RESUMEN
To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer's patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer's patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.
Asunto(s)
Hipermutación Somática de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/inmunología , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Linfocitos B/patología , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/fisiología , Activación Enzimática/genética , Activación Enzimática/inmunología , Técnicas de Sustitución del Gen , Reordenamiento Génico de Cadena Pesada de Linfocito B , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos AnimalesRESUMEN
Cellular DNA is continuously assaulted by chemical and physical agents that arise from both endogenous metabolic processes as well as exogenous insults. Commonly encountered environmental agents include polyaromatic hydrocarbons, polycyclic aromatic amines, the ultraviolet component of sunlight, and ionizing radiation, among many others. Although the kinds of damages and the mechanisms involved in their interaction with DNA vary widely, genotoxic agents alter the structure of DNA in ways that may result in permanent alterations in the DNA sequence or in cell death. To avoid these consequences, cells have evolved countermeasures to reduce the biological consequences of DNA damage. These mechanisms are highly conserved and are present in all eukaryotic cells. In general, cellular responses include the detection of damage, signal transduction to halt cell cycle progression, and the recruitment of repair mechanisms that are tailored to the specific kind of damage. If replication-blocking damage remains when cells enter S-phase, then tolerance mechanisms in the form of complex recombination mechanisms or translesion DNA synthesis using accessory DNA polymerases exist. These mechanisms complete the replication of damaged genomes and suppress cytotoxicity, but at the potential cost of mutagenesis and genomic instability. This review focuses on error-prone mechanisms, including a discussion of the Y-family of DNA polymerases, current concepts of DNA polymerase switching mechanisms, and their relevance to cancer and cancer prevention.
Asunto(s)
Daño del ADN , Replicación del ADN , Genoma , Animales , Benzo(a)pireno , Clonación Molecular , ADN/biosíntesis , ADN/genética , ADN/metabolismo , Reparación del ADN , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Inestabilidad Genómica , Humanos , Mutagénesis , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Fase S , Análisis de Secuencia de ADNRESUMEN
The somatic mutation hypothesis of cancer predicts that reducing the frequency of mutations induced by carcinogens will reduce the incidence of cancer. To examine this, we developed an antimutator strategy based on the manipulation of the level of a protein required for mutagenic bypass of DNA damage induced by the ubiquitous carcinogen benzo[a]pyrene. The expression of this protein, REV1, was reduced in mouse cells using a vector encoding a gene-specific targeting ribozyme. In the latter cells, mutagenesis induced by the activated form of benzo[a]pyrene was reduced by >90%. To examine if REV1 transcripts could be lowered in vivo, the plasmid was complexed with polyethyleneimine, a nonviral cationic polymer, and delivered to the lung via aerosol. The endogenous REV1 transcript in the bronchial epithelium as determined by quantitative real-time PCR in laser capture microdissected cells was reduced by 60%. There was a significant decrease in the multiplicity of carcinogen-induced lung tumors from 6.4 to 3.7 tumors per mouse. Additionally, REV1 inhibition completely abolished tumor formation in 27% of the carcinogen-exposed mice. These data support the central role of the translesion synthesis pathway in the development of lung cancer. Further, the selective modulation of members of this pathway presents novel potential targets for cancer prevention. The somatic mutation hypothesis of cancer predicts that the frequency of cancers will also be reduced.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Carcinógenos/toxicidad , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Nucleotidiltransferasas/fisiología , Animales , ADN Polimerasa Dirigida por ADN , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Mutagénesis , Nucleotidiltransferasas/antagonistas & inhibidores , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Heterocyclic amines such as 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) are dietary carcinogens generated when meats are cooked well-done. Bioactivation includes N-hydroxylation catalyzed by cytochrome P4501A2 (CYP1A2) followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles were treated with IQ or MeIQx to examine the effect of NAT2 genetic polymorphism on IQ- or MeIQx-induced DNA adducts and mutagenesis. MeIQx and IQ both induced decreases in cell survival and significantly (p<0.001) greater number of endogenous hypoxanthine phosphoribosyl transferase (hprt) mutants in the CYP1A2/NAT2*4 than the CYP1A2/NAT2*5B cell line. IQ- and MeIQx-induced hprt mutant cDNAs were sequenced and over 85% of the mutations were single-base substitutions with the remainder exon deletions likely caused by splice-site mutations. For the single-base substitutions, over 85% were at G:C base pairs. Deoxyguanosine (dG)-C8-IQ and dG-C8-MeIQx adducts were significantly (p<0.001) greater in the CYP1A2/NAT2*4 than the CYP1A2/NAT2*5B cell line. DNA adduct levels correlated very highly with hprt mutants for both IQ and MeIQx. These results suggest substantially increased risk for IQ- and MeIQx-induced DNA damage and mutagenesis in rapid NAT2 acetylators.
