RESUMO
The Mre11 nuclease is involved in early responses to DNA damage, often mediated by its role in DNA end processing. MRE11 mutations and aberrant expression are associated with carcinogenesis and cancer treatment outcomes. While, in recent years, progress has been made in understanding the role of Mre11 nuclease activities in DNA double-strand break repair, their role during replication has remained elusive. The nucleoside analog gemcitabine, widely used in cancer therapy, acts as a replication chain terminator; for a cell to survive treatment, gemcitabine needs to be removed from replicating DNA. Activities responsible for this removal have, so far, not been identified. We show that Mre11 3' to 5' exonuclease activity removes gemcitabine from nascent DNA during replication. This contributes to replication progression and gemcitabine resistance. We thus uncovered a replication-supporting role for Mre11 exonuclease activity, which is distinct from its previously reported detrimental role in uncontrolled resection in recombination-deficient cells.
Assuntos
Proteínas de Ligação a DNA , Desoxicitidina , DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Exonucleases/genética , Exonucleases/metabolismo , GencitabinaRESUMO
BACKGROUND: Dispositions for genes encoding opioid receptors may explain some variability in morphine efficacy. Experimental studies show that morphine and morphine-6-glucuronide are less effective in individuals carrying variant alleles caused by the 118 A > G polymorphism in the mu-opioid receptor gene (OPRM1). The purpose of the study was to investigate whether this and other genetic polymorphisms in OPRM1 influence the efficacy of morphine in cancer pain patients. METHODS: We screened 207 cancer pain patients on oral morphine treatment for four frequent OPRM1 gene polymorphisms. The polymorphisms were the -172 G > T polymorphism in the 5'untranslated region of exon 1, the 118 A > G polymorphism in exon 1, and the IVS2 + 31 G > A and IVS2 + 691 G > C polymorphisms, both in intron 2. Ninety-nine patients with adequately controlled pain were included in an analysis comparing morphine doses and serum concentrations of morphine and morphine metabolites in the different genotypes for the OPRM1 polymorphisms. RESULTS: No differences related to the -172 G > T, the IVS2 + 31 G > A and the IVS2 + 691 G > C polymorphisms were observed. Patients homozygous for the variant G allele of the 118 A > G polymorphism (n = 4) needed more morphine to achieve pain control, compared to heterozygous (n = 17) and homozygous wild-type (n = 78) individuals. This difference was not explained by other factors such as duration of morphine treatment, performance status, time since diagnosis, time until death, or adverse symptoms. CONCLUSION: Patients homozygous for the 118 G allele of the mu-opioid receptor need higher morphine doses to achieve pain control. Thus, genetic variation at the gene encoding the mu-opioid receptor contributes to variability in patients' responses to morphine.
Assuntos
Analgésicos Opioides/uso terapêutico , Morfina/uso terapêutico , Neoplasias/complicações , Neoplasias/genética , Dor Intratável/tratamento farmacológico , Dor Intratável/genética , Receptores Opioides mu/genética , Receptores Opioides mu/fisiologia , Idoso , Alelos , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Relação Dose-Resposta a Droga , Feminino , Testes Genéticos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Morfina/administração & dosagem , Morfina/efeitos adversos , Derivados da Morfina/sangue , Neoplasias/patologia , Medição da Dor/efeitos dos fármacos , Polimorfismo Genético , Qualidade de Vida , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have screened a cohort of 239 Norwegian cancer patients for sequence variation in the coding and regulatory regions of the UDP-glucuronosyltransferase 2B7 gene (UGT2B7) and analyzed the impact of gene variants on morphine glucuronidation in vivo. In all, 12 single nucleotide polymorphisms (SNPs) were identified, 10 of which have not been previously described. Only one SNP causes a change in amino acid sequence (H268Y). Seven UGT2B7 genotypes were observed and three main haplotypes predicted. There was no correlation between UGT2B7 genotype or haplotype and morphine glucuronide to morphine serum ratios among 175 patients who received chronic oral morphine therapy, and who had normal renal and hepatic function. The apparent lack of functional polymorphisms fits well with the near unimodal, but broad, distributions of the ratios (morphine 3-glucuronide/morphine: 6.4-309.2; morphine 6-glucuronide/morphine: 0.5-72.8). Our results suggest that factors other than UGT2B7 polymorphism may be more deciding for the variability in morphine glucuronide to morphine serum ratios.
