RESUMO
p53-regulated caspase-independent cell death has been implicated in suppression of tumorigenesis, however, the regulating mechanisms are poorly understood. We previously reported that 8-oxoguanine (8-oxoG) accumulation in nuclear DNA (nDNA) and mitochondrial DNA triggers two distinct caspase-independent cell death through buildup of single-strand DNA breaks by MutY homolog (MUTYH), an adenine DNA glycosylase. One pathway depends on poly-ADP-ribose polymerase (PARP) and the other depends on calpains. Deficiency of MUTYH causes MUTYH-associated familial adenomatous polyposis. MUTYH thereby suppresses tumorigenesis not only by avoiding mutagenesis, but also by inducing cell death. Here, we identified the functional p53-binding site in the human MUTYH gene and demonstrated that MUTYH is transcriptionally regulated by p53, especially in the p53/DNA mismatch repair enzyme, MLH1-proficient colorectal cancer-derived HCT116+Chr3 cells. MUTYH-small interfering RNA, an inhibitor for p53 or PARP suppressed cell death without an additive effect, thus revealing that MUTYH is a potential mediator of p53 tumor suppression, which is known to be upregulated by MLH1. Moreover, we found that the p53-proficient, mismatch repair protein, MLH1-proficient colorectal cancer cell line express substantial levels of MUTYH in nuclei but not in mitochondria, suggesting that 8-oxoG accumulation in nDNA triggers MLH1/PARP-dependent cell death. These results provide new insights on the molecular mechanism of tumorigenesis and potential new strategies for cancer therapies.
RESUMO
Inosine triphosphate pyrophosphatase (ITPase), the enzyme that hydrolyzes ITP and other deaminated purine nucleoside triphosphates to the corresponding purine nucleoside monophosphate and pyrophosphate, is encoded by the Itpa gene. In this study, we established Itpa knockout (KO) mice and used them to show that ITPase is required for the normal organization of sarcomeres in the heart. Itpa(-/-) mice died about 2 weeks after birth with features of growth retardation and cardiac myofiber disarray, similar to the phenotype of the cardiac alpha-actin KO mouse. Inosine nucleotides were found to accumulate in both the nucleotide pool and RNA of Itpa(-/-) mice. These data suggest that the role of ITPase in mice is to exclude ITP from the ATP pool, and the main target substrate of this enzyme is rITP. Our data also suggest that cardiomyopathy, which is mainly caused by mutations in sarcomeric protein-encoding genes, is also caused by a defect in maintaining the quality of the ATP pool, which is an essential requirement for sarcomere function.
Assuntos
Cardiomiopatias/enzimologia , Transtornos do Crescimento/enzimologia , Pirofosfatases/fisiologia , Actinas/genética , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Cardiomiopatias/genética , Cardiomiopatias/patologia , Feminino , Genótipo , Transtornos do Crescimento/genética , Transtornos do Crescimento/mortalidade , Nucleotídeos de Inosina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Miocárdio/patologia , Fenótipo , Pirofosfatases/deficiência , Pirofosfatases/genética , RNA Mensageiro/metabolismo , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Desmame , Inosina TrifosfataseRESUMO
We examined the expression of galectin-1, an endogenous lectin with one carbohydrate-binding domain, in the adult mouse hippocampus after systemic kainate administration. We found that the expression of galectin-1 was remarkably increased in activated astrocytes of the CA3 subregion and dentate gyrus of the hippocampus, and in nestin-positive neural progenitors in the dentate gyrus. Quantitative reverse transcription PCR (RT-PCR) analysis revealed that the galectin-1 mRNA level in hippocampus began to increase 1 day after kainate administration and that a 13-fold increase was attained within 3 days. Western blotting analysis confirmed that the level of galectin-1 protein increased to more than three-fold a week after the exposure. We showed that isolated astrocytes express and secrete galectin-1. To clarify the significance of the increased expression of galectin-1 in hippocampus, we compared the levels of hippocampal cell proliferation in galectin-1 knockout and wild-type mice after saline or kainate administration. The number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells detected in the subgranular zone (SGZ) of galectin-1 knockout mice decreased to 62% with saline, and to 52% with kainate, as compared with the number seen in the wild-type mice. Most of the BrdU-positive cells in SGZ expressed doublecortin and neuron-specific nuclear protein, indicating that they are immature neurons. We therefore concluded that galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the hippocampus.
