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1.
Int J Radiat Biol ; 87(2): 222-30, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21133647

RESUMO

PURPOSE: We analysed the lethal and mutagenic interactions between γ-rays, cisplatin (Pt) and etoposide (E), three agents used in tumour chemoradiotherapy. Corresponding results at cellular and molecular levels could provide additional elements on involved mechanisms and, on antitumour activity and toxicity in combined cancer treatments. MATERIALS AND METHODS: The yeast Saccharomyces cerevisiae SC7K(lys2-3) (auxotrophic for lysine) was used as eukaryotic model. Exponential growing cells were exposed to the mentioned agents, as single and combined treatments. Lethal and mutation interaction equations were determined as a function of doses according to quantitative models. DNA double-strand breaks were evaluated immediately after treatments, through pulsed-field electrophoresis and laser densitometry. RESULTS: All three agents induced significant mutant frequency. The γ +Pt + E combination determined maximal lethal and mutagenic synergism, followed by γ + Pt and γ + E combinations. Meanwhile, Pt + E combination showed lethal additivity and very low mutagenic synergism. Pt + E double combination determined moderate DNA degradation. DNA degradation after γ-exposure, was similar to that of γ + Pt, γ + E and γ + Pt + E combinations. CONCLUSIONS: Synergistic lethal and mutagenic interactions indicate crosstalk between non-homologous end joining, homologous recombination and postreplicative repair pathways. Pt + E additivity indicate independence of involved repair pathways. Furthermore, the quantification of interactive events may be an additional suitable tool in tumour therapy planning.


Assuntos
Cisplatino/farmacologia , Etoposídeo/farmacologia , Células Eucarióticas/efeitos dos fármacos , Células Eucarióticas/efeitos da radiação , Raios gama , Mutagênicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Cisplatino/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Etoposídeo/metabolismo , Células Eucarióticas/metabolismo , Modelos Biológicos , Mutagênicos/toxicidade , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética
2.
J Biol Phys ; 34(1-2): 63-71, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19669493

RESUMO

DNA repair, checkpoint pathways and protection mechanisms against different types of perturbations are critical factors for the prevention of genomic instability. The aim of the present work was to analyze the roles of RAD17 and HDF1 gene products during the late stationary phase, in haploid and diploid yeast cells upon gamma irradiation. The checkpoint protein, Rad17, is a component of a PCNA-like complex-the Rad17/Mec3/Ddc1 clamp-acting as a damage sensor; this protein is also involved in double-strand break (DBS) repair in cycling cells. The HDF1 gene product is a key component of the non-homologous end-joining pathway (NHEJ). Diploid and haploid rad17Delta/rad17Delta, and hdf1Delta Saccharomyces cerevisiae mutant strains and corresponding isogenic wild types were used in the present study. Yeast cells were grown in standard liquid nutrient medium, and maintained at 30 degrees C for 21 days in the stationary phase, without added nutrients. Cell samples were irradiated with (60)Co gamma rays at 5 Gy/s, 50 Gy

3.
Radiat Environ Biophys ; 46(4): 401-7, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17624540

RESUMO

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Delta/chk1Delta and rad17Delta/rad17Delta, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Delta/rad17Delta cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Delta/rad17Delta cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Delta/chk1Delta cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Delta/chk1Delta mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/fisiologia , DNA Fúngico/fisiologia , DNA Fúngico/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem , Dano ao DNA/fisiologia , Reparo do DNA/efeitos da radiação , Genes cdc/fisiologia , Genes cdc/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiação
4.
FEMS Microbiol Lett ; 256(1): 132-6, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16487330

RESUMO

It is known that certain yeast strains, so called 'killers', can produce and excrete proteinaceous toxins that can induce death of other sensitive strains. We obtained a stable fungicidal factor (SKF) through concentration and stabilization of the excretion product of certain killer strains of Saccharomyces cerevisiae (K1 and K2). The isolated proteinaceous complex exhibited activity at broad ranges of pH (4-7.5) and temperatures (20-37.5 degrees C). It was significantly lethal against Candida albicans and Tricophyton mentagrophytes. SKF showed stability and activity after storage, with a mean half-life of 6 months at 4 degrees C or at -20 degrees C.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/farmacologia , Saccharomyces cerevisiae , Trichophyton/efeitos dos fármacos , Antifúngicos/isolamento & purificação , Arthrodermataceae/efeitos dos fármacos , Armazenamento de Medicamentos/normas , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana/métodos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Análise de Sobrevida , Fatores de Tempo
5.
Environ Mol Mutagen ; 45(1): 36-43, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15605356

