RESUMEN
p53-dependent apoptosis is important for the efficacy of cancer treatment, and tumors carrying mutant p53 are often resistant to chemotherapy. Non-small cell lung cancer (NSCLC) cells generally exhibit resistance to apoptosis following treatment with many cytotoxic drugs. The new molecule PRIMA-1 appears to kill human tumor cells by restoring the transcriptional activity to mutated p53. We investigated the induction of apoptosis in response to this drug in three NSCLC cell lines carrying different p53 proteins: A549 (p53wt), LX1 (p53R273H), and SKMes1 (p53R280K). PRIMA-1 alone did not trigger apoptosis but significantly reduced cell viability. However, in combination with adriamycin, PRIMA-1 strengthen the adriamycin-induced apoptosis in A549 and LX1. Interestingly, even in SKMes1 cells, the combined treatment triggered a strong PARP cleavage without DNA fragmentation. Our data suggest that in NSCLC cells, PRIMA-1 may induce cell death through pathways other than apoptosis but may synergize with adriamycin to trigger an apoptotic response.
Asunto(s)
Compuestos Aza/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Doxorrubicina/farmacología , Neoplasias Pulmonares/patología , Proteínas Reguladoras de la Apoptosis/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Rayos UltravioletaRESUMEN
It was previously found that fenarimol, vinclozolin or acephate, three of the most used pesticides worldwide, provoked a marked perturbation of murine cytochrome P450 (CYP)-linked monooxygenases. Here, to more closely mimic human exposure, it was investigated whether different pesticide combinations administered i.p. in male Swiss Albino CD1 mice in single or repeated fashion (daily, for three consecutive days), affect CYP-dependent oxidations. The four simulated mixtures showed a complex pattern of CYP induction and suppression, especially after repeated injection. For example, while fenarimol alone was the most inducing agent--reaching a 79-fold increase over control in testosterone 2alpha-hydroxylase--followed by vinclozolin and acephate, coadministration with the former markedly reduced induction. Coadministration with vinclozolin, determined various positive and negative modulations. An increase of CYP2B1/2 and CYP3A1/2-associated oxidases and a decrease of ethoxycoumarin metabolism was observed in the acephate and vinclozolin mixture. An equivalent or reduced CYP expression, if compared to double combinations, was seen using the complete mixture. Taken as a whole, the unpredictability of the recorded effects with simple mixtures, shrinks the misleading extrapolation performed on a single pesticide. If reproduced in human, such changes, altering either endogenous metabolism or biotransformation of ubiquitous toxins, might have public health implications.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/enzimología , Plaguicidas/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Interacciones Farmacológicas , Inducción Enzimática/efectos de los fármacos , Fungicidas Industriales/toxicidad , Técnicas In Vitro , Insecticidas/toxicidad , Isoenzimas/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Compuestos Organotiofosforados/toxicidad , Oxazoles/toxicidad , Fosforamidas , Pirimidinas/toxicidad , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimologíaRESUMEN
Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.
Asunto(s)
Linfoma de Burkitt/genética , Elementos de Facilitación Genéticos , Cadenas mu de Inmunoglobulina/toxicidad , Mutágenos/toxicidad , Señales de Localización Nuclear/toxicidad , Ácidos Nucleicos de Péptidos/toxicidad , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Células CHO , Cricetinae , Cricetulus , Escherichia coli , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Linfocitos/efectos de los fármacos , Pruebas de Micronúcleos , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Salmonella typhimuriumRESUMEN
The effects of different marine derived agents were studied in A549 cell growth. These drugs induced cell cycle arrest at the G2-M phase associated with the up-regulation of GADD45alpha-gamma and down-regulation of c-Myc. In treated cells, GADD45alpha-gamma and c-Myc were up- and down-regulated, respectively. A cascade of events leading to apoptotic mitochondrial 'intrinsic' pathway was observed in treated cells: (1) dephosphorylation of BAD serine136; (2) BAD dissociation from 14-3-3 followed by its association with BCL-XL; (3) cytochrome c release; (4) caspase-3 activation, and (5) cleavage of vimentin. Caspase(s) inhibitor prevented the formation of cleavage products and, in turn, apoptosis was inhibited through a p53-independent mechanism. Moreover, these compounds did not activate NF-kappaB. Our findings may offer new insights into the mechanisms of action of these agents in A549 cells. The better understanding of their effects might be important to fully exploit the potential of these new drugs.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Toxinas Marinas/farmacología , Proteínas 14-3-3/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/patología , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Vimentina/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Proteína bcl-X/metabolismoRESUMEN
The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in prokaryotes and eukaryotes. Members of this family are genes responsible for cancer predisposition disorders like Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome. Here, we show the sequence homologies between two recently identified mammalian helicases, namely SUVi and BACH1. These two genes also share strong homologies with other members of the RecQ family. On the basis of published data and sequence analysis we suggest that SUVi/BACH1 may represent a novel subfamily of mammalian helicases, functioning in the processing of lesions induced by different genotoxic agents.