Asunto(s)
Arilamina N-Acetiltransferasa/genética , Daño del ADN , Mutagénesis , Quinolinas/toxicidad , Quinoxalinas/toxicidad , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , Humanos , Datos de Secuencia Molecular , Mutágenos/toxicidad , TransfecciónRESUMEN
Comparative mutagenesis of 1,N(6)-ethenoadenine (epsilonA) and 8-oxoguanine (8-oxoG), two endogenous DNA lesions that are also formed by exogenous DNA damaging agents, have been evaluated in HeLa and xeroderma pigmentosum variant (XPV) cell extracts. Two-dimensional gel electrophoresis of the duplex M13mp2SV vector containing these lesions established that there was significant inhibition of replication fork movement past epsilonA, whereas 8-oxoG caused only minor stalling of fork progression. In extracts of HeLa cells, epsilonA was weakly mutagenic inducing all three base substitutions in approximately equal frequency, whereas 8-oxoG was 10-fold more mutagenic inducing primarily G-->T transversions. These data suggest that 8-oxoG is a miscoding lesion that presents a minimal, if any, block to DNA replication in human cells. We hypothesized that bypass of epsilonA proceeded principally by an error-free mechanism in which the undamaged strand was used as a template, since this lesion strongly blocked fork progression. To examine this, we determined the sequence of replication products derived from templates in which a G was placed across from the epsilonA. Consistent with our hypothesis, 93% of the progeny were derived from replication of the undamaged strand. When translesion synthesis occurred, epsilonA-->T mutations increased 3-fold in products derived from the mismatched epsilonA: G construct compared with those derived from the epsilonA: T construct. More efficient repair of epsilonA in the epsilonA: T construct may have been responsible for lower mutation frequency. Primer extension studies with purified pol eta have shown that this polymerase is highly error-prone when bypassing epsilonA. To examine if pol eta is the primary mutagenic translesion polymerase in human cells, we determined the lesion bypass characteristics of extracts derived from XPV cells, which lack this polymerase. The epsilonA: T construct induced epsilonA-->G and epsilonA-->C mutant frequencies that were approximately the same as those observed using the HeLa extracts. However, epsilonA-->T events were increased 5-fold relative to HeLa extracts. These data support a model in which pol eta-mediated translesion synthesis past this adduct is error-free in the context of semiconservative replication in the presence of fidelity factors such as PCNA.
Asunto(s)
Adenina/análogos & derivados , Daño del ADN , Replicación del ADN , Guanina/análogos & derivados , Mutagénesis , Adenina/metabolismo , Extractos Celulares , Línea Celular , ADN Polimerasa Dirigida por ADN/metabolismo , Electroforesis en Gel Bidimensional , Vectores Genéticos , Guanina/metabolismo , Células HeLa , HumanosRESUMEN
The kidney, and to a slight extent the liver, of human fetuses were found to synthesize and secrete the alpha subunit common to glycoprotein hormones. Fetal lung and muscle did not synthesize this protein. Since fetal kidney and liver were previously found to synthesize beta chorionic gonadotropin, their ability to synthesize bioactive chorionic gonadotropin was also determined. The newly synthesized hormone bound to mouse Leydig cells and elicited a biological response: namely, the synthesis of testosterone. These results suggest that the human fetus may participate in metabolic homeostasis during its development.
