RESUMEN
Many epithelial stem cell populations follow a pattern of stochastic stem cell divisions called 'neutral drift'. It is hypothesised that neutral competition between stem cells protects against the acquisition of deleterious mutations. Here we use a Porcupine inhibitor to reduce Wnt secretion at a dose where intestinal homoeostasis is maintained despite a reduction of Lgr5+ stem cells. Functionally, there is a marked acceleration in monoclonal conversion, so that crypts become rapidly derived from a single stem cell. Stem cells located further from the base are lost and the pool of competing stem cells is reduced. We tested whether this loss of stem cell competition would modify tumorigenesis. Reduction of Wnt ligand secretion accelerates fixation of Apc-deficient cells within the crypt leading to accelerated tumorigenesis. Therefore, ligand-based Wnt signalling influences the number of stem cells, fixation speed of Apc mutations and the speed and likelihood of adenoma formation.
Asunto(s)
Carcinogénesis/metabolismo , Transformación Celular Neoplásica/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Células Madre/citología , Células Madre/metabolismo , Vía de Señalización Wnt , Aciltransferasas/antagonistas & inhibidores , Adenoma/etiología , Adenoma/metabolismo , Adenoma/patología , Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Inhibidores Enzimáticos/farmacología , Mucosa Intestinal/efectos de los fármacos , Ligandos , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pirazinas/farmacología , Piridinas/farmacología , Células Madre/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Stem cells are responsible for maintaining differentiated cell numbers during normal physiology and at times of tissue stress. They have the unique capabilities of proliferation, self-renewal, clonogenicity and multi-potentiality. It is a widely held belief that stem-like cells, known as cancer stem cells (CSCs), maintain tumours. The majority of currently identified intestinal stem cell populations appear to be rapidly cycling. However, quiescent stem cell populations have been suggested to exist in both normal intestinal crypts and tumours. Quiescent CSCs may have particular significance in the modern management of colorectal cancer making their identification and characterisation a priority. In this review, we discuss the current evidence surrounding the identification and microenvironmental control of stem cell populations in intestinal crypts and tumours as well as exploring the evidence supporting the existence of a quiescent stem and CSC population in the gut and other tissues.
Asunto(s)
Intestinos/patología , Células Madre Neoplásicas/patología , Neoplasias del Recto/patología , Células Madre/fisiología , Puntos de Control del Ciclo Celular , Neoplasias Colorrectales/patología , Humanos , Neoplasias del Recto/terapiaRESUMEN
Although deregulation of the Wnt signalling pathway has been implicated in urothelial cell carcinoma (UCC), the functional significance is unknown. To test its importance, we have targeted expression of an activated form of ß-catenin to the urothelium of transgenic mice using Cre-Lox technology (UroIICRE(+) ß-catenin(exon3/+)). Expression of this activated form of ß-catenin led to the formation of localized hyperproliferative lesions by 3 months, which did not progress to malignancy. These lesions were characterized by a marked increase of the phosphatase and tensin homologue (PTEN) tumour suppressor protein. This appears to be a direct consequence of activating Wnt signalling in the bladder as conditional deletion of the adenomatous polyposis coli (Apc) gene within the adult bladder led rapidly to coincident ß-catenin and PTEN expression. This PTEN expression blocked proliferation. Next, we combined PTEN deficiency with ß-catenin activation and found that this caused papillary UCC. These tumours had increased pAKT signalling and were dependent on mammalian target of rapamycin (mTOR). Importantly, in human UCC, there was a significant correlation between high levels of ß-catenin and pAKT (and low levels of PTEN). Taken together these data show that deregulated Wnt signalling has a critical role in promoting UCC, and suggests that human UCC that have high levels of Wnt and PI3 kinase signalling may be responsive to mTOR inhibition.