Asunto(s)
Proteínas de Ciclo Celular , ADN Helicasas/química , Proteínas de Unión al ADN , ARN Helicasas/química , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Datos de Secuencia Molecular , ARN Helicasas/genética , ARN Helicasas/metabolismoRESUMEN
By using a lacZ-based gene-trap approach, we identified a mammalian gene induced by UV-C in a Chinese hamster ovary cell clone (Menichini P et al. [1997]: Nucleic Acids Res 25:4803-4807). The activity of the encoded protein fused to a bacterial beta-galactosidase was followed through the hydrolysis of different beta-galactosidase substrates. In this study we describe how the expression of this gene is modulated during the cell cycle and in response to UV-irradiation. We show that the beta-galactosidase activity was virtually undetectable in quiescent cells (G[0]), started to increase when cells progressed in G(1), and reached a maximum in mid-S phase, indicating a possible role of the endogenous protein during DNA synthesis. Following UV-irradiation, besides a delay of the progression through the S phase, a twofold increase of the reporter protein activity in all phases of the cell cycle was observed. The partial sequence analysis showed that this gene, here named SUVi (for S phase UV-inducible), contains a domain that is highly conserved among different helicases. Together, these data suggest that the SUVi gene could be involved in DNA synthesis, a process that takes place both in the S phase and in the processing of UV-induced damage.
Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/genética , Células Clonales/efectos de la radiación , ADN Helicasas/genética , Fase S/genética , Rayos Ultravioleta , Animales , Secuencia de Bases , Células CHO , Línea Celular , Células Clonales/citología , Células Clonales/metabolismo , Clonación Molecular , Cricetinae , ADN Helicasas/biosíntesis , Reparación del ADN , Genes Reporteros/genética , Genes Reporteros/efectos de la radiación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína/genética , ARN/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , beta-Galactosidasa/efectos de la radiaciónRESUMEN
The assumption of molecular epidemiology that carcinogens leave fingerprints has suggested that analysis of the frequency, type, and site of mutations in genes frequently altered in carcinogenesis may provide clues to the identification of the factors contributing to carcinogenesis. In this mini-review, we revise the development, and validation of the yeast-based p53 functional assay as a new tool for molecular epidemiology. We show that this assay has some very interesting virtues but also has some drawbacks. The yeast functional assay can be used to determine highly specific mutation fingerprints in the human p53 cDNA sequence. Discrimination is possible when comparing mutation spectra induced by sufficiently different mutagens. However, we also reported that the same carcinogen may induce distinguishable mutation spectra due to known influencing factors.