Assuntos
Variação Genética , Glucuronídeos/metabolismo , Glucuronosiltransferase/genética , Morfina/metabolismo , Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Feminino , Genótipo , Glucuronosiltransferase/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Células Tumorais CultivadasRESUMO
The human UNG-gene at position 12q24.1 encodes nuclear (UNG2) and mitochondrial (UNG1) forms of uracil-DNA glycosylase using differentially regulated promoters, PA and PB, and alternative splicing to produce two proteins with unique N-terminal sorting sequences. PCNA and RPA co-localize with UNG2 in replication foci and interact with N-terminal sequences in UNG2. Mitochondrial UNG1 is processed to shorter forms by mitochondrial processing peptidase (MPP) and an unidentified mitochondrial protease. The common core catalytic domain in UNG1 and UNG2 contains a conserved DNA binding groove and a tight-fitting uracil-binding pocket that binds uracil only when the uracil-containing nucleotide is flipped out. Certain single amino acid substitutions in the active site of the enzyme generate DNA glycosylases that remove either thymine or cytosine. These enzymes induce cytotoxic and mutagenic abasic (AP) sites in the E. coli chromosome and were used to examine biological consequences of AP sites. It has been assumed that a major role of the UNG gene product(s) is to repair mutagenic U:G mispairs caused by cytosine deamination. However, one major role of UNG2 is to remove misincorporated dUMP residues. Thus, knockout mice deficient in Ung activity (Ung-/- mice) have only small increases in GC-->AT transition mutations, but Ung-/- cells are deficient in removal of misincorporated dUMP and accumulate approximately 2000 uracil residues per cell. We propose that BER is important both in the prevention of cancer and for preserving the integrity of germ cell DNA during evolution.
Assuntos
DNA Glicosilases , N-Glicosil Hidrolases/fisiologia , Timina/análogos & derivados , Animais , Ácido Apurínico/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Domínio Catalítico , Ciclo Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 12/genética , Reparo do DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Desoxirribonuclease (Dímero de Pirimidina) , Nucleotídeos de Desoxiuracil/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Genes , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Família Multigênica , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Pirimidinas/metabolismo , Timina/metabolismo , Uracila-DNA GlicosidaseRESUMO
We examined the cytotoxicity of doxorubicin alone, or in combination with docosahexaenoic acid (22:6 n-3), in glioblastoma cell lines A-172 and U-87 MG and bronchial carcinoma cell lines A-427 and SK-LU-1. For both glioblastoma cell lines we found an enhanced cytotoxicity of doxorubicin when given with concentrations of docosahexaenoic acid that alone are non-toxic. In SK-LU-1 cells no such enhancement was observed, whereas a small increase was observed for A-427 cells. The enhanced cytotoxicity in glioblastoma cells was not caused by lipid peroxidation products. In A-427 cells, however, the modest potentiation could be explained by the formation of cytotoxic lipid peroxidation products. Se-glutathione peroxidase activity increased after doxorubicin exposure and even more after addition of Na-selenite, but this did not reduce the cytotoxicity of doxorubicin. These results demonstrated that the mechanisms of enhancement of cytotoxicity by docosahexaenoic acid are complex and cell-specific and do not require increased lipid peroxidation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Broncogênico/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/farmacologia , Doxorrubicina/farmacologia , Glioblastoma/tratamento farmacológico , Carcinoma Broncogênico/metabolismo , Carcinoma Broncogênico/patologia , Divisão Celular/efeitos dos fármacos , Sinergismo Farmacológico , Ácidos Graxos Ômega-3/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Glutationa Peroxidase/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Uracil DNA glycosylase (UDG) is responsible for the removal of uracil present in DNA after cytosine deamination or misincorporation during replication. Colorectal cancer is widely treated with 5-FU, which leads to thymidylate synthase inhibition; this accounts for increased dUTP intracellular pools and subsequent uracil incorporation into DNA. Uracil misincorporation has also been implicated in the link between folate deficiency and colorectal cancer risk. As there is no information on UDG in colorectal cancer, this study characterized UDG activity and protein expression in a panel of 20 colorectal tumors and 6 colorectal cell lines. UDG activity in colorectal tissue is widely variable and it is statistically higher in tumor tissue (P=0.013) compared to normal bowel. Tumor versus normal activity ratios ranged from 0.49 to 2.2 (median 1.13). Among the six colorectal cell lines tested, UDG activity varied from 40 to 68 units and was markedly (1.7-fold) higher than in tumor tissue (P<0.0001). In both colorectal tissues and cell lines, UDG was expressed as both 29 kDa and 35 kDa forms. Total protein expression varied 3.2-fold in cell lines; variability was also found between patients and between normal and tumoral tissue for the same patient. This study demonstrates UDG protein and functional activity in human colorectal tumors and cell lines. The high tumor:normal tissue ratio supports further interest in base excision repair, through UDG, as a potential source of fluoropyrimidine resistance in colorectal cancer.
