RESUMEN
Female Apc(min/+) mice carrying the BALB/c variant of Prkdc or heterozygous knockout for Xrcc2, were sham- or 2 Gy X-irradiated as adults to compare the effect of mild impairments of double-strand break (DSB) repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR) respectively on spontaneous and radiation-induced mammary and intestinal tumorigenesis. Mice with impaired NHEJ showed no difference in incidence of spontaneous mammary tumours, compared with matched controls, (2.46 fold, P=0.121) and significantly less following irradiation (radiation-induced excess; 0.35 fold, P=0.008). In contrast mice with impaired HR presented with significantly less spontaneous mammary tumours than matched controls (0.33 fold, P=0.027) and significantly more following irradiation (radiation-induced excess; 3.3 fold, P=0.016). Spontaneous and radiation-induced intestinal adenoma multiplicity in the same groups were significantly greater than matched controls for mice with impaired NHEJ (sham; 1.29 fold, P<0.001, radiation-induced excess; 2.55 fold, P<0.001) and mice with impaired HR showed no significant differences (sham; 0.92 fold, P=0.166, radiation-induced excess; 1.16, P=0.274). Genetic insertion events were common in spontaneous tumours from NHEJ impaired mice compared with matched controls. γH2AX foci analysis suggests a significantly faster rate of DSB repair (MANOVA P<0.001) in intestinal than mammary tissue; apoptosis was also higher in irradiated intestine. To conclude, results suggest that pathway of choice for repair of spontaneous and radiation-induced DSBs is influenced by tissue type. NHEJ appears to play a greater role in DSB repair in intestinal tissue since impairment by functional change of Prkdc significantly increases the rate of mis-repair in intestinal but not mammary tissue. HR appears to play a greater role in DSB repair in adult mammary tissue since impaired HR results in significant changes in mammary but not in the intestinal tumorigenesis. This indicates that early DNA damage response and repair is important for cancer susceptibility and plays a role in determining tissue specificity of cancer risk.
Asunto(s)
Carcinogénesis/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Neoplasias Intestinales/genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Inducidas por Radiación/genética , Reparación del ADN por Recombinación , Animales , Apoptosis , Carcinogénesis/efectos de la radiación , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Genes APC , Neoplasias Intestinales/patología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Inducidas por Radiación/patología , Proteínas Nucleares/genéticaRESUMEN
Elevated cataract risk after radiation exposure was established soon after the discovery of X-rays in 1895. Today, increased cataract incidence among medical imaging practitioners and after nuclear incidents has highlighted how little is still understood about the biological responses of the lens to low-dose ionizing radiation (IR). Here, we show for the first time that in mice, lens epithelial cells (LECs) in the peripheral region repair DNA double strand breaks (DSB) after exposure to 20 and 100 mGy more slowly compared with circulating blood lymphocytes, as demonstrated by counts of γH2AX foci in cell nuclei. LECs in the central region repaired DSBs faster than either LECs in the lens periphery or lymphocytes. Although DSB markers (γH2AX, 53BP1 and RAD51) in both lens regions showed linear dose responses at the 1 h timepoint, nonlinear responses were observed in lenses for EdU (5-ethynyl-2'-deoxy-uridine) incorporation, cyclin D1 staining and cell density after 24 h at 100 and 250 mGy. After 10 months, the lens aspect ratio was also altered, an indicator of the consequences of the altered cell proliferation and cell density changes. A best-fit model demonstrated a dose-response peak at 500 mGy. These data identify specific nonlinear biological responses to low (less than 1000 mGy) dose IR-induced DNA damage in the lens epithelium.