Asunto(s)
Carcinoma Papilar/metabolismo , Transformación Celular Neoplásica/metabolismo , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Vejiga Urinaria/inducido químicamente , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Proliferación Celular , Estudios de Cohortes , Femenino , Humanos , Hiperplasia/inducido químicamente , Masculino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Eliminación de Secuencia , Serina-Treonina Quinasas TOR/metabolismo , Vejiga Urinaria/metabolismo , Urotelio/metabolismo , Proteínas Wnt/metabolismoRESUMEN
K-ras mutation occurs in 40-50% of human colorectal adenomas and carcinomas, but its contribution to intestinal tumorigenesis in vivo is unclear. We developed K-ras(V12) transgenic mice that were crossed with Ah-Cre mice to generate K-ras(V12)/Cre mice, which showed beta-naphthoflavone-induction of Cre-mediated LoxP recombination that activated intestinal expression of K-ras(V12) 4A and 4B transcripts and proteins. Only very occasional intestinal adenomas were observed in beta-naphthoflavone-treated K-ras(V12)/Cre mice aged up to 2 years, suggesting that mutated K-ras expression alone does not significantly initiate intestinal tumourigenesis. To investigate the effects of mutated K-ras on DNA mismatch repair (MMR)-deficient intestinal tumour formation, these mice were crossed with Msh2(-/-) mice to generate K-ras(V12)/Cre/Msh2(-/-) offspring. After beta-naphthoflavone treatment, K-ras(V12)/Cre/Msh2(-/-) mice showed reduced average lifespan of 17.3+/-5.0 weeks from 26.9+/-6.8 (control Msh2(-/-) mice) (P<0.01). They demonstrated increased adenomas in the small intestine from 1.41 (Msh2(-/-) controls) to 7.75 per mouse (increased fivefold, P<0.01). In the large intestine, very few adenomas were found in Msh2(-/-) mice (0.13 per mouse) whereas K-ras(V12)/Cre/Msh2(-/-) mice produced 2.70 adenomas per mouse (increased 20-fold, P<0.01). Over 80% adenomas from K-ras(V12)/Cre/Msh2(-/-) mice showed transgene recombination with expression of K-ras(V12) 4A and 4B transcripts and proteins. Sequencing of endogenous murine K-ras showed mutations in two out of 10 tumours examined from Msh2(-/-) mice, but no mutations in 17 tumours from K-ras(V12)/Cre/Msh2(-/-) mice. Expression of K-ras(V12) in tumours caused activation of the mitogen-activated protein kinase and Akt/protein kinase B signaling pathways, demonstrated by phosphorylation of p44MAPK, Akt and GSK3beta, as well as transcriptional upregulation of Pem, Tcl-1 and Trap1a genes (known targets of K-ras(V12) expression in stem cells). Thus, mutated K-ras cooperates synergistically with MMR deficiency to accelerate intestinal tumorigenesis, particularly in the large intestine.
Asunto(s)
Genes ras , Neoplasias Intestinales/etiología , Proteína 2 Homóloga a MutS/fisiología , Mutación , Animales , Reparación de la Incompatibilidad de ADN , Integrasas/fisiología , Neoplasias Intestinales/epidemiología , Neoplasias Intestinales/genética , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Transgénicos , Proteína 2 Homóloga a MutS/deficiencia , Proteínas Proto-Oncogénicas c-akt/fisiología , Timoma/etiología , Neoplasias del Timo/etiologíaRESUMEN
Gain-of-function mutations in the gene encoding the receptor tyrosine kinase RET have been identified as the aetiological factor for multiple endocrine neoplasia type 2A (MEN2A). MEN2A is a dominantly-inherited cancer predisposition syndrome characterized by medullary thyroid carcinoma, a tumour of the calcitonin-producing thyroid C-cells. There are three isoforms of RET: RET9, RET43 and RET51, and although in vitro evidence suggests they vary in cellular transformation activities, little is known about their function in tumorigenesis in vivo. To address this, we used RET51 cDNA to construct mice in which the most frequent MEN2A mutation, Cys-634-Arg, was expressed under the control of the human calcitonin promoter (CT-2A mice). These mice developed C-cell tumours resembling human MTC and follicular tumours resembling human papillary thyroid carcinoma (PTC) depending on the founder line examined. One founder line developed compound MTC/PTC at low frequency (8%) and pancreatic cystadenocarcinoma. CT-2A mice also displayed a developmental defect in thyroid follicular structure, in which much of the thyroid was occupied by large irregular cystic follicles thought to be derived from the ultimobranchial body, a developmental precursor of the thyroid gland. The CT-2A mice will provide a suitable model to further study the effects of the MEN 2A RET mutation in vivo.