Assuntos
Proliferação de Células , Giro Denteado , Galectina 1/metabolismo , Ácido Caínico/metabolismo , Neurônios/fisiologia , Células-Tronco/metabolismo , Animais , Giro Denteado/citologia , Giro Denteado/metabolismo , Galectina 1/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Células-Tronco/citologiaRESUMO
Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To gain more insight into the physiological functions of Elongin A, we generated Elongin A-deficient mice. Elongin A homozygous mutant (Elongin A(-/-)) embryos demonstrated a severely retarded development and died at between days 10.5 and 12.5 of gestation, most likely due to extensive apoptosis. Moreover, mouse embryonic fibroblasts (MEFs) derived from Elongin A(-/-) embryos exhibited not only increased apoptosis but also senescence-like growth defects accompanied by the activation of p38 MAPK and p53. Knockdown of Elongin A in MEFs by RNA interference also dramatically induced the senescent phenotype. A study using inhibitors of p38 MAPK and p53 and the generation of Elongin A-deficient mice with p53-null background suggests that both the p38 MAPK and p53 pathways are responsible for the induction of senescence-like phenotypes, whereas additional signaling pathways appear to be involved in the mediation of apoptosis in Elongin A(-/-) cells. Taken together, our results suggest that Elongin A is required for the transcription of genes essential for early embryonic development and downregulation of its activity is tightly associated with cellular senescence.
Assuntos
Apoptose/genética , Senescência Celular/genética , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Animais , Elonguina , Feminino , Morte Fetal/genética , Feto/anormalidades , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We previously reported that 8-oxoguanine (8-oxoG) accumulates in the cytoplasm of dopamine neurons in the substantia nigra of patients with Parkinson's disease and the expression of MTH1 carrying an oxidized purine nucleoside triphosphatase activity increases in these neurons, thus suggesting that oxidative damage in nucleic acids is involved in dopamine neuron loss. In the present study, we found that levels of 8-oxoG in cellular DNA and RNA increased in the mouse nigrostriatal system during the tyrosine hydroxylase (TH)-positive dopamine neuron loss induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MTH1-null mice exhibited a greater accumulation of 8-oxoG in mitochondrial DNA accompanied by a more significant decrease in TH and dopamine transporter immunoreactivities in the striatum after MPTP administration, than in wild-type mice. We thus demonstrated that MTH1 protects the dopamine neurons from oxidative damage in the nucleic acids, especially in the mitochondrial DNA of striatal nerve terminals of dopamine neurons.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , DNA Mitocondrial/metabolismo , Dopamina/metabolismo , Guanina/análogos & derivados , Neurônios/enzimologia , Transtornos Parkinsonianos/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Corpo Estriado/enzimologia , Corpo Estriado/patologia , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/genética , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Guanina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/patologia , Estresse Oxidativo , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/patologia , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , RNA/metabolismo , Substância Negra/enzimologia , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
We previously identified a novel N-terminally processed form of galectin-1, galectin-1beta (Gal-1beta) whose expression was induced by DeltaFosB. In the present study, the biochemical properties and biological functions of Gal-1beta were compared with the full-length form of galectin-1 (Gal-1alpha). We first purified recombinant mouse Gal-1alpha and beta (rmGal-1alpha, beta) to near homogeneity. The rmGal-1alpha exists as a monomer under oxidized conditions and forms a dimer under reduced conditions, while the rmGal-1beta exists as a monomer regardless of redox conditions. The affinity of rmGal-1beta to beta-lactose was approximately two-fold lower than that of rmGal-1alpha under reduced conditions. The viability of Jurkat cells efficiently decreased when they were exposed to rmGal-1alpha, however, rmGal-1beta barely induced such a reduction. In contrast, both rmGal-1alpha and rmGal-1beta exhibited an equivalent capacity to promote axonal regeneration from the dorsal root ganglion explants. Our results suggest that the biochemical properties of rmGal-1beta determine its biological functions.