Asunto(s)
Proliferación Celular/efectos de la radiación , Forma de la Célula/efectos de la radiación , Ciclina D1/metabolismo , Cristalino/efectos de la radiación , Rayos X , Algoritmos , Animales , Línea Celular , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN , Relación Dosis-Respuesta en la Radiación , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Femenino , Histonas/metabolismo , Humanos , Immunoblotting , Cristalino/citología , Cristalino/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Microscopía Fluorescente , Modelos BiológicosRESUMEN
The growing human exposure to extremely low frequency (ELF) magnetic fields has raised a considerable concern regarding their genotoxic effects. The aim of this study was to evaluate the in vivo effects of ELF magnetic fields irradiation on mutation induction in the germline and somatic tissues of male mice. Seven week old BALB/c×CBA/Ca F1 hybrid males were exposed to 10, 100 or 300µT of 50Hz magnetic fields for 2 or 15h. Using single-molecule PCR, the frequency of mutation at the mouse Expanded Simple Tandem Repeat (ESTR) locus Ms6-hm was established in sperm and blood samples of exposed and matched sham-treated males. ESTR mutation frequency was also established in sperm and blood samples taken from male mice exposed to 1Gy of acute X-rays. The frequency of ESTR mutation in DNA samples extracted from blood of mice exposed to magnetic fields did not significantly differ from that in sham-treated controls. However, there was a marginally significant increase in mutation frequency in sperm but this was not dose-dependent. In contrast, acute exposure X-rays led to significant increases in mutation frequency in sperm and blood of exposed males. The results of our study suggest that, within the range of doses analyzed here, the in vivo mutagenic effects of ELF magnetic fields are likely to be minor if not negligible.
Asunto(s)
Campos Magnéticos , Mutación , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Secuencias Repetidas en TándemRESUMEN
PURPOSE: Following in utero exposure to low dose radiation (10-200 mGy), we recently observed a linear induction of DNA double-strand breaks (DSB) and activation of apoptosis in the embryonic neuronal stem/progenitor cell compartment. No significant induction of DSB or apoptosis was observed following exposure to magnetic fields (MF). In the present study, we exploited this in vivo system to examine whether exposure to MF before and after exposure to 100 mGy X-rays impacts upon DSB repair rates. MATERIALS AND METHODS: 53BP1 foci were quantified following combined exposure to radiation and MF in the embryonic neuronal stem/progenitor cell compartment. Embryos were exposed in utero to 50 Hz MF at 300 µT for 3 h before and up to 9 h after exposure to 100 mGy X-rays. Controls included embryos exposed to MF or X-rays alone plus sham exposures. RESULTS: Exposure to MF before and after 100 mGy X-rays did not impact upon the rate of DSB repair in the embryonic neuronal stem cell compartment compared to repair rates following radiation exposure alone. CONCLUSIONS: We conclude that in this sensitive system MF do not exert any significant level of DNA damage and do not impede the repair of X-ray induced damage.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/efectos de la radiación , Roturas del ADN de Doble Cadena , Reparación del ADN/efectos de la radiación , Campos Magnéticos/efectos adversos , Animales , Encéfalo/embriología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/efectos de la radiación , Femenino , Ventrículos Laterales/embriología , Ventrículos Laterales/metabolismo , Ventrículos Laterales/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Células-Madre Neurales/efectos de la radiación , EmbarazoRESUMEN
The use of X-rays for medical diagnosis is enhancing exposure to low radiation doses. Exposure to extremely low-frequency electromagnetic or magnetic fields is also increasing. Epidemiological studies show consistent associations of childhood leukaemia with exposure to magnetic fields but any causal relationship is unclear. A limitation in assessing the consequence of such exposure is the availability of sensitive assays. The embryonic neuronal stem and progenitor cell compartments are radiosensitive tissues. Using sensitive assays, we report a statistically significant increase in DNA double-strand break (DSB) formation and apoptosis in the embryonic neuronal stem cell compartment following in utero exposure to 10-200 mGy X-rays. Both endpoints show a linear response. We also show that DSB repair is delayed following exposure to doses below 50 mGy compared with 100 mGy. Thus, we demonstrate in vivo consequences of low-dose radiation. In contrast to these impacts, we did not observe any significant induction of DSBs or apoptosis following exposure to 50 Hz magnetic fields (100 or 300 µT). We conclude that any DSB induction by treatment with magnetic fields is lower than following exposure to 10 mGy X-rays. For comparison, certain procedures involving computed tomography scanning are equivalent to 1-5 mGy X-rays.