Asunto(s)
Carcinoma Medular/genética , Carcinoma Papilar/genética , Proteínas de Drosophila , Neoplasia Endocrina Múltiple Tipo 2a/genética , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Neoplasias de la Tiroides/genética , Sustitución de Aminoácidos , Animales , Calcitonina/genética , Carcinoma Medular/patología , Carcinoma Papilar/patología , Transformación Celular Neoplásica/genética , Cistadenocarcinoma/genética , ADN Complementario/genética , Regulación Neoplásica de la Expresión Génica , Genes Sintéticos , Ratones , Ratones Transgénicos , Neoplasia Endocrina Múltiple Tipo 2a/patología , Mutación Missense , Neoplasias Pancreáticas/genética , Regiones Promotoras Genéticas , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-ret , Empalme del ARN , Proteínas Tirosina Quinasas Receptoras/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Neoplasias de la Tiroides/patología , TransgenesRESUMEN
Mice deficient for the mismatch repair (MMR) gene Msh2 show accelerated tumourigenesis and a reduced apoptotic response to DNA damage of methylation type. Here we examine the effect of mutation for Msh2 on in vivo mutation frequencies in the intestine as determined by loss of function at the Dolichos biflorus (Dlb-1) locus. Spontaneous mutation frequencies were scored in cohorts of ageing mice either wild type or mutant for Msh2. In mice less than 1 year old, mutation frequencies were only elevated in Msh2 null mice. However, beyond this age heterozygous Msh2 mice showed significantly higher mutation frequencies than controls. These findings implicate a gene dose dependent requirement for Msh2 in mutation suppression and prompted an analysis of young Msh2 mutants following exposure to DNA damage. Following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Msh2 deficient mice show a reduced apoptotic response and an increase in mutation frequency. Heterozygotes did not differ from controls. Following exposure to cisplatin, no significant elevation was seen in mutation frequencies, even within homozygotes. This is particularly surprising given the association between cisplatin resistance and MMR deficiency. These findings therefore demonstrate a complex reliance upon functional Msh2 in mutation surveillance. We have identified three separate scenarios. First, where retention of both Msh2 alleles over an extended period of time appears critical to the suppression of spontaneous mutation; second, 3 weeks following exposure to MNNG, where only complete loss of Msh2 results in elevated mutation; and finally following cisplatin exposure, where induced levels of mutation are independent of Msh2 status.
Asunto(s)
Disparidad de Par Base , Reparación del ADN , Proteínas de Unión al ADN , Dosificación de Gen , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Cisplatino/toxicidad , Genotipo , Heterocigoto , Metilnitronitrosoguanidina/toxicidad , Ratones , Ratones Noqueados , Proteína 2 Homóloga a MutS , Mutagénesis , Mutación , Proteínas Proto-Oncogénicas/deficienciaRESUMEN
Clinically relevant cancer chemotherapeutic alkylating agents such as temozolomide and dacarbazine induce apoptosis and are mutagenic via the formation of O(6)-alkylguanine adducts in DNA. The DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) functions by dealkylating such adducts and can thus prevent apoptosis and mutagenesis. In attempts to maximize the clinical effectiveness of these alkylating agents, inhibitors of AGT such as O(6)-benzylguanine (BeG) have been developed. We show here that within murine small intestinal crypt cells, BeG administration does not alter the apoptotic response to the direct-acting methylating agents N-methyl-N-nitrosurea (MNU), temozolomide and N-methyl-N'-nitro-N-nitrosoguanidine. Furthermore, we show that BeG pretreatment fails to elevate the mutation frequency at the murine Dlb-1 locus following exposure to MNU. Consistent with these results, we show that intestinal AGT activity is effectively abolished by administration of 100 mg/kg temozolomide, even in the absence of BeG. In contrast, pretreatment with BeG transiently abolished the apoptotic response to the methylating prodrug dacarbazine. Activation of dacarbazine to its reactive intermediate has previously been shown to be cytochrome P450 dependent and we show here that pretreatment of mice with the cytochrome P450 inhibitor metyrapone also inhibits dacarbazine-induced apoptosis. Thus BeG increases neither the prevalence of apoptosis nor mutation frequency in the murine small intestine, but is capable of inhibiting P450-dependent prodrug activation. The positive implication from this study is that BeG treatment may not exacerbate the toxic and mutagenic effects of methylating agents within normal cells, although it may engender other adverse reactions through the suppression of cytochrome P450-dependent processes.