Assuntos
Axônios/efeitos dos fármacos , Axônios/metabolismo , Galectina 1/química , Galectina 1/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Dicroísmo Circular , Dimerização , Galectina 1/genética , Galectina 1/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Células Jurkat , Lectinas/farmacologia , Camundongos , Modelos Moleculares , Oxirredução , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-AtividadeRESUMO
In human cells APE1 is the major AP endonuclease and it has been reported to have no functional mitochondrial targeting sequence (MTS). We found that APE2 protein possesses a putative MTS. When its N-terminal 15 amino acid residues were fused to the N-terminus of green fluorescent protein and transiently expressed in HeLa cells the fusion protein was localized in the mitochondria. By electron microscopic immunocytochemistry we detected authentic APE2 protein in mitochondria from HeLa cells. Western blotting of the subcellular fraction of HeLa cells revealed most of the APE2 protein to be localized in the nuclei. We found a putative proliferating cell nuclear antigen (PCNA)-binding motif in the C-terminal region of APE2 and showed this motif to be functional by immunoprecipitation and in vitro pull-down binding assays. Laser scanning immunofluorescence microscopy of HeLa cells demonstrated both APE2 and PCNA to form foci in the nucleus and also to be co-localized in some of the foci. The incubation of HeLa cells in HAT medium containing deoxyuridine significantly increased the number of foci in which both molecules were co-localized. Our results suggest that APE2 participates in both nuclear and mitochondrial BER and also that nuclear APE2 functions in the PCNA-dependent BER pathway.
Assuntos
Carbono-Oxigênio Liases/metabolismo , Núcleo Celular/enzimologia , Mitocôndrias/enzimologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico/genética , Carbono-Oxigênio Liases/genética , DNA Complementar/química , DNA Complementar/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Regulação Enzimológica da Expressão Gênica , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Células Jurkat , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
O(6)-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the MGMT: gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. MGMT(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD(50) values of MGMT(-/-) and MGMT(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of MGMT(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of MGMT(+/+) and MGMT(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O(6)-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. MGMT(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are MGMT(+/+) mice. Thus, the level of O(6)-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.
Assuntos
Antineoplásicos Alquilantes/toxicidade , O(6)-Metilguanina-DNA Metiltransferase/deficiência , Adenoma/induzido quimicamente , Animais , Morte Celular/efeitos dos fármacos , Ciclofosfamida/toxicidade , Dacarbazina/toxicidade , Feminino , Dose Letal Mediana , Neoplasias Pulmonares/induzido quimicamente , Linfoma/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Nimustina/toxicidade , O(6)-Metilguanina-DNA Metiltransferase/genética , Testículo/efeitos dos fármacos , Neoplasias do Timo/induzido quimicamenteRESUMO
Mice with mutations in both alleles of the Mgmt and the Mlh1 gene, the former encoding a DNA repair methyltransferase and the latter a protein functioning at an early step of mismatch repair, are as resistant to the killing action of alkylating agents as are wild-type mice. These mice yielded a large number of tumors when exposed to alkylating carcinogens, but this characteristic was subdued since they also showed a relatively high level of spontaneous tumorigenicity, as a consequence of the defect in mismatch repair. This complexity is now resolved by introducing the Mlh1(+/-) mutation, instead of Mlh1(-/-), in these methyltransferase-deficient mice. Mgmt(-/-) Mlh1(+/-) mice, with about half the amount of MLH1 protein as Mgmt(-/-) Mlh1(+/+) mice, were resistant to the killing action of N-methyl-N-nitrosourea (MNU), up to the level of 30 mg/kg body wt. Eight weeks after exposure to this dose of MNU, 40% of MNU-treated Mgmt(-/-) Mlh1(+/-) mice had thymic lymphomas and there were no tumors in those mice not given the treatment. It seems that the cellular content of MLH1 protein is a critical factor for determining if damaged cells enter into either one of the two pathways leading to mutation induction or to apototic cell death. Loss of Mlh1 expression was frequently observed in tumors of Mgmt(-/-) Mlh1(+/-) mice and this might be related to progression of the tumors.
