RESUMO
Active DNA-dependent ATPase A Domain inhibitor (ADAADi) is the only known inhibitor of ATP-dependent chromatin remodeling proteins that targets the ATPase domain of these proteins. The molecule is synthesized by aminoglycoside phosphotransferase enzyme in the presence of aminoglycosides. ADAADi interacts with ATP-dependent chromatin remodeling proteins through motif Ia present in the conserved helicase domain, and thus, can potentially inhibit all members of this family of proteins. We show that mammalian cells are sensitive to ADAADi but with variable responses in different cell lines. ADAADi can be generated from a wide variety of aminoglycosides; however, cells showed differential response to ADAADi generated from various aminoglycosides. Using HeLa and DU145 cells as model system we have explored the effect of ADAADi on cellular functions. We show that the transcriptional network of a cell type is altered when treated with sub-lethal concentration of ADAADi. Although ADAADi has no known effects on DNA chemical and structural integrity, expression of DNA-damage response genes was altered. The transcripts encoding for the pro-apoptotic proteins were found to be upregulated while the anti-apoptotic genes were found to be downregulated. This was accompanied by increased apoptosis leading us to hypothesize that the ADAADi treatment promotes apoptotic-type of cell death by upregulating the transcription of pro-apoptotic genes. ADAADi also inhibited migration of cells as well as their colony forming ability leading us to conclude that the compound has effective anti-tumor properties.
Assuntos
Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , DNA/genética , Redes Reguladoras de Genes/genética , Mamíferos/genética , Trifosfato de Adenosina/genética , Aminoglicosídeos/genética , Animais , Linhagem Celular Tumoral , DNA Helicases/genética , Células HeLa , Humanos , Domínios Proteicos/genéticaRESUMO
Ageing is characterized by the perturbation in cellular homeostasis associated with genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intracellular communication. Changes in the epigenome represent one of the crucial mechanisms during ageing and in age-related disorders. The ATP-dependent chromatin remodelers are an evolutionarily conserved family of nucleosome remodelling factors and generally regulate DNA repair, replication, recombination, transcription and cell cycle. Here, we review the chromatin based epigenetic changes that occur in ageing and age-related disorders with a specific reference to chromatin remodelers. We also discuss the link between dietary restriction and chromatin remodelers in regulating age-related processes with a view for consideration in future intervention studies.
Assuntos
Envelhecimento , Cromatina , Trifosfato de Adenosina , Envelhecimento/genética , Senescência Celular , Montagem e Desmontagem da Cromatina , Instabilidade Genômica , HumanosRESUMO
Recent investigations have emphasized the role of miRNA biogenesis proteins in the synthesis of non-coding RNA when double-strand DNA breaks are induced by ionizing radiations. However, the role of these non-coding RNA and their regulation in response to doxorubicin-induced DNA damage is not known. In this paper, BRG1 and SMARCAL1, members of the ATP-dependent chromatin remodelling family, are shown to co-regulate the transcription of DROSHA, DGCR8, and DICER in response to double-strand DNA breaks induced by doxorubicin. Both BRG1 and SMARCAL1 are needed for the upregulation of the three miRNA biogenesis genes as absence of BRG1 results in downregulation of DGCR8 and DICER while absence of SMARCAL1 results in downregulation of DROSHA. These two proteins act in coordination to upregulate expression of DROSHA, DGCR8, and DICER when cells are treated with doxorubicin. This transcriptional regulation of the miRNA biogenesis proteins is needed for the formation of 53BP1 foci as downregulation of either BRG1 or SMARCAL1 reduced the number of 53BP1 foci in DNA damaged cells. The foci formation was restored when the downregulated cells were treated with ncRNA purified from doxorubicin treated HeLa cells. From the results obtained, we conclude that the regulation of miRNA biogenesis proteins by SMARCAL1 and BRG1 is needed for the formation of non-coding RNA and thus, 53BP1 foci in response to doxorubicin-induced DNA damage.
Assuntos
RNA Helicases DEAD-box/genética , Dano ao DNA/genética , DNA Helicases/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Doxorrubicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , MicroRNAs/genética , RNA não Traduzido/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Natural autophagy and autophagic cell death is being studied in the model system, D. discoideum, which has well known genetic and experimental advantages over the other known systems. There is no apoptotic machinery present in this organism which could interfere with the non-apoptotic cell death. The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway which when inhibited by the drug rapamycin induces autophagy. Rapamycin was originally discovered as an anti-fungal agent but its use was abandoned when it was discovered to have potent immunosuppressive and anti-proliferative properties. It is a known drug used today for various cancer treatments and also for increasing longevity in many model organisms. It has a wide usage but its effects on other pathways or molecules are not known. This model system was used to study the action of rapamycin on autophagy induction. Using the GFP-Atg8, an autophagosome marker, it was shown that rapamycin treatment can induce autophagy by an accumulation of reactive oxygen species and intracellular free calcium. Rapamycin suppresses proliferation by induction of cell cycle arrest in the G1 phase. Taken together, the results suggest that the core machinery for autophagy is conserved in D. discoideum and it can serve as a good model system to delineate the action of rapamycin induced autophagy.