RESUMO

Cells react to environmental and endogenous challenges such as high temperature, reactive oxygen species, DNA damage, and nutrient starvation by activating several defense mechanisms known as stress responses. An important feature is the overlap between different stress responses that contributes at least in part to the phenomenon of cross-protection. We previously demonstrated that pretreatment with a heat shock (HS) induces resistance to the lethal and mutagenic effects of the antineoplastic drug Bleomycin (BLM) in wild-type Saccharomyces cerevisiae. At the DNA level, the HS resulted in more efficient repair of BLM-induced DNA damage. In the present study, we have investigated the mechanisms involved in this HS-induced BLM resistance. Since the RAD6 gene is involved in the ubiquitin system and DNA repair, we analyzed the effects of HS on the lethality of BLM in a rad6Delta (ubc2) mutant strain of S. cerevisiae. The rad6Delta mutant was more sensitive to the lethal effects of BLM than wild-type yeast and HS had no effect on the lethality of BLM in the mutant. Analysis of cell proliferation kinetics indicated that the HS-induced cell cycle delay observed in the wild-type yeast was absent in the rad6Delta mutant strain. BLM treatment impaired mutant cell proliferation, and HS had no effect on the delayed cell kinetics of the mutant. In addition, pulsed-field electrophoresis of chromosomes damaged by BLM indicated that there was very little recovery from damage in the mutant after 24 hr of incubation in BLM-free nutrient medium, and that HS had little effect on the recovery. These data indicate that the RAD6 gene is involved in the HS-induced BLM resistance observed in the isogenic wild-type strain.


Assuntos
Bleomicina/toxicidade , Resistência a Medicamentos , Resposta ao Choque Térmico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Enzimas de Conjugação de Ubiquitina/genética , Genes Fúngicos , Temperatura Alta , Mutação
6.
DNA Repair (Amst) ; 3(12): 1591-9, 2004 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-15474420

RESUMO

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Dano ao DNA , Reparo do DNA/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Raios Ultravioleta , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Reparo do DNA/fisiologia , Enzimas Reparadoras do DNA , DNA Fúngico/metabolismo , DNA Fúngico/efeitos da radiação , Proteínas de Ligação a DNA , Fase G1/genética , Fase G1/fisiologia , Fase G2/genética , Fase G2/fisiologia , Interfase/genética , Interfase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Fosforilação , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência/genética , Água/farmacologia
7.
J Altern Complement Med ; 9(3): 379-87, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12816626

RESUMO

We analyzed the antioxidant properties of Ilex paraguariensis infusion (Ip) popularly known as mate (mä'ta), by using two experimental models: the induction of DNA double-strand breaks (DSB) by hydrogen peroxide (H(2)O(2)) and lethality in Saccharomyces cerevisiae, as well as peroxide and lipoxygenase-induced human low-density lipoprotein (LDL) oxidation. Diploid yeast cells were exposed to different concentrations of H(2)O(2) (5-10 mmol/L) in the absence or presence of Ip infusion (10(-1) v/v) or alpha-tocopherol (10(-2) mol/L). Both mate infusion and alpha-tocopherol significantly decreased the dose dependent DSB number, and the lethality induced by H(2)O(2). Peroxynitrite and lipoxygenase-induced human LDL oxidation are inhibited by Ip extracts in a potent, dose-dependent fashion. Dilutions of 5 x 10(-3) v/v provide 50% +/- 10% inhibition. Finally, Ip extracts are potent direct quenchers of the free radical 1,1-diphenyl-2-picrylhydrazyl. Dilutions of 2 x 10(-2) v/v produced quenching of more than 30%, which was comparable to that obtained with 0.5-1 mmol/L alpha-tocopherol or the quercetin aglycone, respectively. For comparison, total polyphenol content of Ip, green, and black tea (Camelia sinensis) were 6.5 +/- 0.8; 1.8 +/- 0.5; and 1.13 +/- 0.3 mmol of quercetin equivalents per liter, respectively. Their respective free radical quenching activities at dilutions of 1 x 10(-1) v/v were 75% +/- 5%; 35% +/- 5%; and 2% +/- 5%. Ip is thus a rich source of polyphenols and has antioxidant properties comparable to those of green tea which merit further in vivo intervention and cross-sectional studies.


Assuntos
Antioxidantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Ilex paraguariensis , Lipoproteínas LDL/metabolismo , Extratos Vegetais/farmacologia , Saccharomyces cerevisiae/genética , Antioxidantes/metabolismo , Quebra Cromossômica , Humanos , Peróxido de Hidrogênio/metabolismo , Lipoproteínas LDL/efeitos dos fármacos , Oxirredução , Extratos Vegetais/metabolismo , Saccharomyces cerevisiae/metabolismo
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