Assuntos
Alquilantes/toxicidade , Alelos , Carcinógenos/toxicidade , Deleção de Genes , Neoplasias Intestinais/genética , Linfoma/genética , Metilnitrosoureia/toxicidade , Proteínas de Neoplasias/genética , O(6)-Metilguanina-DNA Metiltransferase/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Alquilantes/farmacologia , Animais , Carcinógenos/farmacologia , Proteínas de Transporte , Códon/genética , Análise Mutacional de DNA , DNA de Neoplasias/genética , Progressão da Doença , Genes ras/efeitos dos fármacos , Predisposição Genética para Doença , Genótipo , Neoplasias Intestinais/induzido quimicamente , Dose Letal Mediana , Linfoma/induzido quimicamente , Metilnitrosoureia/farmacologia , Camundongos , Camundongos Knockout , Proteína 1 Homóloga a MutL , Proteínas de Neoplasias/fisiologia , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Proteínas Nucleares , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/fisiologia , Neoplasias do Timo/induzido quimicamente , Neoplasias do Timo/genéticaRESUMO
Alkylation of DNA at the O6-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. The enzyme O6-methylguanine-DNA methyltransferase repairs O6-methylguanine as well as a minor methylated base, O4-methylthymine, in DNA. Mouse lines deficient in the methyltransferase (MGMT) gene are hypersensitive to both the killing and to the tumorigenic effects of alkylating agents. We now show that these dual effects of an alkylating agent can be dissociated by introduction of an additional defect in mismatch repair. Mice with mutations in both alleles of the MGMT gene and one of the mismatch repair genes, MLH1, are as resistant to methylnitrosourea (MNU) as are wild-type mice, in terms of survival, but do have numerous tumors after receiving MNU. In contrast to MGMT-/- MLH1(+/+) mice with decrease in size of the thymus and hypocellular bone marrow after MNU administration, no conspicuous change was found in MGMT-/- MLH1(-/-) mice treated in the same manner. Thus, killing and tumorigenic effects of an alkylating agent can be dissociated by preventing mismatch repair pathways.
Assuntos
Alquilantes , Reparo do DNA , Proteínas de Neoplasias/fisiologia , Neoplasias Experimentais/induzido quimicamente , O(6)-Metilguanina-DNA Metiltransferase/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Alquilantes/toxicidade , Animais , Proteínas de Transporte , Feminino , Linfoma/induzido quimicamente , Linfoma/patologia , Masculino , Metilnitrosoureia , Camundongos , Camundongos Knockout , Proteína 1 Homóloga a MutL , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Nucleares , Análise de Sobrevida , Neoplasias do Timo/induzido quimicamente , Neoplasias do Timo/patologiaRESUMO
To investigate the substrate specificity of human O6-methylguanine-DNA methyltransferase (MGMT) for O6-benzylguanine (6BG) derivatives incorporated in oligodeoxynucleotides, we prepared 25-mer lengths of sequences containing various 6BG derivatives and their related compounds and then measured the ability of these derivatives to inactivate MGMT in vitro. Oligodeoxynucleotides containing a 6BG, O6-(2-fluorobenzyl)guanine (2F-6BG), O6-(3-fluorobenzyl)guanine (3F-6BG), O6-(4-fluorobenzyl)guanine (4F-6BG), O6-benzylhypoxanthine (6BH), or O6-methylguanine (6MG) were all good substrates for MGMT, and no obvious differences were observed among them. Oligodeoxynucleotides containing N2-isobutyrylated 6BG and 6MG showed only a slightly reduced capacity for inactivating MGMT compared to N2-nonmodified forms of these derivatives. No obvious differences were observed in the corresponding double-stranded and single-stranded oligodeoxynucleotides. MGMT substrate specificity for the 6BG derivatives in the oligodeoxynucleotide was found to be quite different from that seen in our previous study [Mineura, K., et al. (1994) Int. J. Cancer 58, 706-712; (1995) Int. J. Cancer 63, 148-151. Kohda, K., et al. (1995) Biol. Pharm. Bull. 18, 424-430] and others [Moschel, R. C., et al. (1992) J. Med. Chem. 35, 4486-4491. Chae, M.Y., et al. (1994) J. Med. Chem. 37, 342-347] using the corresponding free bases. In brief, (i) 6BG, 3F-6BG, and 4F-6BG greatly inhibited human MGMT, whereas 2F-6BG, 6BH, and 6MG displayed much weaker activity; (ii) any modifications at the 2-amino group of the 6BG resulted in severe reductions in the ability to inactivate MGMT. These results obtained by the experiments using oligodeoxynucleotides and free bases suggest that human MGMT has low substrate specificity for 6BGs in oligodeoxynucleotides. Conformational changes in human MGMT which favor binding to oligodeoxynucleotides containing 6BG derivatives and the subsequent transfer of their benzyl groups may account for the difference in substrate specificity between the incorporated 6BG derivatives and their free base form.
