been highlighted in SIPS. To gain insight on the potential role of this unique phenomenon invoked upon environmental stress, we sequentially validated the molecular repercussions of this event in ovarian epithelial cells after exposure to methyl isocyanate, an elegant regulator of cellular biotransformation. Persistent accumulation of DNA damage response factors phospho-ATM/γ-H2AX, morphological changes with increased cell size and early yet incremental ß-gal staining, imply the inception of premature senescence. Advent of SASP is attributed by prolonged secretion of pro-inflammatory cytokines along with untimely but significant G1/S cell cycle arrest. Telomeric dysfunction associated with premature senescence is indicative of early loss of TRF2 (telomeric repeat binding factor 2) protein and resultant multiple translocations. Induction of senescence-associated heterochromatic foci formation showcases the chromatin alterations in form of trimethylated H3K9me3 in conjunction with H4 hypoacetylation and altered miRNA expression. Anchorage-independent neoplastic growth observed in treated cells reaffirms the oncogenic transformation following the exposure. Collectively, we infer the possible role of SIPS, as a central phenomenon, to perturbed genomic integrity in ovarian surface epithelium, orchestrated through SASP and chromatin level alterations, a hitherto unknown molecular paradigm. Although translational utility of SIPS as a biomarker for estimating ovarian cancer risk seems evident, further investigations will be imperative to provide a tangible way for its precise validation in clinical settings.


Assuntos
Carcinogênese/patologia , Transformação Celular Neoplásica/efeitos dos fármacos , Senescência Celular/fisiologia , Neoplasias Ovarianas/etiologia , Estresse Fisiológico/fisiologia , Encurtamento do Telômero/fisiologia , Biomarcadores/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Histonas/metabolismo , Humanos , Isocianatos/efeitos adversos , Ovário/citologia , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
7.
Br J Nutr ; 106(8): 1154-68, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21736819

RESUMO

Prevention of cancer through nutritional intervention has gained significant recognition in recent years. Evidence revealed from mechanistic investigations coupled with molecular epidemiology show an inverse association of dietary flavonoids intake with cancer risk. The chemopreventive and anticarcinogenic potential of Selaginella bryopteris, a traditional Indian herb referred to as 'Sanjeevani' in the Ayurvedic system of medicine, was examined in the present study. Comprehensive in vitro and in vivo studies were conducted on the flavonoid-rich benzene fraction of the aqueous extract that demonstrated a significant cytoprotective activity. Biomarkers of chemoprevention such as proliferative index and status of cell-cycle regulatory proteins, antioxidant property, anti-inflammatory effect, reversal of stress-induced senescence and genoprotective effect were investigated in human and murine cell cultures. Chemopreventive potential was assessed in benzopyrene-induced lung carcinogenesis and 7,12-dimethyl benz(a)anthracene-mediated skin papillomagenesis test models. Inhibition of DNA fragmentation, unperturbed cell-cycle regulation, maintenance of intracellular antioxidant defence, anti-inflammatory activity, prevention of stress-induced senescence and genoprotective effects against methyl isocyanate carcinogenicity was observed. Medium-term anticarcinogenicity and two-stage skin papillomagenesis tests strongly substantiated our in vitro observations. Results from the present study provide evidence of anticarcinogenic and chemopreventive activities of S. bryopteris hitherto unreported and reaffirm the nutritional significance of flavonoids in cancer prevention.


Assuntos
Anticarcinógenos/farmacologia , Flavonoides/farmacologia , Plantas Medicinais/química , Selaginellaceae/química , Animais , Animais Recém-Nascidos , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Anticarcinógenos/administração & dosagem , Anticarcinógenos/isolamento & purificação , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Flavonoides/administração & dosagem , Flavonoides/isolamento & purificação , Humanos , Índia , Medicina Tradicional , Camundongos , Papiloma/induzido quimicamente , Papiloma/prevenção & controle , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/prevenção & controle
8.
Int J Toxicol ; 30(2): 225-36, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21300767

RESUMO

We examined the possible molecular mechanisms underlying the cytotoxicity and anticarcinogenic potential of Mentha leaf extracts (petroleum ether, benzene, chloroform, ethyl acetate, methanol, and water extracts) on 6 human cancer (HeLa, MCF-7, Jurkat, T24, HT-29, MIAPaCa-2) and normal (IMR-90, HEK-293) cell lines. Of all the extracts tested, chloroform and ethyl acetate extracts of M piperita showed significant dose- and time-dependent anticarcinogenic activity leading to G1 cell cycle arrest and mitochondrial-mediated apoptosis, perturbation of oxidative balance, upregulation of Bax gene, elevated expression of p53 and p21 in the treated cells, acquisition of senescence phenotype, while inducing pro-inflammatory cytokines response. Our results provide the first evidence of direct anticarcinogenic activity of Mentha leaf extracts. Further, bioassay-directed isolation of the active constituents might provide basis for mechanistic and translational studies for designing novel anticancer drugs to be used alone or as adjuvant for prevention of tumor progression and/or treatment of human malignancies.