Asunto(s)
Gonadotropina Coriónica/biosíntesis , Feto/metabolismo , Animales , Humanos , Riñón/embriología , Células Intersticiales del Testículo/metabolismo , Hígado/embriología , Hormona Luteinizante/biosíntesis , Masculino , Ratones , Placenta/metabolismo , Testosterona/biosíntesisRESUMEN
Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or poleta only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Fase S/efectos de la radiación , Rayos Ultravioleta , Proteínas Portadoras/efectos de la radiación , Núcleo Celular/metabolismo , Células Cultivadas , Daño del ADN/fisiología , Proteínas de Unión al ADN/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Mutagénesis/fisiología , Mutagénesis/efectos de la radiación , Proteínas Nucleares/metabolismo , Proteínas Nucleares/efectos de la radiación , Fotoblanqueo/efectos de la radiación , Unión Proteica/efectos de la radiación , Transporte de Proteínas/efectos de la radiación , Distribución Tisular , Ubiquitina-Proteína Ligasas , Rayos Ultravioleta/efectos adversosRESUMEN
Endogenously generated reactive oxygen species and genotoxic carcinogens can covalently modify bases in cellular DNA. If not recognized and removed prior to S-phase of the cell cycle, such modifications can block DNA replication fork progression. If blocked forks are not are not resolved, they result in double strand breaks and cell death. Recent data indicate that the process of translesion DNA synthesis (TLS) is a highly conserved mechanism for bypassing lesions in template DNA. Although not fully understood, in yeast a ubiquitin ligase (RAD18) signals error-prone Y family polymerases to the blocked fork to bypass the damage with potentially mutagenic consequences. Homologs of the yeast proteins are found in higher eukaryotic cells, including human. We are examining the hypothesis that RAD18 acts as a proximal signal to Y-family polymerases to bypass damage, in a manner analogous to yeast but with additional layers of complexity. Here we report that RAD18 accumulates in nuclear foci after UV irradiation only in cells entering S-phase with DNA damage. These foci co-localize with proliferating cell nuclear antigen (PCNA). In addition, a newly described DNA polymerase, pol iota, also forms nuclear foci in a damage- and S-phase dependent manner. These data support our overall hypothesis that RAD18 accumulates at blocked forks and initiates the signal to recruit TLS polymerases.
Asunto(s)
Daño del ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Fase S , Ciclo Celular/efectos de la radiación , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Fibroblastos/metabolismo , Predicción , Humanos , Pulmón/citología , Pulmón/embriología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Fase S/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , Rayos UltravioletaRESUMEN
Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.
Asunto(s)
Daño del ADN , Mutagénesis/efectos de la radiación , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta/efectos adversos , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular Transformada , Supervivencia Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/efectos de la radiación , Fibroblastos/fisiología , Fibroblastos/efectos de la radiación , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/efectos de la radiación , Masculino , Fosforilación , Serina/metabolismo , Telomerasa/genética , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Metabolically active tissues from second trimester human fetuses were examined for their ability to synthesize the placental hormones chorionic gonadotropin and chorionic somatomammotropin. During short-term incubation studies both placenta and fetal kidney were found to synthesize and secrete the beta-subunit of chorionic gonadotropin, whereas its synthesis was not observed in fetal liver, lung or muscle. In addition, chorionic somatomammotropin synthesis and secretion was demonstrated with placental tissue but could not be detected in any of the fetal tissues examined. These observations constitute the first evidence that the genome of a fetal tissue directs the synthesis of what is considered a placental hormone.
Asunto(s)
Gonadotropina Coriónica/biosíntesis , Feto/metabolismo , Riñón/embriología , Humanos , Metionina/metabolismoRESUMEN
Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C-->T, 30% of the base substitutions consist of C-->A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C-->T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZalpha, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to approximately 37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but approximately 2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C-->A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T-->A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.
