RESUMO
GM1492 human diploid skin fibroblasts derived from a patient with Bloom's syndrome (BS), lack detectable p53 mRNA and protein as shown by Northern and Western blotting, and express an increased RecA-like activity. Here we demonstrate that the p53 gene is grossly intact in GM1492 cells according to Southern blotting. DNA sequencing did not reveal any mutations in the promoter region of p53. A highly sensitive RT-PCR produced a p53 cDNA fragment that was shorter than expected. DNA sequence analysis of p53 cDNA showed that exon 6 was missing, explaining the shorter PCR product. Furthermore, sequencing of genomic DNA revealed a base substitution at the nucleotide preceding the AG splice acceptor site of intron 5. The omission of exon 6 creates a frameshift at the junction of exons 5 and 7, and a premature stop codon in exon 7. The aberrant transcript is predicted to encode a truncated p53 protein containing 189 amino acid residues. Moreover, Western blotting demonstrated elevated HsRAD51 protein levels in GM1492 cells. The lack of sufficient levels of wild-type p53 and increased levels of HsRad51 protein may contribute to the elevated RecA-like activity in the GM1492 fibroblasts.
Assuntos
Processamento Alternativo/genética , Síndrome de Bloom/genética , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Sequência de Bases , Síndrome de Bloom/patologia , Western Blotting , Linhagem Celular , Células Cultivadas , DNA/química , DNA/genética , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Fibroblastos/citologia , Mutação da Fase de Leitura , Regulação da Expressão Gênica , Células HeLa , Humanos , Mutação , Mutação Puntual , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Rad51 Recombinase , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. METHODS AND MATERIALS: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. RESULTS: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. CONCLUSIONS: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.
Assuntos
Carcinoma de Células Escamosas/genética , Dano ao DNA , DNA Ligases/fisiologia , Reparo do DNA , Proteínas de Ligação a DNA , DNA/genética , Proteínas Serina-Treonina Quinases/fisiologia , Sobrevivência Celular/genética , DNA/efeitos da radiação , DNA Ligases/efeitos da radiação , Proteína Quinase Ativada por DNA , Humanos , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/efeitos da radiação , Doses de Radiação , Tolerância a Radiação , Fase S , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
Delayed joining of DNA strand breaks and a high spontaneous level of sister-chromatid exchanges (SCEs) are characteristics of the mutant cell strain EM9 of Chinese hamster ovary (CHO) cells. The introduction of the human gene XRCC1 into EM9 cells reverts the phenotypic properties of EM9 to those of the wild type. We have investigated both DNA ligase activities and a protein which stimulates DNA ligase activity in mutant EM9 cells, XRCC1-transfectant H9T3-7-1 cells and wild-type AA8 cells. Our results, which demonstrate both a decreased DNA ligase activity in EM9 cells using poly(rA).oligo(dT) as substrate and a decreased ability of DNA ligase III to form a covalent DNA ligase III-adenylate intermediate with AMP, clearly indicate an altered DNA ligase III activity in the mutant. Furthermore, the AMP-binding capacity of DNA ligase III and its enzymatic activity with the synthetic polymer were restored after transfection of EM9 with the human XRCC1 gene. Immunoblotting data suggest that the XRCC1 gene does not code for DNA ligase III. In conclusion, the data indicate that the EM9 cell strain has an altered DNA ligase III activity that can be restored by the XRCC1 gene product.
Assuntos
DNA Ligases/metabolismo , Mutação , Monofosfato de Adenosina/metabolismo , Animais , Western Blotting , Células CHO , Cromatografia em Gel , Cricetinae , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/isolamento & purificação , Reparo do DNA , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.
Assuntos
DNA Ligases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO/metabolismo , Clonagem Molecular , Cricetinae , DNA Ligase Dependente de ATP , Reparo do DNA/genética , DNA Complementar/genética , Humanos , Dados de Sequência Molecular , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Transfecção , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
An increased DNA ligase activity is observed in extracts of Bloom's syndrome (BS) fibroblast cell line GM1492. The activity is 2-3-fold higher in this cell line compared to normal human fibroblasts, and 5-20-fold higher than in three other BS cell lines investigated. The DNA ligase activity in GM1492 cells is promoted by a heat-resistant, protease-sensitive factor comigrating with DNA ligases on single-stranded-DNA--cellulose. The factor stimulates DNA ligase I as well as DNA ligase II, and is not identical to the activity-promoting homologous DNA pairing, which is also enhanced in GM1492 cell extracts.
