Asunto(s)
Hidrocarburo de Aril Hidroxilasas/análisis , Placenta/enzimología , Fumar/efectos adversos , Contaminación por Humo de Tabaco/efectos adversos , Adulto , Hidrocarburo de Aril Hidroxilasas/farmacología , Peso al Nacer , Estatura , Femenino , Humanos , Recién Nacido , Intercambio Materno-Fetal , Embarazo , TurquíaRESUMEN
Xeroderma pigmentosum family G from Van, Turkey had two severely affected children: a son with multiple skin cancers who died at age 10 (XP67TMA), and an 8 y old daughter who began developing skin cancer before 3 y of age (XP68TMA). XP67TMA and XP68TMA cells were hypersensitive to killing by ultraviolet and the post-ultraviolet DNA repair level was 12-16% of normal. Host cell reactivation of an ultraviolet-treated reporter plasmid cotransfected with a vector expressing wild-type XPC cDNA assigned XP67TMA to xeroderma pigmentosum complementation group C. The XPC mRNA level was markedly reduced. Sequencing of the 3.5 kb XPC cDNA from XP67TMA showed a C-T mutation in XPC exon 8 at base pair 1840. This mutation converts the CGA codon of arginine at amino acid 579 to a UGA stop codon resulting in marked truncation of the 940 amino acid xeroderma pigmentosum C protein. Restriction fragment length polymorphism analysis of XPC exon 8 DNA in XP67TMA and XP68TMA showed that both affected children had a homozygous mutation and that both parents had heterozygous normal and mutated sequences at the same position consistent with a history of consanguinity in the family. The mutated allele also contained two XPC single nucleotide polymorphisms. The same mutated XPC allele was reported in an Italian family. Studies of 19 microsatellite markers flanking the XPC gene on chromosome 3 suggest that the XPC allele passed between Italy and Turkey approximately 300-500 y ago. This XPC allele containing a nonsense mutation is associated with severe clinical disease with multiple skin cancers and early death.
Asunto(s)
Cromosomas Humanos Par 3 , Codón de Terminación/genética , Proteínas de Unión al ADN/genética , Salud de la Familia , Xerodermia Pigmentosa/genética , Adulto , Alelos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Niño , Codón sin Sentido , Reparación del ADN/efectos de la radiación , Femenino , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Variación Genética , Humanos , Italia , Desequilibrio de Ligamiento , Masculino , Repeticiones de Microsatélite , Linaje , Polimorfismo de Longitud del Fragmento de Restricción , Piel/patología , Neoplasias Cutáneas/genética , Turquía , Rayos Ultravioleta/efectos adversos , Xerodermia Pigmentosa/patologíaRESUMEN
Xeroderma pigmentosum, Cockayne syndrome, the xeroderma pigmentosum-Cockayne syndrome complex, and trichothiodystrophy cells have defects in DNA repair and are associated with clinical and cellular hypersensitivity to ultraviolet radiation (UV). Familial dysplastic nevus syndrome cells have UV hypermutability. Although xeroderma pigmentosum and dysplastic nevus syndrome have markedly increased cancer risk. Cockayne syndrome and trichothiodystrophy do not. At the molecular level, these disorders are associated with several different genetic defects as evidenced by the existence of multiple overlapping complementation groups. Recent progress has been made in identifying the chromosomal location and cloning the defective genes in these disorders. Using plasmid shuttle vectors we have shown abnormal repair and mutagenesis of DNA damaged by 254-nm (UVC) or 295-nm (UVB) radiation or the chemical carcinogen aflatoxin in cells from patients with xeroderma pigmentosum. Although xeroderma pigmentosum cells are defective in repair of all photoproducts, Cockayne syndrome cells appear to be defective in repair of cyclobutane dimers and have normal repair of nondimer photoproducts. DNS cells have post UV plasmid hypermutability. These diseases may serve as models for examining molecular mechanisms of carcinogenesis in humans.
Asunto(s)
Reparación del ADN , Ligamiento Genético , Neoplasias/genética , Xerodermia Pigmentosa/genética , Síndrome de Cockayne/genética , Síndrome del Nevo Displásico/genética , Cabello/anomalías , HumanosRESUMEN
To determine the contribution of a human DNA repair gene, ERCC2 (XPD), to mutagenesis in human cells, two ERCC2 (XPD)-transformed xeroderma pigmentosum complementation group D (XPD) cell lines with increased UV survival compared to XP6BE(SV40), the original XPD line, were studied: D6BE-ER2-2 with slightly increased UV survival; and D6BE-ER2-9 with normal UV survival. ERCC2 (XPD) antibody-reactive protein levels were elevated 4.8-fold in D6BE-ER2-2 and 17.6-fold in D6BE-ER2-9 relative to XP6BE(SV40). DNA repair ability was assessed by measuring the ability of the cells to restore expression to UV-treated plasmids. Transfection of pRSVcat exposed to 1000 J/m2 UV resulted in 0.3% chloramphenicol acetyltransferase activity in XP6BE(SV40) cells but 20-80% in D6BE-ER2-2, D6BE-ER2-9, and repair-proficient cells compared to untreated control plasmids. The UV hypersensitivity of the mutagenesis shuttle vector pSP189 in XP6BE(SV40) cells was partially corrected and the UV hypermutability and excess of G:C-->A:T mutations of pSP189 fell to the normal range in D6BE-ER2-2 and D6BE-ER2-9 cells. However, the frequency of plasmids recovered with multiple base substitution mutations was significantly reduced with XP6BE(SV40) cells and remained low in D6BE-ER2-2 and D6BE-ER2-9 cells, when compared with the normal fibroblasts. The human DNA excision repair gene, ERCC2 (XPD), substantially corrected the plasmid UV hypersensitivity and UV hypermutability of xeroderma pigmentosum complementation group D cells; however, the dose response relationship varied for different end points.