Asunto(s)
Reparación del ADN , Mutación , Xerodermia Pigmentosa/genética , Extractos Celulares , Línea Celular , ADN Polimerasa III/genética , Replicación del ADN , Células HeLa , Humanos , Análisis de Secuencia de ADN , Moldes Genéticos , Rayos UltravioletaRESUMEN
To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.
Asunto(s)
Reparación del ADN , Hipoxantina Fosforribosiltransferasa/genética , Interfase , Mutación , Transcripción Genética , Secuencia de Bases , Línea Celular , Daño del ADN , Fibroblastos/citología , Fibroblastos/efectos de la radiación , Fase G1 , Humanos , Hipoxantina Fosforribosiltransferasa/efectos de la radiación , Cinética , Datos de Secuencia Molecular , Fase S , Rayos Ultravioleta , Xerodermia Pigmentosa/metabolismoRESUMEN
In the recent Falklands campaign four Army Field Surgical Teams were deployed in the two phases of the war. They functioned as Advanced Surgical Centres and operated on 233 casualties. There were 3 deaths. The patterns of wounding and the methods of casualty management are discussed and compared with other recent campaigns.
Asunto(s)
Hospitales Militares/organización & administración , Hospitales de Urgencia , Medicina Militar/historia , Personal Militar , Guerra , Heridas por Arma de Fuego/cirugía , Ambulancias Aéreas , Islas Malvinas , Historia del Siglo XX , Humanos , Medicina Militar/métodos , Transporte de Pacientes , Reino Unido/epidemiología , Heridas por Arma de Fuego/mortalidadRESUMEN
Tobacco smoke contains substances capable of binding iron in an aqueous medium and transferring the metal into both organic solvents and intact mammalian red cells. This iron-binding activity is due to free fatty acids which are abundant in tobacco smoke and form 2:1 (free fatty acid:iron) chelates with ferrous iron. These earlier observations suggested that smoke-borne free fatty acids and the associated delocalization of iron within the lung might contribute to both the chronic pulmonary inflammation and the carcinogenesis associated with smoking. We now report that micromolar concentrations of iron or free fatty acid are not toxic to cultured human lung fibroblasts. However, when combined, the same low concentrations of iron and free fatty acid exert synergistic toxicity. Furthermore, the combination of free fatty acid and iron is highly mutagenic, inducing almost as many selectable mutations in the gene for hypoxanthine/guanine phosphoribosyl transferase as does benzo[a]pyrenediolepoxide, a class I carcinogen generated from benzo[a]pyrene present in cigarette smoke. The combination of free fatty acid and iron also promotes transformation of NIH 3T3 cells into an anchorage-independent phenotype. We conclude that free fatty acids in tobacco smoke may be important contributors to both the pulmonary damage and the carcinogenesis associated with smoking.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Ácidos Grasos no Esterificados/toxicidad , Hierro/toxicidad , Mutágenos/toxicidad , Nicotiana , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Pulmón/citología , Pulmón/efectos de los fármacos , Ratones , Mutación , Células 3T3 NIH/citología , Células 3T3 NIH/efectos de los fármacos , FenotipoRESUMEN
Mutations in DNA are generally considered to have an etiologic role in the development of cancer. If so, it follows that reducing the frequency of such mutations will reduce the incidence of cancer induced by mutagens. Recent advances in elucidating the molecular mechanisms of carcinogen-induced mutagenesis indicate that replication of DNA templates that contain replication-blocking adducts is accomplished with error-prone DNA polymerases. These polymerases have relaxed base-pairing requirements, and can insert bases across from adducted templates, but with potentially mutagenic consequences. In principle, these proteins present new and attractive molecular targets to reduce mutagenesis. If this can be done in vivo without increasing cytotoxic responses to carcinogens, then novel chemopreventive strategies can be designed to reduce the risk of cancer in exposed populations prior to the appearance of disease symptoms.