Assuntos
Síndrome de Bloom/enzimologia , DNA Ligases/genética , Regulação da Expressão Gênica , Polinucleotídeo Ligases/genética , Animais , Bovinos , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Timo/enzimologiaRESUMO
The synthetic pyrethroids cypermethrin, delta-methrin, fenvalerate, permethrin, and the fenvalerate metabolite p-chlorophenylisovaleric acid were investigated for inhibition of gap-junctional intercellular communication in vitro in the Chinese hamster lung fibroblast (V79) metabolic cooperation assay. Fenvalerate was furthermore studied for enhancement of gamma-glutamyl transpeptidase-positive enzyme altered foci incidence in partially hepatectomized, nitrosodiethylamine-initiated male Sprague Dawley rats. The in vitro studies showed that fenvalerate and p-chlorophenylisovaleric acid were inhibitors of intercellular communication at non-cytotoxic concentrations while cypermethrin, deltamethrin, and permethrin were inactive. In the in vivo study in rat liver, fenvalerate administered p.o. (75 mg/kg/day) 5 days a week for 10 weeks induced significantly more foci per cm3 and a larger percentage of liver tissue occupied by foci tissue compared to a vehicle control group. Analysis of size distributions of foci in fenvalerate- and vehicle-treated rats showed elevated foci incidences in fenvalerate-treated rats at all foci sizes. Fenvalerate induced no hepatotoxic effects as judged by plasma transaminase activities and histopathology. The results of this study suggest fenvalerate to be a potential tumour promoter.
Assuntos
Comunicação Celular/efeitos dos fármacos , Inseticidas/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Lesões Pré-Cancerosas/induzido quimicamente , Piretrinas/toxicidade , Animais , Células Cultivadas , Inseticidas/metabolismo , Fígado/enzimologia , Fígado/patologia , Masculino , Nitrilas , Piretrinas/metabolismo , Ratos , Ratos Endogâmicos , gama-Glutamiltransferase/análiseRESUMO
The occurrence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Bloom's syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from phi X174 RFI DNA and fragments of phi X174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4-7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Bloom's syndrome fibroblast cell lines, Fanconi's anemia fibroblasts, virus- (Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.
Assuntos
Anemia Aplástica/genética , Síndrome de Bloom/genética , Reparo do DNA , Anemia de Fanconi/genética , Recombinases Rec A/metabolismo , Recombinação Genética , Animais , Bovinos , Linhagem Celular , Linhagem Celular Transformada , Humanos , Ratos , Distribuição TecidualRESUMO
An in vitro assay measuring inhibition of metabolic cooperation between 6-thioguanine sensitive and 6-thioguanine resistant Chinese hamster (V79) cells in co-culture was used to detect chemically induced inhibition of gap-junctional intercellular communication. Inhibition of this cellular process by xenobiotics has been suggested to be an important event in tumour promotion. This study was undertaken to determine the effect on metabolic cooperation by the bioflavonoid quercetin alone and in co-exposure experiments with two recognized tumour promoters, the phorbol ester TPA and the organochlorine pesticide DDT. Furthermore, co-exposure experiments with TPA and DDT were performed. Quercetin alone did not affect metabolic cooperation at noncytotoxic doses. Treatment of the cells with either TPA, DDT or TPA together with DDT caused significant inhibition of metabolic cooperation. This effect was dose-dependently decreased by addition of quercetin. These findings suggest that quercetin inhibits or compensates a common effect induced by both TPA and DDT. Treatment of the cells with a fixed dose of TPA and increasing doses of DDT, or a fixed dose of DDT and increasing doses of TPA, caused significantly higher recovery of mutant cells than a calculated additive response. The data indicate that TPA and DDT induce a synergistic response with respect to affecting intercellular communication. The results suggest that there are different pathways of action for TPA and DDT.
Assuntos
Comunicação Celular/efeitos dos fármacos , DDT/farmacologia , Flavonoides/farmacologia , Quercetina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Cricetinae , Relação Dose-Resposta a Droga , Sinergismo FarmacológicoRESUMO
The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo[a]pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 microns), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possibly be used as an index of the degree of mitotic disturbances induced by mineral fibers.