Asunto(s)
Antineoplásicos Alquilantes/farmacocinética , Apoptosis/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/fisiología , Dacarbazina/farmacocinética , Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , Intestino Delgado/efectos de los fármacos , O(6)-Metilguanina-ADN Metiltransferasa/fisiología , Animales , Apoptosis/efectos de la radiación , Biotransformación , Cisplatino/farmacología , Daño del ADN , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Guanina/farmacología , Metilnitrosourea/farmacología , Ratones , Mutación , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , TemozolomidaRESUMEN
Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N'-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine. By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.
Asunto(s)
Apoptosis/genética , Reparación del ADN , Proteínas de Unión al ADN , Dacarbazina/análogos & derivados , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Mutagénesis , Mutágenos/toxicidad , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Cisplatino/toxicidad , Dacarbazina/toxicidad , Guanina/análogos & derivados , Guanina/toxicidad , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Intestino Delgado/citología , Intestino Delgado/patología , Metilnitronitrosoguanidina/toxicidad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratones Noqueados , Proteína 2 Homóloga a MutS , Proteínas Proto-Oncogénicas/deficiencia , Células Madre/citología , Células Madre/fisiología , TemozolomidaRESUMEN
We have investigated the mutagenicity of benzo[a]pyrene (B[a]P) in small intestine using the Dlb-1 locus assay in the mouse. Administration of B[a]P by the oral and i.p. routes had markedly different effects on the number of Dlb-I mutations and the pattern of induction of cytochrome P-4501A1 (CYP1A1). In Ahr-responsive animals i.p. injection resulted in marked induction in crypt cells along the length of the small intestine, with some induction in the villus cells. In contrast, after oral administration, CYP1A1 induction was evident only in the villus cells, and this declined distally. The intensity and speed of induction in Ahr-responsive animals was such that the genotoxic effect of a single injection of B[a]P could not be augmented by prior treatment with non-genotoxic inducers such as beta-napthoflavone and TCDD. Oral B[a]P treatment resulted in a decrease in the number of mutations when compared with the i.p. route. Studies in congenic Ahr-non-responsive versus Ahr-responsive mice indicated that induction of CYP1A1 was associated with increased numbers of Dlb-1 mutations. Mutation induction in Ahr-non-responsive mice in the absence of detectable CYP1A1 in either liver or small intestine indicates that an appreciable portion of B[a]P activation to a genotoxin must be by other than a CYP1A1 mediated route. These data show that B[a]P is a potent small intestinal mutagen at the Dlb-1 locus.