Assuntos
Neoplasias Colorretais/enzimologia , DNA Glicosilases , Mucosa Intestinal/enzimologia , N-Glicosil Hidrolases/metabolismo , Proteínas de Neoplasias/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , N-Glicosil Hidrolases/efeitos dos fármacos , Proteínas de Neoplasias/efeitos dos fármacos , Estatísticas não Paramétricas , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Uracila-DNA GlicosidaseRESUMO
Spontaneous deamination of cytosine results in a premutagenic G:U mismatch that may result in a GC-->AT transition during replication. The human UNG-gene encodes the major uracil-DNA glycosylase (UDG or UNG) which releases uracil from DNA, thus, initiating base excision repair to restore the correct DNA sequence. Bacterial and yeast mutants lacking the homologous UDG exhibit elevated spontaneous mutation frequencies. Hence, mutations in the human UNG gene could presumably result in a mutator phenotype. We screened all seven exons including exon-intron boundaries, both promoters, and one intron of the UNG gene and identified considerable sequence variation in cell lines derived from normal fibroblasts and tumour tissue. None of the sequence variants was accompanied by significantly reduced UDG activity. In the UNG gene from 62 sources, we identified 12 different variant alleles, with allele frequencies ranging from 0.01 to 0.23. We identified one variant allele per 3.8kb in non-coding regions, but none in the coding region of the gene. In promoter B we identified four different variants. A substitution within an AP2 element was observed in tumour cell lines only and had an allele frequency of 0.10. Introduction of this substitution into chimaeric promoter-luciferase constructs affected transcription from the promoter. UDG-activity varied little in fibroblasts, but widely between tumour cell lines. This variation did not however correlate with the presence of any of the variant alleles. In conclusion, mutations affecting the function of human UNG gene are seemingly infrequent in human tumour cell lines.
Assuntos
DNA Glicosilases , Variação Genética , N-Glicosil Hidrolases/genética , Células Cultivadas , Éxons/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Íntrons/genética , Masculino , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Células Tumorais Cultivadas , Uracila-DNA GlicosidaseRESUMO
Base excision repair (BER) of DNA corrects a number of spontaneous and environmentally induced genotoxic or miscoding base lesions in a process initiated by DNA glycosylases. An AP endonuclease cleaves at the 5' side of the abasic site and the repair process is subsequently completed via either short patch repair or long patch repair, which largely require different proteins. As one example, the UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) uracil DNA glycosylase and prevents accumulation of uracil in the genome. BER is likely to have a major role in preserving the integrity of DNA during evolution and may prevent cancer.
