RESUMEN
It has been demonstrated recently that certain repetitive sequences and even expressed single-copy genes are capable of retrotransposition, but little is known about the endogenous or exogenous modifiers of this process in human cells. Retrotransposition may contribute to gene inactivation and genetic instability in cancer development. We have used the human cell line MCF-7 to generate a method for investigating de novo retrotransposition in breast cancer cells. The strategy employs a reporter construct transfected into MCF-7 cells that encodes neomycin phosphotransferase gene (neoR) sequences interrupted by an intron derived from the gamma-globin gene and sandwiched between two promoters in opposite orientation; the phosphotransferase is not produced in transfected cells expressing the plasmid until transposition via a spliced antisense neoR RNA intermediate has occurred, conferring a functional gene product and thereby resistance to G418. A stable transfectant line that showed presence of reporter plasmid DNA and expression of reporter antisense neoR was obtained and used to demonstrate spontaneous retrotransposition of neoR sequences: tester cells were subjected to selection in G418 medium, and neomycin-resistant clones were isolated at a frequency of 10(-7). A simple PCR-based prescreening of colonies fixed and stained in Petri dishes can be used to verify intronless neoR DNA. Expanded populations of G418-resistant colonies were determined to be derived from reporter sequences that had transposed via an RNA intermediate by Southern blot genotyping. This experimental assay may be used for exploring endogenous and environmental factors that influence host cell-mediated retrotransposition of unbiased cellular sequences in breast tumor cells. Genes Chromosomes Cancer 26:84-91, 1999.
Asunto(s)
Neoplasias de la Mama/genética , Retroelementos/genética , Secuencia de Bases , Neoplasias de la Mama/patología , Globinas/genética , Humanos , Intrones/genética , Kanamicina Quinasa/genética , Datos de Secuencia Molecular , Empalme del ARN , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Esophageal tumors from 29 patients residing in Guangzhou, China were examined for mutations in exons 5-8 of the p53 tumor suppressor gene and for p53 protein accumulation in tumor cell nuclei. Anamnestic data for each patient, which included information on family history of cancer, tobacco smoking, drinking of alcoholic beverages, and dietary habits such as consumption of pickled vegetables, were recorded. Screening of DNA from tumor cells microdissected from biopsies was performed by PCR amplification of p53 gene exons 5-8, denaturing gradient gel electrophoresis analysis, and DNA sequencing. Mutations were identified in 20 of 29 tumors (69%). All tumors harboring a missense mutation in the p53 gene also showed nuclear accumulation of the tumor suppressor protein by immunohistochemistry. The most common p53 mutations in these tumors were guanine to adenine (G-->A) transitions (10 of 20 tumors; 50%). We did not find multiple mutations at codon 176, in contrast to Lung et al. in their recent study of esophageal cancer patients from Guangzhou (M. L. Lung et al., Cancer Epidemiol. Biomark. Prev., 5: 277-284, 1996). The mutation prevalence was high both in smokers (13 mutations in 20 smokers; 65%) and in nonsmokers (7 of 9 tumors with mutations; 78%), an observation that differs from that of studies in European and North American patients, which demonstrate a much higher prevalence of p53 mutations in smokers than in nonsmokers (reviewed in R. Montesano et al., Int. J. Cancer Predict. Oncol., 69: 225-235, 1996.). Our findings in this pilot study of tumor suppressor gene mutations in patients from Guangzhou support a large body of epidemiological observations pointing to dietary mutagenic carcinogens peculiar to populations in China at high risk of esophageal cancer.
Asunto(s)
Neoplasias Esofágicas/epidemiología , Neoplasias Esofágicas/genética , Genes p53/genética , Mutación , Adenocarcinoma/epidemiología , Adenocarcinoma/genética , Adulto , Anciano , Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Escamosas/genética , China/epidemiología , Análisis Mutacional de ADN , Dieta/efectos adversos , Exones , Femenino , Humanos , Inmunohistoquímica , Incidencia , Masculino , Persona de Mediana Edad , Proyectos Piloto , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Fumar/efectos adversosAsunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 17 , Genes p53 , Mutación , Neoplasias/genética , Neoplasias/prevención & control , Aflatoxinas , Alelos , Composición de Base , Carcinógenos , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/genética , Mapeo Cromosómico , Análisis Mutacional de ADN , Elementos Transponibles de ADN , Femenino , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/genética , Neoplasias/patología , Neoplasias Inducidas por Radiación/genética , Mutación Puntual , Eliminación de Secuencia , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/genética , Fumar/efectos adversos , Luz Solar , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Serum antibodies reacting with the tumor suppressor protein p53 have been detected previously in cancer patients with a variety of neoplasms. Two initial (although insufficient) prerequisites for a B-cell response to occur have been proposed: p53 protein accumulation in the tumor or a mutant p53 gene, or both. We have examined 65 esophageal cancer cases (42 from Guangzhou and Shenyang, People's Republic of China, and 23 from Paris, France) to obtain a prevalence estimate of anti-p53 antibodies for this type of cancer and to define the relationship of p53 tumor status to B-cell immune response. Sera were analyzed in a triplicate assay (enzyme-linked immunoassay, immunoprecipitation, and immunoblot) for anti-p53 antibodies. Tumor DNA was screened for mutations in exons 5-8, and tumor tissue was examined by immunohistochemistry for abnormal p53 protein accumulation. p53 mutations were found in 36 (58%) of 62 cases analyzed. Sixteen patients (25%) had circulating antibodies to the tumor suppressor protein. All but two (88%) of the tumors from seropositive cases had a mutation in the DNA binding region of the p53 gene, and with one exception, these tumors also showed nuclear accumulation of the p53 protein. In contrast, tumor mutations were found in just 22 (46%) of the 48 individuals in whom we did not detect anti-p53 antibodies. Among the 22 seronegative cases for which we found no tumor mutations, 11 revealed p53 protein accumulation by immunohistochemical analysis. Thus, circulating anti-p53 antibodies may be present in one-fourth of esophageal cancer patients, most of whom also would be expected to have a p53 gene mutation in their tumors. Patients without such mutations appear considerably less likely to mount a B-cell response to the p53 tumor suppressor protein than those that do (P < 0.01).