Asunto(s)
Benzo(a)pireno/toxicidad , Genes/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Mutagénesis , Mutágenos/toxicidad , Animales , Benzo(a)pireno/farmacocinética , Biotransformación , Cruzamientos Genéticos , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , Daño del ADN , Inducción Enzimática/efectos de los fármacos , Femenino , Intestino Delgado/química , Intestino Delgado/enzimología , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Microvellosidades/enzimología , Pruebas de Mutagenicidad , Mutágenos/farmacocinética , Receptores de Hidrocarburo de Aril/genética , Receptores Mitogénicos/genéticaRESUMEN
Comparison of results derived from transgenic animal gene-mutation assays with those from mutation analyses in endogenous genes is an important step in the validation of the former. We have used lambda lacZ transgenic mice to study alkylation-induced mutagenesis in vivo in (a) lacZ and hprt in spleen cells, and (b) lacZ and Dlb-I in small intestine from lambda lacZ+/0/Dlb-Ia/b mice. Induction of mutations by ethyl- and methylnitrosourea (ENU, MNU) and ethyl methanesulphonate (EMS) was investigated at 7 weeks after a single i.p. dose of each of these chemicals. In the small intestine, treatment with various dosages of ENU (10-150 mg/kg) resulted in a linear dose-response in both lacZ and Dlb-I. MNU (30 mg/kg) was also mutagenic in lacZ and Dlb-I, while EMS (250 mg/kg) did not significantly induce mutations in either gene. In spleen, ENU gave a linear dose-related response in both lacZ and hprt, MNU induced mutation sin both lacZ and hprt, and EMS was only positive for lacZ. No differences in response were observed between single and split-dose treatment with ENU (1 x 50 or 5 x 10 mg/kg with a 1- or 7-day interval), both in spleen and small intestine, except for lacZ in small intestine, where the single high dose gave a significantly higher induction than the split dose with the 7-day interval. The overall results suggest that mutagenic effects of fractionated doses are generally additive. In most cases, the induction factor (ratio treated over controls) for mutations in lacZ was lower than that for hprt and Dlb-I, presumably due to a higher background in lacZ and/or a lower mutability of lacZ. The general concordance between the data for lacZ and the endogenous genes indicates that lambda lacZ transgenic mice are a suitable model to study induction of gene mutations in vivo.
Asunto(s)
Operón Lac , Mutágenos/toxicidad , Alquilantes/toxicidad , Animales , Metanosulfonato de Etilo/toxicidad , Etilnitrosourea/toxicidad , Marcadores Genéticos , Hipoxantina Fosforribosiltransferasa/genética , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Metilnitrosourea/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de Mutagenicidad , Mutación , Bazo/citologíaRESUMEN
The ability to process damaged DNA may vary between cells depending on their differentiated status. However, there is little in vivo data available and it is not intuitively obvious how the activity of specific repair pathways may vary between different subpopulations (e.g. stem cells and proliferative, committed and differentiated cells) of a particular tissue. To obtain such information for the intestinal epithelium, we have developed an assay that detects differences in the way different regions of the crypt (stem, proliferative and maturation zones) respond to DNA damage. The assay is a variant of the 'comet' assay, which detects DNA strand breaks by measuring the proportion of DNA migrating from individual cells, or in this case intact isolated crypts, in an electrophoretic field. The method is quantitative, with the amount of migrating DNA being proportional to the number of strand breaks. Isolated crypts are repair competent and spatial differences are apparent with some agents. The assay has the potential to characterize the repair properties of cells at different stages of differentiation within the crypt, determine the characteristics that might predispose them to damage and may help in understanding the route of stem cell mutation.
Asunto(s)
Daño del ADN , Reparación del ADN , Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Animales , Diferenciación Celular , Línea Celular , Humanos , Mucosa Intestinal/patología , Mutagénesis , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Normal function of the p53 gene is integral to the cellular response to genotoxic stress. One prediction arising from this is that p53 deficiency results in an increased mutation frequency. However, limited evidence has been produced in support of this idea. In order to further investigate the in vivo role of p53 in surveillance against mutation, and particularly to address the significance of p53-dependent apoptosis, we scored mutation frequency at the Dlb-1 locus within cells of the intestinal epithelium of animals which were wild type, heterozygous or null for p53 and heterozygous (a/b) at the Dlb-1 locus. Using this assay we have shown that loss of a p53-dependent apoptotic pathway is associated with the detectable acquisition of mutations, but only at high levels of DNA damage. These results question the significance of the immediate 'wave' of p53-dependent apoptosis seen in this tissue, particularly as there was a delayed p53-independent apoptotic pathway. We conclude that loss of p53 function only becomes relevant to the in vivo acquisition of mutations and thus tumorigenesis in certain circumstances.