Asunto(s)
Saccharomyces cerevisiae/genética , Proteína p53 Supresora de Tumor/genética , Alquilantes/farmacología , Humanos , Epidemiología Molecular/métodos , Pruebas de Mutagenicidad/métodos , Mutágenos/farmacología , Mutación , Neoplasias/inducido químicamente , Neoplasias/epidemiología , Neoplasias/genética , Piel/metabolismo , Piel/efectos de la radiación , Neoplasias Cutáneas/genética , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/efectos de la radiación , Rayos UltravioletaRESUMEN
8-Methoxypsoralen (8-MOP) plus UVA irradiation (PUVA therapy) has been used for the treatment of psoriasis. PUVA therapy has been associated with an increased risk of developing skin squamous cell carcinoma (SCC). In order to determine the PUVA-induced p53 mutation spectrum, a yeast expression vector harbouring a human wild-type p53 cDNA was incubated with 8-MOP, and UVA irradiated in vitro. PUVA-damaged and undamaged DNA was transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. An 8-MOP concentration-dependent decrease in survival and increase in mutant frequency were observed. At a fixed 8-MOP concentration, survival decreased and mutant frequency increased as UVA irradiation increased. Eleven mutant clones contained 11 mutations: 10 were single base pair substitutions, the remaining one being a complex mutation. All eight T:A-targeted mutations were at 5'-TpA sites, hallmark mutations of PUVA mutagenesis. Through a rigorous statistical test, the PUVA-induced p53 mutation spectrum appears to differ significantly (P < 0.0002) from that observed in SCC in PUVA-treated patients. The present work demonstrates that a specific PUVA-induced mutational fingerprint could be obtained and recognized on human p53 cDNA. This result may suggest that PUVA therapy can be a risk factor for the development of SCC in psoriasis patients through a mechanism not involving the induction of p53 mutations.
Asunto(s)
Genes p53 , Metoxaleno/toxicidad , Mutación , Terapia PUVA , Neoplasias Cutáneas/genética , Rayos Ultravioleta , Análisis Mutacional de ADN , Humanos , Plásmidos , TransfecciónRESUMEN
Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been synthesized in an attempt to minimize carcinogenic activity while favoring antineoplastic activity. CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence-selective DNA alkylation inducing thermolabile N3-alkyladenines (N3-Alkyl-As) at lex equilibrium binding sites. CENU-lex sequence specificity for DNA alkylation was determined using 32P-end-labeled restriction fragments of the p53 cDNA. The adducted sites were converted into single-strand breaks by sequential heating at neutral pH and exposure to piperidine. To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutants were isolated from independent ade- transformants. The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of which are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lex p53 mutations (45 of 49) were bp substitutions, 29 of which were GC-->AT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-->TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutation hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was associated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed. Hence, it is tempting to conclude that, in this experimental system, N3-Alkyl-A is more lethal than mutagenic, whereas O6-alkylguanine is a common premutational lesion formed at non-lex binding sites. These results suggest that CENU derivatives with virtually absolute specificity for A residues would make targeting of lethal, nonmutagenic lesions at A+T-rich regions possible, and this may represent a new strategy for the development of new chemotherapeutic agents with a higher therapeutic index.
Asunto(s)
Antineoplásicos/farmacología , ADN Complementario/efectos de los fármacos , Etilnitrosourea/análogos & derivados , Genes p53/efectos de los fármacos , Mutágenos/farmacología , Netropsina/análogos & derivados , Alquilación , Antineoplásicos/toxicidad , Secuencia de Bases , ADN Complementario/genética , ADN Complementario/metabolismo , Etilnitrosourea/química , Etilnitrosourea/farmacología , Etilnitrosourea/toxicidad , Humanos , Datos de Secuencia Molecular , Mutágenos/toxicidad , Netropsina/química , Netropsina/farmacología , Netropsina/toxicidad , Relación Estructura-Actividad , TransfecciónRESUMEN
Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin.
Asunto(s)
Citosina/análogos & derivados , Desoxirribonucleasa HpaII/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/efectos de la radiación , Levaduras/genética , Levaduras/efectos de la radiación , 5-Metilcitosina , Codón , Islas de CpG , Citosina/metabolismo , Metilación de ADN/efectos de la radiación , Humanos , Mutación , Neoplasias Cutáneas/genética , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta , Levaduras/metabolismoRESUMEN
To determine the influence of some bacterial DNA repair pathways on the mutagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr-ada+ogt+, and uvr-ada-ogt-). Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains. One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced. All mutants were characterized by single base-pair substitutions, most of which (more than 96%) were GC-->AT transitions (the mutated G being almost exclusively preceded 5' by a purine). Mutation distribution was not random. Position 160 (5'-GGT-3', nontranscribed (NT) strand) was a uvr+ada+ogt(+)-specific hot-spot. Position 123 (5'-GGG-3', NT strand) was a common hot-spot but significantly more mutable in repair-proficient strains than in repair-deficient strains. Conversely, position 168 (5'-GGA-3', transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains. By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr- strains, whereas in the uvr- background, no difference was observed between mutation spectra in ada+ogt+ versus ada-ogt- strains. Our results are consistent with the hypothesis that O6-alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of GC-->AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr- strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E. coli.