Asunto(s)
Apoptosis/efectos de la radiación , Encéfalo/embriología , Roturas del ADN de Doble Cadena/efectos de la radiación , Embrión de Mamíferos/metabolismo , Células-Madre Neurales/metabolismo , Animales , Encéfalo/metabolismo , Relación Dosis-Respuesta en la Radiación , Embrión de Mamíferos/patología , Femenino , Campos Magnéticos , Ratones , Células-Madre Neurales/patología , Rayos X/efectos adversosRESUMEN
PURPOSE: To compare the development of intestinal adenomas following neutron and X-ray exposure of Apc(Min/+) mice (Apc - adenomatous polyposis coli; Min - multiple intestinal neoplasia). MATERIALS AND METHODS: Adult mice were exposed to acute doses of X-rays or fission neutrons. Tumour counting was undertaken 200 days later and samples were taken for Loss of Heterozygosity (LOH) analysis. RESULTS: Tumour numbers (adenomas and microadenomas) increased by 1.4-fold, 1.7-fold, 2.7-fold and 9-fold, after 0.5, 1, 2 and 5 Gy X-rays, respectively, and by 2.4-fold and 5.7-fold, after 0.5 and 1 Gy fission neutrons, respectively. LOH analysis of tumours from neutron-exposed mice showed that 63% had lost Apc and 90% (cf. 53% in controls) had lost D18mit84, a marker for Epb4.1l4a/NBL4 (erythrocyte protein band 4.1-like 4a/novel band 4.1-like 4), known to be involved in the Wnt (wingless-related mouse mammary tumour virus integration site) pathway. Some tumours from neutron-exposed mice appeared to have homozygous loss of some chromosomal markers. CONCLUSIONS: X-ray or fission neutron irradiation results in strongly enhanced tumour multiplicities. Comparison of tumour yields indicated a low Relative Biological Effectiveness of around 2-8 for fission neutrons compared with X-rays. LOH in intestinal tumours from neutron-exposed mice appeared to be more complex than previously reported for tumours from X-irradiated mice.
Asunto(s)
Neoplasias Intestinales/etiología , Neoplasias Inducidas por Radiación/etiología , Animales , Relación Dosis-Respuesta en la Radiación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neutrones , Dosis de Radiación , Efectividad Biológica Relativa , Rayos XRESUMEN
XRCC2 has an important role in repair of DNA damage by homologous recombination. Adult Apc(min/+) (min, multiple intestinal neoplasia) mice, wild-type or heterozygous for Xrcc2 deficiency, were sham-irradiated or 2-Gy X-irradiated. Spontaneous mammary and intestinal tumor incidences are lower in Apc(min/+) Xrcc2(+/-) mice than in Apc(min/+) Xrcc2(+/+) mice (mammary tumors: 14% and 38%, respectively, χ(2) P = 0.03; intestinal adenomas in mice reaching full life span: 108.6 and 130.1, respectively, t-test P = 0.005). Following irradiation, the increase in mammary tumors was greatest in female mice heterozygous for Xrcc2 (7.25 ± 0.50-fold in Apc(min/+) Xrcc2(+/-) mice compared with 2.57 ± 0.35-fold in Apc(min/+) Xrcc2(+/+) mice; t-test P < 0.001). The increase in intestinal tumor multiplicity following irradiation was significantly greater in Apc(min/+) Xrcc2(+/-) mice (Apc(min/+) Xrcc2(+/-), 4.14 ± 0.05-fold, versus Apc(min/+) Xrcc2(+/+), 3.30 ± 0.05-fold; t-test P < 0.001). Loss of heterozygosity of all chromosome 18 markers was greater in intestinal tumors from Apc(min/+) Xrcc2(+/-) mice than in tumors from Apc(min/+) Xrcc2(+/+) mice. These findings indicate that Xrcc2 haploinsufficiency reduces spontaneous tumor incidence on an Apc(min/+) background but increases the tumorigenic response to radiation.
Asunto(s)
Poliposis Adenomatosa del Colon/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Intestinales/patología , Lesiones Precancerosas/metabolismo , Animales , Femenino , Neoplasias Intestinales/genética , Pérdida de Heterocigocidad/genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Lesiones Precancerosas/patología , Rayos XRESUMEN
PURPOSE: To investigate microsatellite instability (MSI) in radiation-induced murine tumours, its dependence on tissue (haemopoietic, intestinal, mammary, brain and skin) and radiation type. MATERIALS AND METHODS: DNA from spontaneous, X-ray or neutron-induced mouse tumours were used in Polymerase Chain Reactions (PCR) with mono- or di-nucleotide repeat markers. Deviations from expected allele size caused by insertion/deletion events were assessed by capillary electrophoresis. RESULTS: Tumours showing MSI increased from 16% in spontaneously arising tumours to 23% (P = 0.014) in X-ray-induced tumours and rising again to 83% (P << 0.001) in neutron-induced tumours. X-ray-induced Acute Myeloid Leukaemias (AML) had a higher level of mono-nucleotide instability (45%) than di-nucleotide instability (37%). Fifty percent of neutron-induced tumours were classified as MSI-high for mono-nucleotide markers and 10% for di-nucleotide markers. Distribution of MSI varied in the different tumour types and did not appear random. CONCLUSIONS: Exposure to ionising radiation, especially neutrons, promotes the development of MSI in mouse tumours. MSI may therefore play a role in mouse radiation tumourigenesis, particularly following high Linear Energy Transfer (LET) exposures. MSI events, for a comparable panel of genome-wide markers in different tissue types, were not randomly distributed throughout the genome.