Assuntos
Pareamento Incorreto de Bases , DNA Glicosilases , Reparo do DNA , N-Glicosil Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , Dano ao DNA , Humanos , Mamíferos , Dados de Sequência Molecular , N-Glicosil Hidrolases/química , Uracila-DNA GlicosidaseRESUMO
Acrolein is a highly reactive unsaturated aldehyde formed endogenously and present in the environment. Acrolein efficiently reduces glutathione-contents and is highly cytotoxic in two lung carcinoma cell lines (A-427 and SK-LU-1) and the glioblastoma cell line A-172. A-427, which has the lowest GSH content of the cell lines, is also more sensitive to growth inhibition and more depleted in GSH after acrolein exposure. A-427 is also highly sensitive to docosahexaenoic acid (22:6 n-3, DHA) and acrolein potentiates the cytotoxic effect of DHA in this cell line, but not in the DHA-resistant cell lines SK-LU-1 and A-172. Surprisingly, the cytotoxic effect of acrolein was partially reversed by vitamin E, selenite and 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen, a Se-glutathione peroxidase mimic) in A-427 cells, but not in SK-LU-1 and A-172 cells. Using the TUNEL assay a strong nuclear fluorescence was observed in DHA-treated A-427 cells, indicating death by apoptosis, whereas acrolein apparently did not induce apoptosis.
Assuntos
Acroleína/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Glutationa/metabolismo , Apoptose/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Glutationa Peroxidase/metabolismo , Humanos , Peroxidação de Lipídeos , Ácido Pirrolidonocarboxílico , Tiazóis/farmacologia , Tiazolidinas , Células Tumorais CultivadasRESUMO
DNA repair is of fundamental importance for protection of the genetic material against mutations in an interplay with mechanisms that regulate the cell cycle, gene expression, and programmed cell death. Defects in DNA repair, or in processes in tegrated with DNA repair, may give cells a hyper mutable phenotype that increases the likelihood of mutations in genes controlling cell growth. Two principally different DNA repair mechanisms are known; (a) direct repair of a damaged base by a single enzyme without using information from the complementary strand, and (b) excision repair, in which DNA containing the damage is removed and replaced by new DNA using DNA repair synthesis. Mechanisms for excision repair are complex and comprise base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and recombination repair. In addition, the cell has mechanisms for repair of strand breaks. It has recently become clear that defective MMR is the cause of hereditary nonpolyposis colon cancer (HNPCC), and probably some 15% of the cases of sporadic colon cancer. There is also evidence that defective repair may be a primary cause of certain other forms of cancer.
Assuntos
Dano ao DNA , Reparo do DNA , Enzimas/genética , Análise Mutacional de DNA , HumanosRESUMO
We studied the repair of cyclobutane pyrimidine dimers (CPDs) in the 5' terminal part of the transcriptionally inactive O6-methylguanine-DNA methyltransferase (MGMT) gene of MGMT-deficient human cell lines (A172, A-253 and WI-38 VA13) and in a proficient cell line (HaCaT), in which the MGMT gene was transcribed. Repair rates in the MGMT gene were compared with those in the active uracil-DNA glycosylase (UNG) and c-myc genes, and those in the repressed X-linked 754 locus and the RNA polymerase I-transcribed ribosomal gene cluster. In the active MGMT gene, there was a distinct strand specificity with more repair in the template (transcribed) strand (TS) than in the non-template strand (NTS). In contrast, no apparent strand bias in the repair of CPDs was observed in the inactive MGMT gene in the MGMT deficient cell lines, although the rates of repair varied between different cell lines. Repair in the inactive MGMT gene was consistently lower than repair in the NTSs of the expressed genes, and approached the generally poor repair of the repressed 754 locus. Whereas repair in the UNG gene was strand-specific in HaCaT, A-172 and WI-38 VA13 cells, no clear strand bias in repair of this gene was evident in A253 cells and repair was relatively inefficient. Although the repair kinetics was essentially similar in the two strands of the c-myc gene in all cell lines examined, the rate and extent of repair were in general significant, probably due to an observed transcription of both strands in the c-myc region. In conclusion, our results indicate that the relative rates of repair in inactive MGMT genes are comparable to those of repressed loci and are lower than repair rates in the NTSs of active genes, but the absolute rate of repair varies between different transformed cells.