Asunto(s)
Carcinógenos/toxicidad , Daño del ADN , Reparación del ADN , ADN Bacteriano/efectos de los fármacos , Escherichia coli/genética , Lomustina/toxicidad , Mutágenos/toxicidad , Secuencia de Bases , Análisis Mutacional de ADN , ADN Bacteriano/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
Treatment of cells with DNA damaging agents leads to induction of a variety of genes involved in different cellular processes. We have applied a lacZ-based gene trap strategy to search for new mammalian genes induced by genotoxic stress. A population of 32 x 10(3) neo r clones stably transfected with a gene trap vector was obtained, stained with fluorescein di-beta-d-galactopyranoside and analyzed by flow activated cell sorting and replica plating. This strategy allowed isolation of 30 neo r 'putative inducible' cell lines expressing lacZ only after a DNA damaging treatment. For three clones the site of integration and the degree of inducibility after UV treatment were determined by Southern blot and beta-galactosidase measurement respectively. One cell line (clone VI) showed a single integration site and a reproducible 3-fold induction of beta-galactosidase activity following UV irradiation. Fused transcripts were isolated from induced cells and a portion of the trapped gene was amplified by rapid amplification of cDNA ends. Sequence analysis and comparison with available gene and protein databanks revealed that the gene was novel.
Asunto(s)
Daño del ADN , Técnicas Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Células CHO , Clonación Molecular , Cricetinae , ADN/genética , ADN/aislamiento & purificación , Vectores Genéticos , Hibridación Fluorescente in Situ , Operón Lac , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Transfección , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.
Asunto(s)
Aneuploidia , Astrocitoma/genética , Neoplasias Encefálicas/genética , Deleción Cromosómica , Genes p53/genética , Glioblastoma/genética , Mutación Puntual/genética , Adulto , Anciano , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 15/genética , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Exones/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Análisis de RegresiónRESUMEN
Using the pZ189 shuttle vector approach, we determined two chloroethyl-cyclohexyl-nitrosourea (CCNU)-induced mutation spectra (3 and 6 mM) in African green monkey kidney cells (CV1). One hundred and twenty-one independent clones (101 CCNU induced, 45 at 3 mM and 56 at 6 mM; 20 spontaneous) showing functional inactivation of the supF gene were analyzed. One hundred and five plasmids (91 CCNU induced, 41 at 3 mM and 50 at 6 mM; 14 spontaneous), showing no large deletion/rearrangements, were sequenced. Ninety mutants (81 CCNU induced and 9 spontaneous) showed at least one mutation in the supF region. The analysis of the 122 CCNU-induced mutations (56 and 66 at 3 and 6 mM, respectively) revealed that: (a) the majority of the mutations were GC-targeted base pair substitutions; (b) AT-targeted mutations were significantly more frequent in the CCNU-induced (6 mM) than in the spontaneous mutational spectrum (P < 0.0006, Fisher's exact test); (c) mutational spectra obtained at 3 and 6 mM CCNU were significantly different (P < 0.008); (d) induced mutations were nonrandomly located in both spectra and generated either a common hot spot (position 123, 5'-GGG-3') or hot spots exclusive for each CCNU concentration (3 mM: position 159, 5'-AGG-3'; 6 mM: position 109, 5'-GGG-3'); (e) the occurrence of GC-->AT transitions was significantly different as a function of CCNU concentration (P < 0.02, Fisher's exact test), the mutated G being almost exclusively preceded by a purine (5'Pu G) at 6 mM and by either Pu or Py at 3 mM; and (f) by applying Calladine's rules, we found that sequences encompassing the three CCNU hot spots shared identical helix parameters for no more than 2 bp steps 5' (or 3 bp steps 3') to the mutated G. Our results are consistent with the hypothesis that O6-alkylguanine is responsible, either directly or indirectly, for the majority of GC-targeted mutations, while O4-alkylthymine and/or N3-alkyladenine are probably responsible for AT-targeted mutations. The results suggest also that, in CV1 cells, the efficiency of the repair mechanism(s) involved in the removal of O6-alkylguanine is influenced by the DNA sequence context. All of these factors determine the CCNU mutational fingerprint. CCNU has been implicated in the induction of therapy-related leukemias.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Antineoplásicos Alquilantes/toxicidad , Lomustina/toxicidad , Mutágenos , Animales , Secuencia de Bases , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Chlorocebus aethiops , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Plásmidos , Mutación Puntual , Relación Estructura-ActividadRESUMEN