Asunto(s)
Replicación del ADN , Neoplasias/prevención & control , Animales , Transformación Celular Neoplásica/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , MutagénesisRESUMEN
In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Mutagénesis/efectos de la radiación , Nucleotidiltransferasas/metabolismo , ARN Catalítico/metabolismo , Secuencia de Bases , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Frecuencia de los Genes , Humanos , Masculino , Proteínas Nucleares , Conformación de Ácido Nucleico , Nucleotidiltransferasas/genética , ARN Catalítico/química , ARN Catalítico/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae , Rayos UltravioletaRESUMEN
The REV1 gene encodes a Y-family DNA polymerase that has been postulated to have both catalytic and structural functions in translesion replication past UV photoproducts in mammalian cells. To examine if REV1 is implicated in DNA damage tolerance mechanisms after exposure of human cells to a chemical carcinogen, we generated a plasmid expressing REV1 protein fused at its C-terminus with green fluorescent protein (GFP). In transient transfection experiments, virtually all of the transfected cells had a diffuse nuclear pattern in the absence of carcinogen exposure. In contrast, in cells exposed to benzo[a]pyrenediolepoxide, the fusion protein accumulated in a focal pattern in the nucleus in 25% of the cells, and co-localized with PCNA. These data support the idea that REV1 is present at stalled replication forks. We also examined the mutagenic response at the HPRT locus of human cells that had greatly reduced levels of REV1 mRNA due to the stable expression of gene-specific ribozymes, and compared them to wild-type cells. The mutant frequency was greatly reduced in the ribozyme-expressing cells. These data indicate that REV1 is implicated in the mutagenic DNA damage tolerance response to BPDE and support the development of strategies to target this protein to prevent such mutations.
Asunto(s)
Núcleo Celular/enzimología , Daño del ADN , Mutagénesis , Nucleotidiltransferasas/análisis , Nucleotidiltransferasas/fisiología , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Línea Celular , Humanos , Mutágenos/toxicidad , Proteínas Nucleares , Nucleotidiltransferasas/genética , Proteínas Recombinantes de Fusión/análisisRESUMEN
The detection of an increase in the frequency of mutants in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of circulating T-cells has been proposed as a method to evaluate the biological effects of human exposure to environmental mutagens. We exposed adult human T-cells in vitro to 1-nitrosopyrene (1-NOP), a partially reduced metabolite of 1-nitropyrene, a ubiquitous environmental carcinogen. In populations of T-cells from two unrelated donors, a dose of 1-NOP that reduced survival to 40% of the untreated cells increased the HPRT mutant frequency 6 to 7 times over the background frequency of 5 x 10(-6). The coding region of 35 independent mutants was amplified by polymerase chain reaction and sequenced. Single base substitutions were found in 63% of the mutants (22 of 35). These were distributed randomly throughout the gene. Most of the substitutions (82%) involved G-C base pairs, mainly G.C-->A.T transitions and G.C-->T.A transversions. Fifteen mutants were lacking one or more exons; 9 of the 15 were lacking exons 2 and 3. Examination showed that at least four of the latter had resulted from V(D)J recombinase acting illegitimately to recombine sites located in introns 1 and 3 of the HPRT gene. T-cells from a second unrelated donor were exposed to 1-NOP and 38 additional independent mutants were analyzed. The results indicated that such mutations occurred at a frequency of 2.4 x 10(-6) compared to a background frequency of less than 0.3 x 10(-6). This recombinase, which plays an important role in leukemogenesis, is normally present in developing, but not mature, B- and T-cells such as those used here as target cells for 1-NOP. The present study is the first report showing that exposure to an environmental carcinogen can cause mutations induced by the action of this enzyme.