Asunto(s)
Apoptosis , Genes p53 , Intestino Delgado/metabolismo , Mutación , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/genética , Células Epiteliales , Epitelio/metabolismo , Genotipo , Intestino Delgado/citología , Ratones , Ratones Noqueados , Especificidad de Órganos , Receptores Mitogénicos/genética , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
We have developed a method to quantitate DNA strand breaks as a measure of DNA damage and repair in intact, isolated intestinal crypts. The assay is a modified form of the single-cell gel electrophoresis or 'comet' assay. By maintaining the spatial relationship between the cells we were able to characterise the repair response and the susceptibility to DNA damage of cells as a function of their position in the crypt. All cells were equally repair competent over the first 30 minutes of the repair of UV-C and gamma-radiation induced lesions. DNA damage was equally distributed following gamma-radiation but following incubation with the topoisomerase II inhibitor etoposide, damage was greater in the lower crypt with an unusual component to the comet tall which was tapered, implying an incremental change in susceptibility by cell position. This tapered component of the comet tail resolved rapidly after removal of etoposide. The pattern of damage produced by hydrogen peroxide was dose dependent with lower doses producing more strand breaks in the base of the crypt-an effect lost at higher doses. The assay has the ability to detect differences between cells in their susceptibility to DNA damage and their subsequent repair response which may vary with their proliferative or differentiative status.
Asunto(s)
Daño del ADN , Reparación del ADN , Intestinos/citología , Animales , Electroforesis en Gel de Poliacrilamida , Etopósido/farmacología , Femenino , Peróxido de Hidrógeno/farmacología , Ratones , Ratones Endogámicos C57BLRESUMEN
Mutations in long lived stem cells are critical events in carcinogenesis. The Dlb-1 assay detects intestinal stem cell mutation at the Dlb-1 locus in Dlb-1a/b heterozygous mice by visualizing mutated clones of epithelial cells in situ which do not bind the lectin Dolichos biflorus agglutinin. We have used this assay to show that the food-derived heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent intestinal mutagen when administered either i.p. or p.o. This contrasts with the inactivity of the structurally related mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the assay which we have described previously. Immunocytochemical localization of the P-450 enzyme CYP1A2, which is responsible for the primary activation of these mutagens, shows that in untreated mice it is present in liver hepatocytes and in occasional villus epithelial cells but is absent from the target intestinal stem cell population. In addition, liver microsomes, unlike intestinal microsomes, were able to convert PhIP to the proximate mutagen N-hydroxy-PhIP. CYP1A2 immunoreactivity in beta-napthoflavone-induced animals was elevated in liver hepatocytes and increased to a lesser extent in duodenal villus epithelial cells. Treatment with beta-napthoflavone produced an unexpected 46% decrease in the number of Dlb-1 mutations in response to PhIP. Following treatment with PhIP, there was no difference in the number of Dlb-1 locus mutations between the proximal and distal ends of the small intestine in uninduced animals, indicating that the bile duct is unlikely to be responsible for transport of mutation inducing metabolites of PhIP to the small intestine. Our results demonstrate that metabolic activation of an indirect acting genotoxic agent can occur at a site other than the target tissue, and absence of the enzymes required for activation of a mutagen does not necessarily protect that tissue from its genotoxic effects.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Imidazoles/toxicidad , Intestino Delgado/efectos de los fármacos , Microsomas Hepáticos/enzimología , Mutágenos/toxicidad , Oxidorreductasas/biosíntesis , Animales , Citocromo P-450 CYP1A2 , Femenino , Heterocigoto , Hidroxilación , Imidazoles/metabolismo , Inmunohistoquímica , Intestino Delgado/enzimología , Intestino Delgado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Microsomas/efectos de los fármacos , Microsomas/enzimología , Microsomas Hepáticos/efectos de los fármacos , Mutagénesis , Mutágenos/metabolismo , Quinoxalinas/toxicidadRESUMEN
The ability of four mutagenic/carcinogenic chemicals administered as single doses to induce a programmed form of cell death (apoptosis) in the BDF1 mouse large bowel was studied and compared with a previous study on the small intestine using the same mice. The number of apoptotic cells was counted following treatment with the direct-acting agents N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) and two agents which require metabolic activation 1,2-dimethylhydrazine (DMH) and N-nitrosodimethylamine (NDMA). DMH (80 mg/kg) was the most effective at inducing acute cell death and this was closely followed by NMU (200 mg/kg). The least effective agent in the large bowel was NDMA. The peak yield of apoptosis occurred between 4 h (NEU) and 8 h (DMH) after treatment. An analysis of the changing shapes of the frequency plots of apoptosis at each cell position in the crypt at various times after exposure permits an estimate to be made of the position in the crypt of the primary target cells for the cytotoxic action at time t = 0. For the agents studied, this is in the range of the 5th to the 10th position from the base of the crypt. This distribution for the target cells for apoptotic cell death is not coincident with that for the presumptive stem cells, which is at cell position 1 or 2. Comparisons with results previously obtained in the small intestine (ileum) of the same mice show that the relative cytotoxic effectiveness of the four agents differs. Furthermore, the position of the target cells is at about the 4th position from the bottom of the crypt in the ileum, and here the distribution is coincident with that presumed for the stem cells. Our interpretation of the data is that damaged cells in the stem cell region of the small bowel are removed by the activation of a cell suicide programme, which effectively removes potentially harmful genetic alterations. In contrast, in the large bowel, cell death is not initiated particularly strongly in the stem cell region but tends to occur higher in the crypt. The absence of this selective deletion process may result in the perpetuation of deleterious mutations in the colonic stem cell population and this may explain in part, the higher incidence of cancers observed in the large bowel.