Assuntos
Reparo do DNA , O(6)-Metilguanina-DNA Metiltransferase/genética , Dímeros de Pirimidina/genética , Cromossomo X/genética , Linhagem Celular , HumanosRESUMO
We have studied the effect of low levels of paracetamol (0.3 and 1.0 mM) on gene-specific DNA repair, recovery of total RNA synthesis and cytotoxicity after exposure of human keratinocyte cells (HaCaT) to ultraviolet (UV) irradiation. Repair of cyclobutane pyrimidine dimers (CPDs) was measured in the transcriptionally active uracil-DNA glycosylase (UNG) and c-MYC loci. Repair of both strands in the UNG gene was consistently lower in the presence of paracetamol, but this reduction reached significance only at 8 h after irradiation and no dose-response was observed. For the c-MYC gene, we found no significant effect of paracetamol on the repair of CPDs, possibly because UV-irradiation is known to induce transcription of the c-MYC gene and enhanced transcription coupled repair might counteract a negative effect of paracetamol on global genome repair. A dose-dependent delay in the recovery of total RNA synthesis after UV exposure was observed in the presence of paracetamol, which also caused a 20% increase in UV-induced cytotoxicity after 24 h. Paracetamol had no significant effect on either RNA synthesis or cell survival in the absence of UV after 24 h, but reduced cell survival by approximately 10% (at 0.3 mM) and 50%, (at 1.0 mM) after 96 h exposure. Our results demonstrate that paracetamol may inhibit gene-specific repair of CPDs by affecting global genome repair and that different genes may be differentially affected.
Assuntos
Acetaminofen/farmacologia , Analgésicos não Narcóticos/farmacologia , DNA Glicosilases , Reparo do DNA/efeitos dos fármacos , Queratinócitos/efeitos da radiação , N-Glicosil Hidrolases/genética , Raios Ultravioleta/efeitos adversos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Reparo do DNA/genética , Relação Dose-Resposta a Droga , Genes myc/efeitos dos fármacos , Genes myc/genética , Humanos , Queratinócitos/efeitos dos fármacos , Dímeros de Pirimidina/metabolismo , RNA/biossíntese , Uracila-DNA GlicosidaseRESUMO
Promoters PA and PBin the UNG gene and alternative splicing are utilized to generate nuclear (UNG2) and mitochondrial (UNG1) forms of human uracil-DNA glycosylase. We have found the highest levels of UNG1 mRNA in skeletal muscle, heart and testis and the highest UNG2 mRNA levels in testis, placenta, colon, small intestine and thymus, all of which contain proliferating cells. In synchronized HaCaT cells mRNAs for both forms increased in late G1/early S phase, accompanied by a 4- to 5-fold increase in enzyme activity. A combination of mutational analysis and transient transfection demonstrated that an E2F-1/DP-1-Rb complex is a strong negative regulator of both promoters, whereas 'free' E2F-1/DP-1 is a weak positive regulator, although a consensus element for E2F binding is only present in PB. These results indicate a central role for an E2F-DP-1-Rb complex in cell cycle regulation of UNG proteins. Sp1 and c-Myc binding elements close to transcription start areas were positive regulators of both promoters, however, whereas overexpression in HeLa cells of Sp1 stimulated both promoters, c-Myc and c-Myc/Max overexpression had a suppressive effect. CCAAT elements were negative regulators of PB, but positive regulators of PA. These results demonstrate differential expression of mRNAs for UNG1 and UNG2 in human tissues.