Assuntos
Antineoplásicos/metabolismo , Guanina/análogos & derivados , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Oligonucleotídeos/metabolismo , DNA/química , Guanina/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , O(6)-Metilguanina-DNA Metiltransferase/isolamento & purificação , Especificidade por SubstratoRESUMO
The enzyme O6-methylguanine-DNA methyltransferase repairs alkylation-induced DNA damage, O6-methylguanine and O4-methylthymine, the former being formed more frequently. Previously, by means of gene targeting, we generated mice in which alleles for methyltransferase were disrupted. We now use these mouse lines, which are totally deficient in methyltransferase activity, to examine protective effects of the enzyme against tumor formation. In gene-targeted female mice given an i.p. injection of 5 mg/kg of dimethylnitrosamine, a larger number of liver and lung tumors occurred, as compared with normal female mice treated in the same manner. In male mice given a lower dose of carcinogen, the difference between normal and gene-targeted mice was statistically insignificant although more tumors did form in the gene-targeted mice. Methyltransferase apparently afforded protection from nitrosamine-induced tumorigenesis.
Assuntos
Neoplasias Hepáticas/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Metiltransferases/fisiologia , Animais , Dimetilnitrosamina , Feminino , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Masculino , Metiltransferases/deficiência , Camundongos , Camundongos Knockout , O(6)-Metilguanina-DNA Metiltransferase , Fatores SexuaisRESUMO
Gene targeting was used to obtain mice defective in the MGMT gene, encoding O6-methylguanine-DNA methyltransferase [Tsuzuki et al., Carcinogenesis (Lond.), 17: 1215-1220, 1996]. These MGMT-/- mice were most sensitive to the alkylating carcinogen, methylnitrosourea; when varied doses of methylnitrosourea were administered to 6-week-old mice and survivals at the 30th day were determined, LD50s of MGMT-/- and MGMT+/+ mice were 20 and 240 mg/kg of body weight, respectively. MGMT+/- mice were as resistant as MGMT+/+ mice, but some difference in survival time was noted when the two genotypes of mice were exposed to a relatively high dose of methylnitrosourea. A large number of thymic lymphomas, as well as lung adenomas, occurred in MGMT-/- mice exposed to methylnitrosourea at a dose of 2.5 mg/kg of body weight. In case of exposure to the same dose of drug, no or few tumors occurred in the MGMT+/+ and MGMT+/- mice. It appears that the DNA repair methyltransferase protein protected these mice from methylnitrosourea-induced tumorigenesis.
Assuntos
Carcinógenos , Neoplasias Pulmonares/induzido quimicamente , Metilnitrosoureia , Metiltransferases/fisiologia , Neoplasias do Timo/induzido quimicamente , Animais , Dosagem de Genes , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/patologia , Linfoma/patologia , Metiltransferases/genética , Camundongos , Camundongos Knockout , O(6)-Metilguanina-DNA Metiltransferase , Taxa de Sobrevida , Neoplasias do Timo/patologiaRESUMO
Alkylation of DNA at the O6-position of guanine is one of the most critical events leading to induction of mutation as well as cancer. An enzyme, O6-methylguanine-DNA methyltransferase, is present in various organisms, from bacteria to human cells, and appears to be responsible for preventing the occurrence of such mutations. The enzyme transfers methyl groups from O6-methylguanine and other methylated moieties of the DNA to its own molecule, thereby repairing DNA lesions in a single-step reaction. To elucidate the role of methyltransferase in preventing cancer, animal models with altered levels of enzyme activity were generated. Transgenic mice carrying extra copies of the foreign methyltransferase gene showed a decreased susceptibility to alkylating carcinogens, with regard to tumor formation. By means of gene targeting, mouse lines defective in both alleles of the methyltransferase gene were established. Administration of methylnitrosourea to these gene-targeted mice led to early death while normal mice treated in the same manner showed no untoward effects. Numerous tumors were formed in the gene-defective mice exposed to a low dose of methylnitrosourea, while none or only few tumors were induced in the methyltransferase-proficient mice. It seems apparent that the DNA repair methyltransferase plays an important role in lowering a risk of occurrence of cancer in organisms.