Asunto(s)
ADN Helicasas , Reparación del ADN , Proteínas de Unión al ADN , Mutación , Proteínas/genética , Factores de Transcripción , Xerodermia Pigmentosa/genética , Adulto , Secuencia de Bases , Línea Celular , Supervivencia Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Femenino , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Plásmidos , Rayos Ultravioleta , Proteína de la Xerodermia Pigmentosa del Grupo DRESUMEN
Cytochrome P-450 mRNAs were quantitated by in vitro translation of liver RNA followed by immunoprecipitation with antibodies specific for cytochromes P-450. The kinetics of cytochrome P-450 mRNA induction by 3-methylcholanthrene and by phenobarbital were examined, and differences in the types of cytochrome P-450 mRNAs induced by 3-methylcholanthrene (MC), beta-naphthoflavone (BNF), and phenobarbital (PB) in male and female rats were determined. Phenobarbital strongly induced the mRNA encoding a single peptide antigenically related to the phenobarbital-induced cytochrome P-450PB-B. Male rat liver contained 62% more translatable mRNA for this peptide than did female. 3-Methylcholanthrene and beta-naphthoflavone, but not phenobarbital, induced mRNAs encoding three peptides that were immunologically related to cytochrome P-450BNF/MC-B, which is induced by 3-methylcholanthrene. The levels of translatable mRNA coding for these peptides were twice as high in females as in males. Striking sex differences were observed in the levels of translatable mRNAs for peptides related to cytochrome P-450PB/PCN-E, which is induced by phenobarbital and by pregnenolone-16-alpha-carbonitrile. In females, only RNA preparations from the livers of phenobarbital-treated rats had significant levels of mRNAs encoding these peptides. In contrast, significant levels of these RNAs were observed even in untreated males, and the levels of these mRNAs increased markedly following phenobarbital treatment. All cytochrome P-450 inducers examined caused a 50 to 70% decrease in translatable albumin mRNA. This effect was specific for albumin mRNA, since levels of total translatable mRNA were not generally altered by these inducers. The kinetics of induction of cytochrome P-450 mRNA differed from those of induction of aryl hydrocarbon hydroxylase (AHH) activity. Translatable cytochrome P-450 mRNA was increased as early as 4 h after phenobarbital treatment, peaked between 24 and 36 h, and dropped back to control levels by 120 h. The induction of AHH lagged behind the increase in translatable mRNA, remaining at control levels well after levels of translatable mRNA began to increase but then decreasing roughly in parallel with translatable mRNA. These findings suggest that transcription was not rate limiting for regulation of PB-inducible cytochrome P-450 activity. 3-Methylcholanthrene caused parallel increases in AHH activity and translatable cytochrome P-450 mRNA, but when translatable mRNA began to decrease after about 24 h, AHH activity remained high, suggesting that this P-450 mRNA was less stable than the enzyme for which it coded.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , ARN Mensajero/biosíntesis , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Autorradiografía , Benzoflavonas/farmacología , Precipitación Química/métodos , Sistema Enzimático del Citocromo P-450/biosíntesis , Densitometría , Electroforesis en Gel de Poliacrilamida , Femenino , Inmunoquímica , Cinética , Masculino , Metilcolantreno/farmacología , Fenobarbital/farmacología , Fotofluorografía , Biosíntesis de Proteínas , Ratas , Ratas Endogámicas , Caracteres Sexuales , beta-naftoflavonaRESUMEN
An improved high-pressure liquid chromatography system was used to analyze the amount of benzo[a]pyrene metabolites formed in reconstituted microsomal mixed-function oxidase systems containing different cytochromes P-450. We separated twelve identified and seven unknown metabolites of BP which included three diols: the 9,10-, 4,5-and 7,8-dihydrodiols; four phenols, 9-,7-, 1-, and 3-hydroxybenzo[a]pyrene (OH-BP); and three quinones: the 1,6-. 3,6-, and 6,12-quinones. Two additional peaks co-migrated with synthetic 4-OH-BP and 5-OH-BP, respectively. The former, designated fraction 1, was shown by u.v. spectra to contain primarily the 4,5-epoxide with small amounts of 4-OH-BP. The total metabolism of BP was found to be approximately 20-fold greater with the cytochrome P-450 from the 3-methylcholanthrene (P-450 3-MC) and beta-naphthoflavone (P-450 BNF) treated rats than with the phenobarbital preinduced cytochrome P-450 (P-450 BP). 3-OH-BP ad 9-OH-BP were the major phenolic products for both P-450 3-MC and P-450 BNF whereas the 3-OH-BP and 1-OH-BP were the major phenolic products for P-450 BP. The ratio of total phenols to diols was found to be 3.34, 4.85 and 0.70 for P-450 3-MC, P-450 BNF and P-450 PB. The major dihydrodiol generated by P-450 3-MC and P-450 BNF was 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, whereas the 9,10-diol was the major diol from P-450 PB. The amount of 1,6- and 3,6-quinones produced was greater than the 6,12-quinone with the P-450 3-MC and P-450 BNF but all three quinones were produced in low and equal amounts by the P-450 PB. In respect to the percent metabolites formed at a given region of the BP, P-450 3-MC and P-450 BNF preferred oxidation at the 1, 3 positions, 6 position and the 7, 8 positions, whereas the P-450 PB preferred oxidation at the 4, 5 position. This study demonstrates the unique positional specificity of different forms of cytochrome P-450 which may regulate the balance between activation and detoxification pathways of polycyclic aromatic hydrocarbon metabolism.