Asunto(s)
Inestabilidad de Microsatélites/efectos de la radiación , Repeticiones de Microsatélite/efectos de la radiación , Neoplasias Inducidas por Radiación/etiología , Neoplasias Inducidas por Radiación/genética , Neutrones/efectos adversos , Rayos X/efectos adversos , Animales , Línea Celular Tumoral , ADN/aislamiento & purificación , ADN/metabolismo , ADN/efectos de la radiación , Electroforesis Capilar , Fibroblastos/patología , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias Inducidas por Radiación/clasificación , Neoplasias Inducidas por Radiación/patología , Reacción en Cadena de la PolimerasaRESUMEN
Ionising radiation is a carcinogen capable of inducing tumours, including colorectal cancer, in both humans and animals. By backcrossing a recombinant line of Apc(Min/+) mice to the inbred BALB/c mouse strain, which is unusually sensitive to radiation-induced tumour development, we obtained panels of 2Gy-irradiated and sham-irradiated N2 Apc(Min/+) mice for genotyping with a genome-wide panel of microsatellites at approximately 15 cM density and phenotyping by counting adenomas in the small intestine. Interval and composite interval mapping along with permutation testing identified five significant susceptibility quantitative trait loci (QTLs) responsible for radiation induced tumour multiplicity in the small intestine. These were defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (log of odds, LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p<0.00001, 6.2, p<0.00001) and two regions within chromosome 16 respectively (LOD 4.1, p = 4x10(-5), 4.8, p<0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p<0.005). Genes containing BALB/c specific non-synonymous polymorphisms were identified within Mrip regions and prediction programming used to locate potentially functional polymorphisms. Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in Apc(Min/+) mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Casc5, and Bub1), DNA repair (Recc1 and Prkdc) or inflammation (Duox2, Itgb2l and Cxcl5). Our study demonstrates use of in silico analysis in candidate gene identification as a way of reducing large-scale backcross breeding programmes.
Asunto(s)
Adenoma/genética , Adenoma/patología , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Fosfolipasas A2 Grupo II/metabolismo , Neoplasias Inducidas por Radiación/genética , Proteínas/metabolismo , Sitios de Carácter Cuantitativo/genética , Aminoácidos/genética , Animales , Cromosomas de los Mamíferos/genética , Biología Computacional , Marcadores Genéticos , Genoma/genética , Intestino Delgado/patología , Escala de Lod , Ratones , Mutación/genética , Polimorfismo Genético , Selección GenéticaRESUMEN
Apc(Min) mice have provided an example of a locus (Modifier of Min1; Mom1) modifying adenoma numbers in the intestines of inbred strains. Linkage analysis located Mom1 on chromosome 4, and further investigation identified secretory phospholipase A2 (Pla2g2a) as a candidate gene. Because of unknown variation introduced by a single founding male mouse, our Min stock, although Pla2g2a(Mom1-s), was not on a pure C57BL/6J background and exhibited several polymorphic loci, including a region on chromosome 18 distal to Apc. Through selective breeding for homozygosity for distal chromosome 18 markers, six recombinant lines that presented with limited intraline variation in adenoma numbers were established. One line (V) showed a particularly severe phenotype (mean adenoma number +/- SEM, 370 +/- 21) compared with the other lines that recorded significantly lower means (3- to 5-fold; P < 10(-3), t test). Intercrosses between lines I and V showed suppression of the severe phenotype in the N1 generation. In N2 (and subsequent) backcrosses, tumor multiplicity depended on the origins of the WT and Min Apc alleles. Mice carrying both alleles from line V had a severe phenotype; others had mild disease very similar to line I (likelihood ratio statistic > 49.0; likelihood of odds > 10; P < 10(-5)). Frequency of allele loss at Apc was increased significantly in adenomas of mice with more severe disease. We propose that a modifier gene close to Apc or structural variation on chromosome 18 modifies polyp numbers in our mice, possibly by altering the frequency of WT Apc allele loss.
