Genomic alterations may initiate cancer development as the consequence of endogenous or exogenous DNA damaging factors. Defects in DNA repair mechanisms may also facilitate cancer progression as well as accumulation of mutations which favor cancer cell survival. However, DNA repair pathways in cancer cells can be considered as their Achilles heel which are possible targets in order to compromise their survival. For instance, it has been demonstrated recently that inhibition of a protein called MTH1 via RNA interference (RNAi) or chemical inhibitors can stop tumor growth and triggers cell death by increasing the load of oxidative DNA damage. MTH1 is a hydrolase which converts 8-oxo-dGTP into 8-oxo-dGMP in order to prevent incorporation of oxidatively damaged nucleotides into DNA. In addition, DNA glycosylases which recognize and remove mismatched or damaged nucleotide pairs in DNA can also participate in repair of 8-oxo-dG, such as MUTYH repairing A:8-oxo-dG pair. The goal of the current study was to investigate the importance of MUTYH activity upon MTH1 depletion. The current study tried to answer whether simultaneous knock-down of MTH1 and MUTYH sensitizes cancer cells to oxidative stress and increases cell death. Both enzymes were simultaneously depleted in T cell acute lymphoblastic leukemia cells using RNAi. Then, we analyzed the efficiency of gene and protein knock-down by quantitative real-time-PCR and western blotting, respectively. Induction of cell death was also assessed by flow cytometric analysis of cell cycle. Afterwards, the effect of the treatments on DNA repair pathways was studied by analysis of gene expression of several DNA glycosylases and DNA polymerases using qRT-PCR. The results showed that concurrent depletion of both enzymes led to synergistic induction of cell death. Down-regulation of NEIL1 DNA glycosylase as well as POLQ and POLH DNA polymerases mRNAs adapted their DNA repair pathways to cope with induced damages under these conditions. Finally, the results of this study suggest that dual suppression of MTH1 and MUTYH may provide a new approach to reduce survival of T cell ALL.

Made available in DSpace on 2015-03-26T13:36:46Z (GMT). No. of bitstreams: 1 texto completo.pdf: 250559 bytes, checksum: dfc57be40260157fcf945d27d2d9744b (MD5) Previous issue date: 2007-03-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aluminum (Al) effects on the growth and enzymatic and non-enzymatic antioxidative mechanisms involved in the scavenging of reactive oxygen species (ROIs) in two rice cultivars: Fernandes (CNA-1158) and Maravilha (CNA-6843-1) with differential tolerance to Al were studied. The plants, grown in pH 4.0 nutrient solution, for ten days in nutrient solution, pH 4,0, were treated with Al 0 and 1 mM for ten days. Exposure to Al did not affect growth in Fernandes cultivar, while in Maravilha cultivar significant reductions were observed in the evaluated growth parameters. Aluminum increased the activities of the enzymes, catalases (CATs), peroxidases (POXs), glutathione reductases (GRs) and glutathione peroxidases (GPXs) in the roots of both cultivars. The activity of superoxide dismutases (SODs), however, increased only in the roots of the Fernandes cultivar, while in Maravilha cultivar it remained unchanged. Independent of the applied treatment and of the plant analyzed, the activity of the ascorbate peroxidases (APXs) was always larger in Fernandes cultivar. In the leaves, Al did not change any enzyme activity in Fernandes cultivar, except the POXs activity which reduced. In Maravilha cultivar, on the other hand, SODs, POXs and GPXs activities reduced in the presence of Al. Among the studied enzymes, SODs, APXs and GPXs in the roots and of CATs, POXs and APXs in the leaves, showed responses to Al treatment consistent with the differential tolerance to Al of these two rice cultivars. Ascorbate (AA) contents increased in the leaves and dehydroascorbate (DHA) decreased in the roots, in response to the Al treatment in both cultivars. The reduced form (AA) content was much higher in the leaves, where the AA/DHA ratio reached values thirty times larger times than in the roots. In the roots, Fernandes cultivar showed lower AA contents, but higher APXs activity and the Maravilha cultivar the opposite, indicating an important role of this metabolite as substrate for the reaction catalyzed by APXs. Total glutathione contents, also, seem be important to the non-enzymatic defense system of both cultivars, but probably will be necessary to discriminate between reduced (GSH) and oxidized (GSSG) form to understand the role of this substance in sustaining antioxidative enzyme activities. In general, the activity of the antioxidative system, enzymatic and non-enzymatic, both in roots and leaves, were suggestive of having Fernandes cultivar a more efficient defense mechanism to the ROIs produced by Al treatments. / Os efeitos do alumínio (Al) sobre o crescimento e sobre os sistemas de defesa antioxidativos enzimáticos e não-enzimáticos envolvidos na eliminação dos intermediários reativos de oxigênio (ROIs) foram avaliados em dois cultivares de arroz: Fernandes (CNA-1158) e Maravilha (CNA-6843-1) com tolerância diferencial ao Al. As plantas, cultivadas em solução nutritiva, pH 4,0, foram tratadas com Al nas concentrações de 0 e 1 mM, durante dez dias. A exposição das plantas ao Al não afetou o crescimento no cultivar Fernandes, enquanto, no Maravilha, reduções significativas foram observadas nos parâmetros de crescimento avaliados. Nas raízes dos dois cultivares, a presença do Al aumentou a atividade das enzimas catalases (CATs), peroxidases (POXs), redutases da glutationa (GRs) e peroxidases da glutationa (GPXs). A atividade das superóxido dismutases (SODs) aumentou apenas nas raízes do cultivar Fernandes, não sendo modificada para o cultivar Maravilha. Independente do tratamento aplicado e da parte da planta analisada, a atividade das peroxidases do ascorbato (APXs) foi sempre maior no cultivar Fernandes. Nas folhas, o Al não alterou a atividade de nenhuma das enzimas no cultivar Fernandes, exceto a das POXs que sofreu redução. No cultivar Maravilha, por outro lado, as atividades das SODs, POXs e GPXs foram reduzidas na presença de Al. Dentre as enzimas estudadas, as SODs, APXs e GPXs nas raízes e as CATs, POXs, APXs nas folhas, exibiram resposta consistente com a tolerância diferencial ao Al apresentada pelos dois cultivares de arroz estudados. Nos dois cultivares, o teor de ascorbato (AA) aumentou nas folhas e o de desidroascorbato (DHA) reduziu nas raízes, em resposta ao tratamento com Al. O teor da forma reduzida (AA) foi muito mais elevado nas folhas, na qual a relação AA/DHA atingiu valores 30 vezes maiores que nas raízes. Nas raízes, o cultivar Fernandes apresentou menores teores de AA, porém maior atividade das APXs, enquanto no cultivar Maravilha foi observado o oposto, demonstrando o importante papel desse metabólito como substrato para a reação catalisada pelas APXs. A concentração de glutationa total, também, parece ser importante no sistema de defesa não enzimático, mas provavelmente seria necessário discriminar entre as suas formas, reduzida (GSH) e oxidada (GSSG), para entender seu papel como substrato das enzimas antioxidativas. De modo geral, as atividades dos sistemas de defesa antioxidativos enzimático e não-enzimático, tanto nas raízes como nas folhas dos dois cultivares, indicaram ter o cultivar Fernandes mecanismos de defesa mais eficientes no combate aos ROIs produzidos durante o tratamento com Al.