Assuntos
Proteínas de Transporte , Núcleo Celular/enzimologia , DNA Glicosilases , Proteínas de Ligação a DNA , Regulação Enzimológica da Expressão Gênica , Mitocôndrias/enzimologia , N-Glicosil Hidrolases/genética , Processamento Alternativo , Sequência de Bases , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Feminino , Células HeLa , Humanos , Isoenzimas/genética , Masculino , Mutagênese Sítio-Dirigida , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Distribuição Tecidual , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Uracila-DNA GlicosidaseRESUMO
We examined effects of polyunsaturated fatty acids (PUFA), their corresponding hydroperoxy fatty acids (hp-PUFA), as well as various pro- and antioxidants on the growth of tumor cells in culture. When cultured in RPMI 1640 medium, A-427 and WEHI clone 13 cells were both highly sensitive to hydroperoxy docosahexaenoic acid (hp-DHA), but they were far less sensitive in minimum essential medium (MEM). In contrast, A-427 cells were also sensitive to DHA in both culture media, while WEHI clone 13 cells, as well as other cell lines, tested in their respective media, were resistant. The lower sensitivity of the cell lines to hp-DHA in MEM-medium was apparently due to a more rapid reduction of hp-DHA to the corresponding hydroxy-DHA in MEM-medium. Addition of glutathione (GSH) to the culture medium abolished the effects of hp-DHA, but not the effects of DHA, while depletion of intracellular GSH levels by L-buthionine-S,R-sulfoximine strongly enhanced the cytotoxic effect of hp-DHA, but not the cytotoxic effect of DHA. alpha-Tocopherol protected A-427 cells against the toxic effect of DHA and abolished the induced lipid peroxidation, while it did not protect against the toxic effects of hp-DHA in A-427 or WEHI clone 13 cells. Ascorbic acid reduced the cytotoxic effect of DHA, but potentiated the toxic effect of hp-DHA while selenite essentially abolished the toxicity of both DHA and hp-DHA. These results indicate that sensitivity of tumor cell lines to PUFA and their oxidation products depends on their antioxidant defense mechanisms, as well as culture conditions, and establishes hp-DHA as a major, but probably not the sole, metabolite responsible for cytotoxicity of DHA.
Assuntos
Divisão Celular/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Peróxidos Lipídicos/farmacologia , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Butionina Sulfoximina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Meios de Cultura/química , Ácidos Docosa-Hexaenoicos/toxicidade , Ácidos Graxos Ômega-6 , Glutationa/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Peróxidos Lipídicos/toxicidade , Maleatos/farmacologia , Camundongos , Células Tumorais Cultivadas , Vitamina E/farmacologiaRESUMO
The human lung adenocarcinoma cell line A-427 is significantly more sensitive to cytotoxic lipid peroxidation products of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) than the human lung adenocarcinoma cell line SK-LU-1, and the glioblastoma cell lines A-172 and U-87 MG. The cytotoxic effect as well as lipid peroxidation were abolished by vitamin E. The differential sensitivities of the cell lines were not correlated to the levels of lipid peroxidation products (measured as the end product malondialdehyde), indicating differences in sensitivities to products of lipid peroxidation. The high sensitivity of A-427 is apparently due to a low level of selenium-dependent glutathione peroxidase (GSH-Px), because pretreatment with sodium selenite (250 nM) increased the GSH-Px activity 3- to 4-fold and protected the cells almost completely against the growth inhibitory effect of DHA. Furthermore, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) a seleno-organic GSH-Px mimic, suppressed the cytotoxic action of DHA to A-427 in a dose dependent manner. Northern analysis demonstrated that pretreatment with sodium selenite (250 nM) was accompanied by an increased level of GSH-Px mRNA (1.8-fold) in A-427 cells, while the level remained unchanged under the same conditions in DHA/EPA-resistant A-172 cells. In addition, the level of selenophosphate synthetase mRNA (SelD), a key intermediate in tRNA(Sec) formation, increased 1.2- to 1.7-fold in A-427 and A-172 cells after pretreatment with sodium selenite. These results indicate that upregulation of GSH-Px activity by sodium selenite in the EPA/DHA sensitive cell line A-427 may be due to an increase in mRNAs for GSH-Px and a precursor important for formation of tRNA(Sec) which is required for incorporation of selenocysteine in GSH-Px during translation. These results demonstrate an important role for GSH-Px in the cellular defence against cytotoxic lipid peroxidation products. Furthermore, measurement of GSH-Px activities in tumour cells may be one useful biochemical predictor for their sensitivities to polyunsaturated fatty acids.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Proteínas de Drosophila , Ácido Eicosapentaenoico/farmacologia , Glutationa Transferase/metabolismo , Fosfotransferases , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Proteínas de Bactérias/metabolismo , Divisão Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Malondialdeído/metabolismo , Selenito de Sódio/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast two-hybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single- or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair.