Asunto(s)
Adenoma/genética , Genes APC , Neoplasias Intestinales/genética , Alelos , Animales , Mapeo Cromosómico , Femenino , Ligamiento Genético , Predisposición Genética a la Enfermedad , Pólipos Intestinales/genética , Masculino , Ratones , Ratones Endogámicos C57BL , FenotipoRESUMEN
Ionizing radiation (IR) is a well-characterized carcinogen in humans and mice. The BALB/c mouse strain is unusually sensitive to IR-induced tissue damage and cancer development in a range of organs, suggestive of a partial defect in DNA damage response. This has been confirmed by finding BALB/c-specific functional polymorphism in Prkdc, a gene on mouse chromosome 16 that encodes the catalytic subunit of DNA-dependent protein kinase. Prkdc(BALB) has been associated with increased susceptibility to IR-induced mammary and lymphatic neoplasia. Here, we provide evidence that chromosome 16 segments from BALB/c interact with Apc(Min) (multiple intestinal neoplasia) and specifically enhance IR-induced adenoma development in the upper part of the small intestine.
Asunto(s)
Adenoma/genética , Proteínas de Unión al ADN , Neoplasias Intestinales/genética , Neoplasias Inducidas por Radiación/genética , Proteínas Serina-Treonina Quinasas/genética , Adenoma/enzimología , Adenoma/etiología , Poliposis Adenomatosa del Colon/enzimología , Poliposis Adenomatosa del Colon/genética , Animales , Mapeo Cromosómico , Cromosomas de los Mamíferos/genética , Cocarcinogénesis , Proteína Quinasa Activada por ADN , Femenino , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/etiología , Intestino Delgado/enzimología , Intestino Delgado/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Inducidas por Radiación/enzimología , Neoplasias Inducidas por Radiación/etiología , Proteínas NuclearesRESUMEN
Cytogenetic and molecular studies have provided evidence of the clustering of chromosome 2 deletion breakpoints in radiation-induced murine acute myeloid leukemia (AML). Moreover, clustering occurs in at least two fragile domains rich in telomere-like arrays. Here we describe a physical map of the distal breakpoint cluster and confirm the presence of inverted head-to-head telomeric sequence arrays. These potentially recombinogenic sequences were not, however, the direct focus for post-irradiation chromosome breakage in AML. Instead, the two arrays bordered a 2.5-kb sequence with properties expected of a nuclear matrix attachment region (MAR). The putative MAR co-localized in the fragile domain with genes important to the hemopoietic system (leukocyte tyrosine kinase, zinc finger protein 106, erythrocyte protein band 4.2, and beta(2)-microglobulin (beta2m)); the beta2m subdomain was a particular focus of breakage. On the basis of these and other data, we suggest that AML-associated chromosome 2 fragility in the mouse is a consequence of domain-specific fragility in genomic domains containing numerous genes critical to the hemopoietic system. Recorded with the permission of the controller of Her Majesty's Stationery Office. Published by Wiley-Liss, Inc.
Asunto(s)
Leucemia Mieloide/genética , Neoplasias Inducidas por Radiación/genética , Enfermedad Aguda , Animales , Secuencia de Bases , Cromosomas Artificiales Bacterianos , Cromosomas Artificiales de Levadura , Clonación Molecular , ADN de Neoplasias , Leucemia Mieloide/etiología , Ratones , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The long arm of human chromosome 7 between 7q22 and 7q36 has been identified as a region harboring one or more tumor-suppressor genes (TSGs) inactivated in acute myeloid leukemia (AML). Additional TSGs mapping to other chromosomes may well be involved in the etiology of this disease. For example, experiments using a mouse model system have indicated the possible presence of an AML TSG at 11p11-12. Microcell-mediated chromosome transfer (MMCT) has been used to introduce human chromosomes 7 and 11 into a murine myeloid leukemia cell line. A proportion of MMCT hybrid clones containing either whole chromosome 7 or fragments of chromosome 11 showed a significant delay in leukemogenic onset when injected into syngeneic mice. Screening of hybrid clones did not associate any human microsatellite markers with decreased leukemogenic potential in vivo. However, preliminary evidence was obtained of allelic loss at chromosomal regions homologous with human 7q22 in murine F1 hybrid AMLs. Our data provide functional evidence of AML-associated TSGs localized to human chromosomes 7 and 11 in support of previously published studies on cytogenetic and allelic losses associated with AML development.