Assuntos
Reparo do DNA , Metiltransferases/fisiologia , Mutagênese , Alquilantes/toxicidade , Alquilação , Sequência de Aminoácidos , Animais , Carcinógenos/toxicidade , Escherichia coli/enzimologia , Escherichia coli/genética , Feminino , Humanos , Masculino , Metiltransferases/química , Metiltransferases/deficiência , Metiltransferases/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , O(6)-Metilguanina-DNA Metiltransferase/fisiologiaRESUMO
Alkylation of DNA at the O(6)-position of guanine is one of the most critical events leading to induction of mutation as well as to cancer. The enzyme O(6)-methylguanine-DNA methyltransferase repairs this and related lesions in DNA. By means of gene targeting, we established mouse lines deficient in the methyltransferase gene and tissues from these mice contained no methyltransferase activity. Administration of methylnitrosourea to these gene-targeted mice led to early death, and normal mice treated in the same manner showed no untoward effects. In mice given methylnitrosourea treatment, the bone marrow became hypocellular and there was a drastic decrease in the number of leukocytes and platelets, thereby indicating an impaired reproductive capacity of hematopoietic stem cells. Methyltransferase apparently protected these mice from the pancytopenia caused by the alkylating agent.
Assuntos
Alquilantes/toxicidade , Carcinógenos/toxicidade , Hipersensibilidade a Drogas/etiologia , Hipersensibilidade a Drogas/genética , Metilnitrosoureia/toxicidade , Metiltransferases/genética , Animais , Hipersensibilidade a Drogas/enzimologia , Masculino , Metiltransferases/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , O(6)-Metilguanina-DNA MetiltransferaseRESUMO
The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Letais , Camundongos Endogâmicos C57BL/embriologia , Alelos , Animais , Sequência de Bases , Linhagem Celular , Desenvolvimento Embrionário/genética , Feminino , Heterozigoto , Homozigoto , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , Gravidez , Rad51 RecombinaseRESUMO
Alkylation of DNA at the 0(6) position of guanine is regarded as one o f the most critical events leading to induction of mutations and cancers in organisms. Once 0(6)-methylguanine is formed, it can pair with thymine during DNA replication, the result being a conversion of the guanine.cytosine to an adenine.thymine pair in DNA, and such mutations are often found in tumors induced by alkylating agents. To counteract such effects, organisms possess a mechanism to repair 0(6)-methylguanine in DNA. An enzyme, 0(6)-methylguanine-DNA methyltransferase, is present in various organism, from bacteria to human cells, and appears to be responsible for preventing the occurrence of such mutations. The enzyme transfers methyl groups from 0(6)-methylguanine and other methylated moieties of the DNA to its own molecule, thereby repairing DNA lesions in a single-step reaction. To elucidate the role of methyltransferase in preventing cancers, animal models with altered levels of enzyme activity were generated. Transgenic mice carrying the foreign methyltransferase gene with functional promoters had higher levels of methyltransferase activity and showed a decreased susceptibility to N-nitroso compounds in regard to liver carcinogenesis. Mouse lines deficient in the methyltransferase gene, which were established by gene targeting, exhibited an extraordinarily high sensitivity to an alkylating carcinogen.
Assuntos
Reparo do DNA , Metiltransferases/fisiologia , Neoplasias/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cricetinae , DNA Complementar/genética , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , O(6)-Metilguanina-DNA Metiltransferase , Coelhos , Ratos , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Using transgenic substrates, we found that the immunoglobulin kappa gene 3' enhancer (E3') acts as a negative regulator in V kappa-J kappa joining. Although the E3' was originally identified as a transcriptional enhancer, it acts in a suppressive manner for recombinational regulation. Base substitution analysis has shown that the PU.1-binding site within the E3' regulates the B/T specificity of V kappa-J kappa joining. In a substrate with a mutated PU.1-binding site (GAGGAA to TCTTCG), V kappa-J kappa joining occurred not only in B cells, but also in T cells. The E3' region is also responsible for determining the pro-B/pre-B specificity of V kappa-J kappa joining. When the E3' region was deleted, kappa gene rearrangement actively occurred at the early pro-B stage of B cell development: nongermline (N) nucleotides were common at recombination junctions.