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1.
Teratog Carcinog Mutagen ; 20(6): 357-86, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-11074520

RESUMEN

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.


Asunto(s)
Análisis Mutacional de ADN/métodos , Pruebas de Mutagenicidad/métodos , Mutación , Secuencia de Bases , Línea Celular , Colorantes Fluorescentes , Genes p53 , Antígenos HLA-A/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Pérdida de Heterocigocidad , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Repeticiones de Minisatélite , Datos de Secuencia Molecular , Mutación Puntual , Proteínas Proto-Oncogénicas c-bcl-2/genética , Mapeo Restrictivo , Sensibilidad y Especificidad , Translocación Genética
2.
J Invest Dermatol ; 115(4): 687-93, 2000 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10998144

RESUMEN

We have assessed the ability of xeroderma pigmentosum and normal keratinocytes grown out from skin biopsies to undergo apoptosis after irradiation with ultraviolet B. Keratinocytes have been studied from xeroderma pigmentosum complementation groups A (three biopsies), C (three biopsies), D (one biopsy), xeroderma pigmentosum variant (two biopsies), and Cockayne syndrome (one biopsy). The three xeroderma pigmentosum group A and the xeroderma pigmentosum group D samples were at least six times more sensitive than normal cells to ultraviolet B-induced apoptosis. The xeroderma pigmentosum variant samples showed intermediate susceptibility. Xeroderma pigmentosum group C samples proved heterogeneous: one showed high sensitivity to apoptosis, whereas two showed near normal susceptibility. The Cockayne syndrome sample showed the high susceptibility of xeroderma pigmentosum groups A and D only at a higher fluence. These results suggest that the relationships between repair deficiency, apoptosis, and susceptibility to skin cancer are not straightforward. Ultraviolet B-induced skin cancer is also thought to be due in part to ultraviolet B-induced impairment of immune responses. The release of the inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha from cultured xeroderma pigmentosum keratinocytes tended to occur at lower fluences than in normals, but was less extensive, and was more readily inhibited at higher fluences of ultraviolet B.


Asunto(s)
Queratinocitos/citología , Rayos Ultravioleta , Xerodermia Pigmentosa/patología , Apoptosis/efectos de la radiación , Células Cultivadas , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de la radiación , Humanos , Etiquetado Corte-Fin in Situ , Recién Nacido , Interleucina-6/metabolismo , Queratinocitos/efectos de la radiación , Masculino , Factor de Necrosis Tumoral alfa/metabolismo
3.
Carcinogenesis ; 21(6): 1087-95, 2000 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10836995

RESUMEN

We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.


Asunto(s)
Apoptosis/efectos de la radiación , Citocinas/metabolismo , Queratinocitos/efectos de la radiación , Apoptosis/efectos de los fármacos , Línea Celular Transformada , ADN/efectos de la radiación , Rayos gamma , Células HeLa , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Mitoxantrona/farmacología , Rayos Ultravioleta
4.
Hum Immunol ; 60(11): 1018-27, 1999 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-10599998

RESUMEN

The appearance of a population of dual-staining CD4+CD8+ cells in human T-lymphocyte cultures has been reported by various authors, including our own observation that they are always seen in simple phytohaemagglutinin-stimulated cultures from several different donors. The purpose of the present study was to investigate factors involved in the dual-staining (DS) phenotype, and to clarify some apparent inconsistencies between published observations. Our findings can be summarised as follows. 1. A population of DS CD4+CD8+ cells always appears in PHA-stimulated T-cell cultures if they contain both CD4 and CD8 subsets. The incidence of DS cells is related to PHA concentration, but other factors are involved since DS cells are not seen in PHA-stimulated cultures of purified CD4+ or CD8+ cells. Stimulation with PHA is not a prerequisite since very similar results are seen with ConA. 2. Direct physical contact between CD4+ and CD8+ cells is required for the appearance of the DS phenotype; soluble factors alone, including IL-4, appear nor to be responsible. 3. The DS phenotype in these conditions is always CD4+ cells weakly expressing CD8 and is a consequence of de novo synthesis of the CD8alpha molecule by the CD4+ cells.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/biosíntesis , Linfocitos T CD8-positivos/inmunología , Apoptosis , Secuencia de Bases , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Antígenos CD8/química , Antígenos CD8/genética , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Cartilla de ADN/genética , Humanos , Interleucina-4/farmacología , Fenotipo , Fitohemaglutininas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo
5.
Cancer Res ; 59(14): 3454-60, 1999 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-10416610