We had previously reported that an oligonucleotide containing a site-specifically incorporated O4-methylthymine (m4T) was replicated under kinetic conditions by the Klenow fragment of E. coli DNA polymerase I (Kf) (Dosanjh et al., 1993). Using other polymerases for complete replication, but with limiting enzyme, a pause site before the m4T was observed. In order to investigate whether such a pause could be due to enzyme dissociation or stalling, trapping experiments were designed to aid in differentiating the two mechanisms. Rather than the generally used heparin or sheared DNA trap, these experiments utilized as the acceptor the same oligonucleotide containing unmodified thymine. It was observed that, under enzyme-limiting conditions, the nature of the enzyme played a major role in replication of m4T. With a running start, Kf and calf-thymus polymerase alpha-primase allowed replication beyond the m4T, while Sequenase and T7 showed a strong pause site at the base before m4T. When the oligonucleotide trap was added after different times of replication, it was found that Sequenase remained bound to the template-primer, regardless of whether T or m4T was present. In contrast, Kf dissociated and re-associated rapidly. Thus, m4T appears to be a strong replication block when using limiting amounts of a highly processive enzyme such as Sequenase or T7. This may imply that such enzymes discriminate against forming a poor basepair but remain bound to the primer-template or become inactivated.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Timina/análogos & derivados , Secuencia de Bases , Cinética , Datos de Secuencia Molecular , Mutación , Oligodesoxirribonucleótidos/química , Timina/metabolismoRESUMEN
The molecular analysis of mutations affecting mRNA processing may contribute to a better understanding of the splicing mechanism through the identification of genomic sequences necessary for the recognition of splice sites. In this paper we report the sequence analysis of 14 splice mutants induced by 4-nitroquinoline 1-oxide (4NQO) at the hamster hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus. We show that mutations at the 3' acceptor splice site or at the first or fifth base of the 5' donor splice site are responsible for exon skipping. In addition, mutations in exon sequences also determine the skipping of one or more exons. Our data indicate that point mutations in intron regions at either side of an internal exon may induce the skipping of the same exon, supporting a model where the exon is the unit of early spliceosome assembly. Furthermore, they suggest that the splicing of hprt mRNA precursors may proceed through a clustering of exons 2, 3 and 4 which are then spliced in a concerted way.
Asunto(s)
4-Nitroquinolina-1-Óxido/toxicidad , Hipoxantina Fosforribosiltransferasa/genética , Empalme del ARN/efectos de los fármacos , Animales , Secuencia de Bases , Células CHO , Cricetinae , ADN , Análisis Mutacional de ADN , Exones , Humanos , Intrones , Datos de Secuencia MolecularRESUMEN
Mutation spectra induced by 4-nitroquinoline 1-oxide (4NQO) at the hprt locus for both normal (AA8) and 4NQO-sensitive (UV5) Chinese hamster ovary cells were determined to investigate the effect of DNA repair on the nature of induced mutations. The UV5 cell line is three times more sensitive to 4NQO than the AA8 parental cell line. In UV5 cells, the dGuo-N2-AQO adduct, which is considered to be the most toxic and mutagenic adduct in Escherichia coli, is poorly repaired. The molecular nature of 30 hprt mutants isolated from AA8 and 20 isolated from UV5 cells was determined by sequence analysis of in vitro amplified hprt cDNA. Both similarities and differences emerged. In both cell lines we found that (i) 4NQO is basically a base substitution mutagen acting almost exclusively at G residues and (ii) G transversions are prevalent over G transitions in both cell lines, independently from the ability to repair dGuo-N2-AQO. A high proportion (13/25) of splice mutations was observed in AA8 cells, statistically different (P < 0.04, Fisher's exact test) from the incidence of splice mutants in UV5 cells (4/20). In AA8 mutants, all but two of the point mutations were due to lesions localized on the non-transcribed strand, suggesting preferential repair of the transcribed strand. Compared with AA8, the proportion of mutants due to lesions present on the transcribed strand was higher in UV5 cells, as expected if a preferential repair mechanism was impaired in the sensitive cell line. Our data are consistent with the molecular defect in DNA repair recently characterized in UV5.