Asunto(s)
Carcinógenos/toxicidad , Intestino Grueso/efectos de los fármacos , 1,2-Dimetilhidrazina , Animales , Apoptosis/efectos de los fármacos , Biotransformación , Dimetilhidrazinas/farmacocinética , Dimetilhidrazinas/toxicidad , Etilnitrosourea/toxicidad , Intestino Grueso/patología , Intestino Delgado/efectos de los fármacos , Masculino , Metilnitrosourea/toxicidad , Ratones , Ratones Endogámicos , Compuestos Nitrosos/farmacocinética , Compuestos Nitrosos/toxicidadRESUMEN
We have identified a minor population of crypts in small intestine which do not appear to export cells to villi. These crypts can be observed in whole-mounts of small intestine prepared from C57BL/6J<-->SWR mouse aggregation chimaeras stained with a peroxidase conjugate of the lectin Dolichos biflorus agglutinin (DBA-Px). In preparations where by chance the C57BL/6J epithelium (positive staining) forms only a minor component of the chimaera, occasional crypts occur which are isolated from larger patches of C57BL/6J epithelium and are surrounded by SWR (non-staining) epithelium. Fifty-one of 383 isolated C57BL/6J crypts (13%) did not appear to export cells to villi, although the crypt mouth is surrounded by a small patch of C57BL/6J epithelium on the intervillus gut floor.
Asunto(s)
Intestino Delgado/anatomía & histología , Animales , Agregación Celular , Movimiento Celular , Quimera , Células Epiteliales , Epitelio/anatomía & histología , Intestino Delgado/citología , Ratones , Ratones Endogámicos C57BLRESUMEN
Two direct-acting mutagens, N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU), and two agents requiring metabolic activation, 1-2-dimethylhydrazine (DMH) and N-nitrosodimethylamine (NDMA), were administered i.p. to mice. Sections of crypts of the small intestine were assayed for acute histological cell death at various times up to 12 h after treatment. Dead or dying cells exhibited the typical light microscopic morphological features of apoptosis. The incidence of apoptosis at each cell position along the side of longitudinal crypt sections was recorded and frequency plots of the incidence against cell position were determined. NEU (50 mg/kg) produced the highest incidence of cell death but this was closely followed by NDMA (50 mg/kg) and NMU (200 mg/kg). DMH (40 or 80 mg/kg) was the least cytotoxic but even here significantly elevated levels of cell death were observed. The highest incidence of cell death occurred 4-5 h after treatment with NEU, NMU and DMH and at 6 h after NDMA. The data obtained at 4 h after NEU suggest that approximately 22 cells out of a total crypt population of 250 cells are killed, but that for some cell positions near the crypt base (stem cell regions) up to 24% of the cells may be killed. Analysis of the changing shape of the frequency plots with time after treatment enabled the target cell position in the crypts for cytotoxicity to be estimated. This was at cell position 4 for NEU, NMU and DMH and at cell position 5 for NDMA. The stem cells in the crypts are believed to be located at the fourth cell position and hence at least NEU, NMU and DMH are targeting the stem cells with some specificity.