Assuntos
Anticarcinógenos/farmacologia , Citotoxinas/farmacologia , Mentha/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Apoptose , Western Blotting , Caspase 3/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Regulação da Expressão Gênica , Glutationa Redutase/metabolismo , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
9.
Reprod Toxicol ; 30(3): 377-86, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20685613

RESUMO

Ovarian surface epithelium is under constant physiological pressure to maintain its integrity. Environmental toxic exposure can contribute to degenerative pathologies including ovarian cancer. Based on our current understanding, we aimed at listing mechanistic insights that contribute to ovarian carcinogenesis after exposure to methyl isocyanate, an ubiquitous environmental pollutant. Ovarian epithelial cells manifested a persistent DNA damage response along with increased accumulation of GADD45, p21, p16(INK4A) and pRb proteins upon treatment. Increase in cell size and ß-gal positive staining showing inception of premature senescence with morphological transformation and structural and numerical chromosomal abnormalities were also observed. Immuno-FISH analysis illustrated early loss of TRF2 protein suggestive of telomeric dysfunction due to premature senescence and plausible association with chromosomal and microsatellite instability. Soft-agar assay displayed neoplasticity in treated cells demonstrating onset of malignant transformation. These results indicate that isocyanate exposure alters ovarian epithelial cell proliferation and might lead to ovarian dysfunction and carcinogenesis.


Assuntos
Transformação Celular Neoplásica , Poluentes Ambientais/toxicidade , Células Epiteliais/efeitos dos fármacos , Isocianatos/toxicidade , Ovário/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Aberrações Cromossômicas/induzido quimicamente , Análise Citogenética , Dano ao DNA , Reparo do DNA , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Hibridização in Situ Fluorescente , Camundongos , Instabilidade de Microssatélites/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Ploidias , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
10.
J Environ Pathol Toxicol Oncol ; 29(3): 213-34, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21303328

RESUMO

Male reproductive health is exquisitely sensitive to environmental insults as evidenced by the rising incidence of testicular cancers and low and probably declining semen quality. Isocyanates, such as methyl isocyanate (MIC), with their wide industrial applications, are known to exert severe ill health effects. The present study was performed to find out the pathophysiological implications of isocyanate exposure on the male germ line. The investigations were performed in the cultured mouse spermatogonial GC-1 spg cell line using N-succinimidyl N-methylcarbamate, a surrogate chemical to MIC. DNA damage, oxidative stress and apoptosis response parameters increased with time of exposure and dose after treatment. Treated cells also displayed elevated levels of inflammatory cytokines as well as morphological transformation and stress-responsive senescence. Chromosomal aberrations, telomere anomaly, aneuploidy and variable amplification of microsatellite repeats additionally indicated induced genomic instability. This was accompanied by evidence of a deregulation of cell cycle progression, such as substantial fold-changes in the expression of proliferating cell nuclear antigen, Cyclin D1, Bcl-2 and Bax genes; and aberrant expression of p53, cyclin A, cyclin E, CDK-2 and aurora kinase-B proteins. Our results demonstrate that MIC in the form of N-succinimidyl N-methylcarbamate promotes germ-line genomic instability in vitro. We envisage that understanding the interplay between environmental toxin-induced signaling and predisposition to testicular cancers would spur identification of meaningful targets for useful therapeutic translational modalities.


Assuntos
Instabilidade Genômica/efeitos dos fármacos , Isocianatos/toxicidade , Espermatozoides/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Aberrações Cromossômicas , Citocinas/análise , Dano ao DNA , Masculino , Camundongos , Instabilidade de Microssatélites , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura
11.
Cell Biol Int ; 33(6): 675-83, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19376255

RESUMO

The toxic response of cultured human colon epithelial-FHC cells to methyl isocyanate was investigated with regard to genomic instability. Qualitative and quantitative assessments of the extent of phosphorylation of DNA damage signaling factors such as ATM, gammaH2AX and p53, was increased in treated cells compared to controls. At the same time, many treated cells were arrested at the G2/M phase of the cell cycle, and had an elevated apoptotic index and increased inflammatory cytokine levels. Cytogenetic analyses revealed varied chromosomal anomalies, with abnormal expression of pericentrin protein. Analysis through ISSR PCR demonstrated increased microsatellite instability. The results imply that isocyanates can cause genomic instability in colonocytes.


Assuntos
Colo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Isocianatos/farmacologia , Antígenos/metabolismo , Apoptose , Linhagem Celular , Colo/citologia , Citocinas/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , Fase G2 , Instabilidade Genômica/genética , Humanos , Cariotipagem , Metáfase , Transdução de Sinais
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