Asunto(s)
ADN Nucleotidiltransferasas/fisiología , Hipoxantina Fosforribosiltransferasa/genética , Mutación Puntual/genética , Pirenos/toxicidad , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Eliminación de Gen , Humanos , Masculino , Datos de Secuencia Molecular , Tioguanina/farmacología , VDJ RecombinasasRESUMEN
BACKGROUND: ACE inhibition (ACEI) attenuates post-myocardial infarction (MI) LV remodeling, but the effects of angiotensin II type 1 receptor (AT(1)) antagonism alone or in combination with ACEI are unclear. Accordingly, we investigated the effects of AT(1) antagonism, ACEI, and their combination in a well-characterized ovine postinfarction model. METHODS AND RESULTS: Beginning 2 days after transmural anteroapical MI, 62 sheep were treated with 1 of 5 treatment regimens: no therapy (control, n=12), standard-dose ACEI (sACEI; ramipril 10 mg/d, n=14), high-dose ACEI (hACEI; ramipril 20 mg/d, n=8), AT(1) blockade (losartan 50 mg/d, n=13), and combination therapy with sACEI+AT(1) blockade (CT; ramipril 10 mg/d+losartan 50 mg/d, n=15). MRI was performed before and 8 weeks after MI to quantify changes in LV end-diastolic and end-systolic volume indices (DeltaEDVI, DeltaESVI) and ejection fraction (DeltaEF). Change in regional percent intramyocardial circumferential shortening in noninfarcted segments adjacent to the infarct (Adj Delta%S) was measured by tagged MRI. CT resulted in the most marked blunting of LV remodeling: DeltaESVI (+1.0+/-0.4, +0.7+/-0.4, +0.6+/-0.3, +0.9+/-0.5, and +0.4+/-0.2* mL/kg); DeltaEDVI (+0.9+/-0.4, +0.7+/-0.5, +0.6+/-0.5, +0.9+/-0.5, and +0.4+/-0.3 mL/kg); DeltaEF (-24+/-7, -18+/-6, -14+/-7, -18+/-10, and -11+/-9* %); and Adj Delta%S (-8+/-4, -7+/-3, -5+/-3, -5+/-3, and -2+/-3* %) for Control, sACEI, hACEI, AT(1) blockade, and CT, respectively (*P<0.04 versus sACEI, AT(1) blockade, and control; P<0.05 versus control; P<0.002 versus AT(1) blockade and control). EDVI and ESVI at 8 weeks after MI were smallest with CT (P<0.02 versus all). CONCLUSIONS: Combination therapy with sACEI+AT(1) blockade shows promise in attenuating postinfarction LV remodeling but was not clearly superior to hACEI in the present study.
Asunto(s)
Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/fisiopatología , Remodelación Ventricular/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Electrocardiografía , Femenino , Procesamiento de Imagen Asistido por Computador , Losartán/farmacología , Imagen por Resonancia Cinemagnética , Infarto del Miocardio/diagnóstico , Miocardio/metabolismo , Miocardio/patología , Ramipril/farmacología , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Ovinos , Volumen Sistólico/efectos de los fármacos , Sístole , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/fisiologíaRESUMEN
Earlier, we reported that second trimester human fetal kidney and, to a much lesser extent, human fetal liver were capable of synthesizing and secreting the beta-subunit of hCG. Recently, we also have shown that these tissues, likewise, synthesize and secrete the alpha-subunit of hCG. The hCG produced is biologically active. To determine the cellular localization of these peptides, immunocytochemical studies were performed on human fetal tissues using antibodies against beta hCG, alpha hCG, and the intact hormone. Placental syncytiotrophoblast served as an immunopositive control. In the human fetal kidney, the ascending (thick) limb of the loop of Henle, distal convoluted tubule, and occasional cells in the collecting ducts were distinctly immunopositive for both beta hCG and the alpha-subunit. Small amounts of light positive staining occurred in only a few hepatocytes. Placental syncytiotrophoblast was routinely positive for both subunits, but fetal lung and striated muscle were negative. These immunocytochemical results indicate that immunoreactive beta hCG as well as the alpha-subunit are present in placental syncytiotrophoblast, in the distal renal nephron, and in a limited population of hepatocytes. The qualitative number and intensity of immunopositive cells closely correlate with the quantitative amounts of their hCG subunit synthesis. Taken together with our previous biosynthetic data, the immunocytochemical localization reported here indicates the probable cellular sites of alpha- and beta hCG synthesis in these tissues. The presence of comparable alpha- and beta-subunit staining in identical cell populations suggests that both hCG subunits and, therefore, perhaps intact hCG are produced at these same cellular sites during fetal life.