Assuntos
DNA Glicosilases , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , N-Glicosil Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Núcleo Celular/enzimologia , Proteínas de Ligação a DNA/química , Humanos , Camundongos , Dados de Sequência Molecular , N-Glicosil Hidrolases/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ratos , Proteína de Replicação A , Uracila-DNA Glicosidase , Xeroderma Pigmentoso/genética , Proteína de Xeroderma Pigmentoso Grupo ARESUMO
A distinct nuclear form of human uracil-DNA glycosylase [UNG2, open reading frame (ORF) 313 amino acid residues] from the UNG gene has been identified. UNG2 differs from the previously known form (UNG1, ORF 304 amino acid residues) in the 44 amino acids of the N-terminal sequence, which is not necessary for catalytic activity. The rest of the sequence and the catalytic domain, altogether 269 amino acids, are identical. The alternative N-terminal sequence in UNG2 arises by splicing of a previously unrecognized exon (exon 1A) into a consensus splice site after codon 35 in exon 1B (previously designated exon 1). The UNG1 sequence starts at codon 1 in exon 1B and thus has 35 amino acids not present in UNG2. Coupled transcription/translation in rabbit reticulocyte lysates demonstrated that both proteins are catalytically active. Similar forms of UNG1 and UNG2 are expressed in mouse which has an identical organization of the homologous gene. Constructs that express fusion products of UNG1 or UNG2 and green fluorescent protein (EGFP) were used to study the significance of the N-terminal sequences in UNG1 and UNG2 for subcellular targeting. After transient transfection of HeLa cells, the pUNG1-EGFP-N1 product colocalizes with mitochondria, whereas the pUNG2-EGFP-N1 product is targeted exclusively to nuclei.
Assuntos
Processamento Alternativo , Núcleo Celular/enzimologia , Núcleo Celular/genética , DNA Glicosilases , Mitocôndrias Hepáticas/enzimologia , N-Glicosil Hidrolases/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma Embrionário , Humanos , Camundongos , Mitocôndrias Hepáticas/metabolismo , Dados de Sequência Molecular , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/metabolismo , Neurônios , Homologia de Sequência de Aminoácidos , Células-Tronco , Células Tumorais Cultivadas , Uracila-DNA GlicosidaseRESUMO
O6-Methylguanine-DNA methyltransferase (MGMT) plays an important role in protecting cells from the mutagenic potency of alkylating agents. This study addresses the role of DNA methylation in the expression of the human MGMT gene. Southern blot analysis of DNA from human Mer+ (MGMT proficient) and Mer- (MGMT deficient) cell lines demonstrated that the methylation state of a unique SmaI site in the MGMT gene promoter, previously shown by others to be invariably unmethylated in Mer+ cells and methylated in Mer- cells, did not correlate with the Mer phenotype. Neither was there any significant difference in the density of CpG methylation in the MGMT gene 5'-flanking sequences between Mer+ and Mer- cells. On the other hand, the body of the MGMT gene was less methylated in most Mer- cells relative to Mer+ cells, and in three of six Mer- cell lines the gene was essentially methylation-free. Interestingly, the Mer- cells that were hypomethylated in the MGMT gene also tended to be less methylated at other loci. Widespread hypomethylation is a frequent trait in carcinogenesis, and may be involved in development of the frequently found Mer- phenotype.
Assuntos
Metiltransferases/genética , Sequência de Bases , Linhagem Celular , Humanos , Metilação , Metiltransferases/deficiência , O(6)-Metilguanina-DNA MetiltransferaseRESUMO
We have examined the effects of linoleic acid, LA (18:2 n-6) and its naturally occurring conjugated derivatives (CLA) on the growth of three different lung adenocarcinoma cell lines (A-427, SK-LU-1, A549) and one human glioblastoma cell line (A-172). CLA exerted a dose dependent reduction in proliferation of the lung adenocarcinoma cell lines with A-427 being the most sensitive one, but had virtually no effect on A-172. In contrast, LA had no inhibitory effect on either cell line. A significant increase in lipid peroxidation (measured as formation of malondialdehyde, MDA) was observed after exposure of the lung adenocarcinoma cell lines to 40 microM CLA. This level was approximately 2-fold higher than after exposure to 40 microM LA. The formation of MDA was completely abolished by 30 microM vitamin E, but the growth rates were only partially restored, indicating that cytotoxic lipid peroxidation products are only in part responsible for the growth inhibitory effects of CLA.