RESUMEN

Severe immunodeficiency characterized by lymphopenia was found in two siblings, one of whom was examined in detail. The calcium flux, pattern of tyrosine phosphorylation of proteins, and interleukin 2 (IL-2) production and proliferation in response to mitogens suggested that the peripheral blood T cells activated normally. The peripheral blood T cells were shown to have an activated phenotype with increased expression of CD45RO+ and CD95/Fas. Increased spontaneous apoptosis occurred in unstimulated lymphocyte cultures. The elevated apoptosis was not due to alterations in expression or to mutations in Bcl-2, Bcl-X(L), or Flip, nor could the spontaneous apoptosis be prevented by blocking Fas, suggesting that it was independent of Fas signaling. This is the first inherited combined immunodeficiency associated with impaired lymphocyte survival. Fibroblasts derived from the patient showed appreciable radiosensitivity in clonal assays, but apoptosis was not elevated. Our results show that the fibroblasts represent a new radiosensitive phenotype not associated with cell cycle checkpoint defects, V(D)J recombination defects, or elevated chromosome breakage. We suggest that the affected gene plays a role in an undetermined damage response mechanism that results in elevated spontaneous apoptosis in lymphoid cells and radiosensitivity in fibroblasts.


Asunto(s)
Apoptosis , Fibroblastos/efectos de la radiación , Síndromes de Inmunodeficiencia/patología , Linfocitos/efectos de la radiación , Inmunodeficiencia Combinada Grave/patología , Apoptosis/efectos de la radiación , Niño , Preescolar , Inversión Cromosómica , Cromosomas Humanos Par 7/ultraestructura , Daño del ADN , Reparación del ADN , ADN Complementario/genética , Femenino , Fibroblastos/patología , Rayos gamma , Humanos , Linfocitos/patología , Masculino , Tolerancia a Radiación , Inmunodeficiencia Combinada Grave/genética , Transducción de Señal/fisiología , Translocación Genética
6.
Int J Radiat Biol ; 72(3): 271-83, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9298107

RESUMEN

We have studied the intrinsic radiosensitivity, repair of potentially lethal damage (PLD) and the repair rate of radiation-induced DNA double-strand breaks (DSB) in 11 non-transformed human fibroblast cell lines, four of which were homozygous for the A-T mutation and two that were heterozygous (A-TH). All the experiments were done on cells in plateau phase of growth (97-99% of cells in G0/G1). With a dose of 30 Gy delivered at 4 degrees C, the A-T cell lines had faster repair rates of up to 6 h, after which the repair curve crossed that of the control so that the residual damage at 24 h was higher in the A-T cells. Irradiation at 37 degrees C at low dose rate 1 cGy.min-1) produced even more marked differences between the A-T cells and controls: the residual DSB level was always higher in A-T cells than controls at doses of 5-40 Gy, due to defective repair of a small fraction of DSB in A-T cells. The two protocols showed DSB repair rates for the A-TH cell lines that were intermediate between those of the A-T and control cells. There was a quantitative relationship between the residual DSB after irradiation at 37 degrees C and the intrinsic radiosensitivity, and with the extent of PLD repair. There were very few apoptotic cells in the non-transformed control and A-T cell line, both before and after irradiation. In combination, these result support the contention that the defective repair of DSB is a mechanism of the hypersensitivity linked to the A-T mutation.


Asunto(s)
Ataxia Telangiectasia/patología , Daño del ADN , ADN/efectos de la radiación , Fibroblastos/efectos de la radiación , Tolerancia a Radiación , Línea Celular , ADN/metabolismo , Relación Dosis-Respuesta en la Radiación , Fibroblastos/metabolismo , Fibroblastos/fisiología , Humanos , Cinética , Temperatura
7.
Environ Mol Mutagen ; 30(2): 97-111, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9329634

RESUMEN

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.


Asunto(s)
Accidentes de Trabajo , Contaminantes Atmosféricos/toxicidad , Monitoreo del Ambiente/métodos , Adulto , Aductos de ADN/sangre , Exposición a Riesgos Ambientales , Hemoglobinas/análisis , Hemoglobinas/genética , Humanos , Hidrocarburos Aromáticos/metabolismo , Hipoxantina Fosforribosiltransferasa/efectos de los fármacos , Hipoxantina Fosforribosiltransferasa/genética , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Mutágenos/toxicidad , Mutación , Petróleo/toxicidad , Radioisótopos de Fósforo , Proyectos Piloto , Escocia
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