RESUMEN
Two-color fluorescence in situ hybridization (FISH) in combination with digital image analysis was used to develop an automatic system for the detection and classification of chromosome aberrations. Algorithms were developed for the automatic thresholding of the three digitized images: an FITC image representing specific painted chromosomes, a TRITC image representing the centromeres of all chromosomes, and a DAPI image representing all the counterstained chromosomes. A further algorithm was developed for the automatic classification of the different types of chromosome aberrations, such as translocations, dicentrics, and fragments. For this study, a dataset of 252 metaphases were digitized and analyzed automatically as well as manually. Of these metaphases, 81.3% could be correctly classified by the algorithm. The error rate was reduced to 9.3% by automatically excluding the detected clusters and artifacts. The average analysis time per metaphase was 34.5 s without any user intervention.
Asunto(s)
Aberraciones Cromosómicas/genética , Sondas de ADN , Diagnóstico por Imagen/métodos , Hibridación Fluorescente in Situ/métodos , Expansión de Repetición de Trinucleótido/genética , Adenocarcinoma , Algoritmos , Árboles de Decisión , Relación Dosis-Respuesta en la Radiación , Humanos , Procesamiento de Imagen Asistido por Computador , Neoplasias Pulmonares , Metafase , Expansión de Repetición de Trinucleótido/efectos de la radiación , Células Tumorales CultivadasRESUMEN
In order to find an explanation for the eventual disappearance of all chromosome aberrations in two radiosensitive human tumour cell lines, the type and stability of different aberration types was investigated in more detail. To classify the aberrations into unstable and stable types, three-colour fluorescence in situ hybridization was performed, including a whole-chromosome probe, a pancentromere probe, and a stain for total DNA. This technique enables the appropriate classification of the aberrations principally by the presence (stable) or not (unstable) of a single centromere per chromosome. Unstable-type aberrations were found to disappear within 7 days (several divisions) in the two radiosensitive and the two radioresistant tumour lines investigated. Stable-type aberrations were found to remain at an approximately constant level over the duration of the experiment (14 days; 8-10 divisions) in the two radioresistant lines. In contrast, the majority of these stable-type aberrations had disappeared by 14 days in the two radiosensitive lines. The previous findings of disappearance of total aberrations in radiosensitive cells was therefore not due to a reduced induction of stable-type aberrations, but the complete disappearance of cells with this aberration type. These results could not be explained by differences in apoptosis or G1 blocks. Two possible explanations for these unexpected findings involve non-random induction of unstable-type aberrations, or lethality of stable-type aberrations. The results suggest caution in the use of stable-type aberration numbers as a predictor for radiosensitivity.
Asunto(s)
Aberraciones Cromosómicas , Tolerancia a Radiación , Células Tumorales Cultivadas/efectos de la radiación , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular , Cromosomas Humanos/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Hibridación Fluorescente in Situ , Tolerancia a Radiación/genética , Factores de Tiempo , Células Tumorales Cultivadas/ultraestructuraRESUMEN
Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer cell lines that cover a range of drug-resistance levels, and we have analyzed the MRP amplicon. In the SW-1573-derived, weakly resistant cell line 30.3M, MRP mRNA is elevated 3-fold in the absence of gene amplification. Run-on analysis shows that the increased MRP gene expression in this cell line is due to transcriptional activation. In the highly resistant GLC4/ADR and COR-L23/R cells, MRP gene amplification predominates, whereas in the moderately resistant MOR/R cells, gene amplification is combined with a mechanism resulting in an additional increase in the level of MRP mRNA. Fluorescence in situ hybridization shows that, in the GLC4/ADR cells, amplified MRP sequences are present both in double minute chromosomes (DM) and in homogeneously staining regions (HSR). By pulsed-field gel electrophoresis we show that the MRP-containing DM are 1 Mb in length. Chromosome-16-specific repetitive sequences adjacent to the MRP gene are also present in the DM and HSR, compatible with the involvement of these sequences in recombination events underlying MRP gene amplification. Our results show that low levels of drug resistance may arise by transcriptional activation of the MRP gene, whereas at high levels of drug resistance amplification of the MRP gene predominates, possibly facilitated by the presence of recombination-prone sequences.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/genética , Resistencia a Múltiples Medicamentos/genética , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Transportadoras de Casetes de Unión a ATP/biosíntesis , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Pequeñas/metabolismo , Carcinoma de Células Pequeñas/patología , Cromosomas Humanos Par 16 , ADN Complementario/genética , ADN de Neoplasias/genética , Genes , Humanos , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Miosinas/biosíntesis , Miosinas/genética , Proteínas de Neoplasias/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/genética , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
This paper describes the kinetics of fibroblastic colony forming units (CFU-f) in murine blood after phenylhydrazine-induced haemolytic anaemia and their subsequent migration into haemopoietic organs. Murine blood contained 5.3 +/- 0.8 CFU-f per 10(6) nucleated cells. Absence of particle ingestion and factor VIII-related antigen in addition to the enzyme pattern in CFU-f-derived cells confirmed that these cells did not have a macrophage-like or endothelial nature. Phenylhydrazine treatment of mice resulted in a 3-fold increase in blood CFU-f numbers which was accompanied by increases in blood cellularity and granulocyte-macrophage progenitor numbers. When both partners of CBA/N and CBA/T6T6 mice in parabiosis had been treated with phenylhydrazine, spleens and femoral bone marrow of both mice were shown to contain partner-derived CFU-f. These data suggest that circulating CFU-f represent a stromal cell population which can migrate into haemopoietic organs.
Asunto(s)
Anemia Hemolítica/sangre , Fibroblastos/citología , Células Madre Hematopoyéticas/citología , Anemia Hemolítica/inducido químicamente , Animales , Médula Ósea/patología , Células de la Médula Ósea , Fibroblastos/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Cinética , Ratones , Ratones Endogámicos CBA , Ratones Endogámicos , Fenilhidrazinas , Bazo/citología , Bazo/patologíaRESUMEN
The recovery of fibroblastic colony-forming units (CFU-F) in murine bone marrow hemopoietic stroma was studied during eighteen months after 9 Gy lethal total-body irradiation and reconstitution with syngeneic bone marrow cells. After an initial depletion, CFU-F numbers increased from 10% of normal values at three months to 40% at 18 months after treatment, irrespective of graft size and presence of CFU-F in the graft. Fourteen months after treatment 35% of all CFU-F present in the recipients' bone marrow was donor-derived independent of graft size. When mice were treated with high-dose lipopolysaccharide-W three months after irradiation and bone marrow transplantation, CFU-F numbers decreased to hardly detectable levels within one day, and then recovered to normal numbers four weeks later--whereas radiation control mice still had low CFU-F numbers. These data suggest that after lethal total-body irradiation the stroma still contained viable fibroblastic cells that had lost their in vitro colony-forming capacity as a result of radiation damage. In consequence there was no need for replacement of these fibroblastic cells by donor-derived or host-derived CFU-F. Only depletion of CFU-F from the bone marrow, as was induced with lipopolysaccharide, stimulated repopulation of the stroma with colony-forming fibroblastic cells.