Asunto(s)
Dimetilhidrazinas/toxicidad , Dimetilnitrosamina/toxicidad , Etilnitrosourea/toxicidad , Intestino Delgado/efectos de los fármacos , Metilnitrosourea/toxicidad , 1,2-Dimetilhidrazina , Animales , Femenino , Intestino Delgado/citología , Masculino , Ratones , Células Madre/efectos de los fármacosRESUMEN
The ability of three model carcinogens, 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, to induce mutation in a novel in vivo assay in mouse intestine has been examined. The assay is based on mutations at the Dlb-1 locus which determines the tissue specific pattern of expressio of the binding site for the lectin Dolichos biflorus agglutinin. In C57BL/6J x SWR F1 mice Dlb-1 mutants are recognized as clones of epithelial cells not staining with a peroxidase conjugate of D. biflorus agglutinin. Chronic administration of 1,2-dimethylhydrazine (20 mg/kg/week s.c. for 10 weeks) induced Dlb-1 mutants, whereas administration of a single dose did not. Similarly, chronic dimethylnitrosamine treatment p.o. (0.001% in drinking water for 8 weeks) induced Dlb-1 mutants, but acute administration did not. In contrast, neither chronic nor acute treatment of the mice with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline induced Dlb-1 mutations. The activities of 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the Dlb-1 assay more accurately reflect their carcinogenic potential than do many in vitro bioassays.
Asunto(s)
Carcinógenos/toxicidad , Dimetilhidrazinas/toxicidad , Dimetilnitrosamina/toxicidad , Intestino Delgado/patología , Mutagénesis , Quinoxalinas/toxicidad , 1,2-Dimetilhidrazina , Animales , Mapeo Cromosómico , Epitelio/efectos de los fármacos , Epitelio/patología , Femenino , Intestino Delgado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Quinoxalinas/farmacología , Salmonella typhimurium/efectos de los fármacosRESUMEN
We have investigated stem-cell organization in mouse small intestine (SI) by using a cellular marker induced by somatic mutation. In small intestinal whole mounts from heterozygous Dlb-1b/Dlb-1a mice stained with a peroxidase conjugate of Dolichos biflorus agglutinin (DBA-Px), mutations of Dlb-1b in stem cells result in loss of DBA-Px binding and so are recognizable as wholly or partly unstained crypts. The frequency of these clonal patterns can be measured during the accumulation of spontaneous mutations in untreated mice, or after treatment with ethylnitrosourea (ENU). The results show that there is a single infrequently dividing stem cell that maintains the epithelium of each crypt through a population of transit stem cells. The entire crypt epithelium is renewed approximately every 12 weeks.
Asunto(s)
Intestino Delgado/citología , Células Madre/citología , Animales , División Celular , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Marcadores Genéticos , Intestino Delgado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , MutaciónRESUMEN
We have previously proposed that an in vivo mutagenicity assay could be based on the detection of mutations affecting the Dlb-1 locus in the small intestine of C57BL/6J x SWR F1 mice. F1 mice are heterozygous Dlb-1b/Dlb-1a and have only a single allele (Dlb-1b) which specifies expression of the binding site for the lectin Dolichos biflorus agglutinin (DBA) in intestinal epithelia. In whole-mount preparations of small intestine stained with a DBA--peroxidase conjugate, mutated stem cells and their progeny can be recognized as ribbons of unstained cells against a stained background. These DBA-negative ribbons can be quantified. The present investigation characterizes the responses of F1 mice to high- and low-dose rate gamma radiation (1.8 and 0.01 Gy/min respectively) and shows that the induction of ribbons is dose dependent in both cases. Dose sparing is evident at the lower dose rate: 20 ribbons/10(4) villi can be induced by 4 Gy at high-dose rate and by 7.1 Gy at low-dose rate, a dose sparing of 1.76. Age-matched untreated mice had a background level of 3.7 ribbons/10(4) villi. As expected, no ribbons were observed in homozygous (Dlb-1b/Dlb-1b) C67BL/6J mice, in which each stem cell has two alleles specifying lectin binding. The results further validate this system as a sensitive in vivo mutagenicity assay and suggests that the target cells are capable of repairing radiation damage.