Asunto(s)
4-Nitroquinolina-1-Óxido/toxicidad , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Hipoxantina Fosforribosiltransferasa/genética , Mutación , Animales , Secuencia de Bases , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cartilla de ADN/genética , ADN Complementario/genética , Datos de Secuencia Molecular , Mutación Puntual , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genéticaRESUMEN
The mutagenic potential of O4-methylthymine (m4T) and O4-ethylthymine (e4T) was determined by a primer extension assay on a 25mer oligonucleotide containing a single site-specifically incorporated modified thymine. The e4T-containing oligonucleotide was prepared by using a new synthetic procedure suitable for large alkyl groups on thymine. The second-order rate constants, K(app)m and V(rel)max, permitted calculation of the frequency of formation and extension of modified base pairs compared to Watson-Crick pairing. With both m4T and e4T, the T.G type pairing was formed at least 10-fold more frequently than the nonmutagenic alkyl T.A pairing. However, there was a small but reproducible preference for m4T.G pairing. In both cases T-->C transitions would result. There was no evidence for formation of alkyl T.C or T.T. These data suggest that reported T-->A transversions by ethylation are not likely to result from O4-alkylthymine. In contrast to insertion, extension beyond alkylthymine under kinetic conditions did not occur with alkyl T.A. but only with the alkyl T.G termini. For this latter T.G type pairing, the larger ethyl group did not hinder extension compared to that of the methyl group, in the sequence studied. Under non-limiting conditions of dNTP concentration and time, complete replication could be demonstrated for both methyl- and ethyl-containing oligonucleotides. We conclude that the increase in size of the alkyl group from methyl to ethyl does not significantly affect the mutagenic potential and type of mutations of O4-alkylthymine in vitro.
Asunto(s)
Adenosina Trifosfato/metabolismo , Composición de Base , Replicación del ADN , Nucleótidos de Desoxiguanina/metabolismo , Guanina/metabolismo , Timina/análogos & derivados , Timina/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/químicaRESUMEN
To understand molecular mechanisms of the mutation fixation process induced by a mutagen and carcinogen, a multi-system approach is suggested to reduce the probability that the results are biased by the assay used. In this light we described our different approaches to answer basic questions on the mutagenesis induced by the chemical carcinogen 4-Nitroquinoline-1-oxide. We determined mutations at the molecular level in three experimental systems: a) in prokaryotes (ss M13mp19 lacZ'/E. coli F'lacZ delta M15); b) in eukaryotes (i) ss and ds pZ189 supF/CV1-P/E.coli lacZam and (ii) HPRT in CHO cells with different repair capacity. We think this type of approach can be used to study the genetic effects of new cancer drugs for which the molecular mechanisms of action at the molecular level are still not well understood. We think to apply the know-how to study mutational spectra in tumor derived tumor suppressor genes.
Asunto(s)
4-Nitroquinolina-1-Óxido/toxicidad , Daño del ADN , Mutagénesis , Animales , Células CHO , Línea Celular , Cricetinae , Células Eucariotas/efectos de los fármacos , Células Procariotas/efectos de los fármacosRESUMEN
In the attempt to determine the possible influence of excision repair processes on 4-NQO mutational spectra in mammalian cells, 4-NQO-induced mutants at the hprt locus were isolated in excision repair proficient (AA8) and deficient (UV5) CHO cell lines. DNA sequencing data on these mutants revealed that DNA repair may indeed modulate the induced mutational spectrum. In particular, more splice mutations were found in the repair proficient than in the repair deficient cells. This can be interpreted by a difference in repairability of the two principal 4-NQO G-adducts or by the existence of a transcription-coupled DNA preferential repair process.