Assuntos
Amianto/toxicidade , Ovário/citologia , Tioguanina/farmacologia , Animais , Asbesto Crocidolita , Benzo(a)pireno/toxicidade , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Resistência a Medicamentos , Sinergismo Farmacológico , Feminino , Hipoxantina Fosforribosiltransferase/genética , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Mutação , Ovário/efeitos dos fármacos , Ovário/enzimologia , Fatores de TempoRESUMO
A DNA recombining protein has been partly purified from cell lines derived from patients suffering from the hereditary disease, Bloom's syndrome. The protein induces the formation of displacement loops in phi X174 RFI DNA molecules after the addition of single-stranded DNA fragments. A filter binding method and electron microscopy were used to determine the reaction. The recombinogenic protein is dependent on divalent cations and ATP for activity.
Assuntos
DNA Recombinante/metabolismo , DNA Viral/metabolismo , Proteínas/metabolismo , Recombinação Genética , Bacteriófago phi X 174 , Síndrome de Bloom , Linhagem Celular , Cromatografia de Afinidade , Humanos , Microscopia Eletrônica , Peso Molecular , Pele/metabolismoAssuntos
Desoxirribonucleases/isolamento & purificação , Endonucleases/isolamento & purificação , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Desoxirribonucleases/metabolismo , Estabilidade de Medicamentos , Endonucleases/metabolismo , Cinética , Especificidade por SubstratoRESUMO
Contradictory data have recently been published from two different laboratories on the presence vs absence of an intrinsic endonucliolytic activity of E. coli exonuclease III at apurinic sites in double-stranded DNA. It is shown here that an endonuclease activity of this specificity co-chromatographs exactly with exonuclease III on phosphocellulose and Sephadex G-75 columns, indicating that the endonuclease and exonuclease activities are due to the same enzyme. In addition, another E. coli endonuclease specific for apurinic sites exists, which can be separated from exonuclease III by the same chromatographic procedures.
Assuntos
Ácido Apurínico , DNA , Desoxirribonucleases/metabolismo , Endonucleases/metabolismo , Escherichia coli/enzimologia , Exonucleases/metabolismo , Polinucleotídeos , Desoxirribonucleases/isolamento & purificação , Endonucleases/isolamento & purificação , Exonucleases/isolamento & purificação , Especificidade por SubstratoRESUMO
A new DNA endonuclease has been purified 3000-fold from Escherichia coli. The enzyme specifically catalyzes the formation of single strand breaks at apurinic and apyrimidinic sites in DNA, but has no activity on intact or single-stranded DNA. Further, the enzyme shows little or no activity on heavily ultraviolet-irradiated DNA, but cleaves x-irradiated DNA, presumably at apurinic and apyrimidinic sites introduced by the radiation treatment. The enzyme, which is tentatively named endonuclease IV, has no detectable associated exonuclease or DNA N-glycosidase activity and does not seem to be identical with any previously known E. coli endonuclease. Endonuclease IV has no Mg2+ requirement, and is fully active in the presence of EDTA. Enzyme activity is stimulated by 0.2 to 0.3 M NaCl and is unusually salt-resistant. Further, the enzyme is fairly heat-stable, and is not inhibited by tRNA. The sidimentation coefficient, S(o)20,w, is 3.4 S. It seems that endonuclease IV is active in DNA repair.
Assuntos
DNA Bacteriano/metabolismo , DNA Viral/metabolismo , Desoxirribonucleases/isolamento & purificação , Endonucleases/isolamento & purificação , Escherichia coli/enzimologia , Ácido Apurínico/metabolismo , Bacteriófagos , Reparo do DNA , DNA Circular/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Viral/efeitos da radiação , Desoxirribonucleases/metabolismo , Estabilidade de Medicamentos , Endonucleases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Relação Estrutura-Atividade , Raios Ultravioleta , Raios XAssuntos
Reparo do DNA , Escherichia coli/enzimologia , Glicosídeo Hidrolases/metabolismo , Aminoácidos/análise , Cromatografia de Afinidade , Cromatografia em Gel , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/isolamento & purificação , Cinética , Peso Molecular , Fosfatos/análise , Espectrofotometria Ultravioleta , Reagentes de Sulfidrila/farmacologia , Uracila/metabolismoRESUMO
A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.