RESUMEN
The tumour suppressor genes, TP53 and RB1, and four genes involved in their regulation, INK4a, ARF, MDM2 and MDMX, were analysed in a series of 36 post-radiotherapy radiation-induced sarcomas. One-third of the tumours developed in patients carrying a germline mutation of RB1 that predisposed them to retinoblastoma and radiation-induced sarcomas. The genetic inactivation of RB1 and/or TP53 genes was frequently observed in these sarcomas. These inactivations were owing to an interplay between point mutations and losses of large chromosome segments. Radiation-induced somatic mutations were observed in TP53, but not in RB1 or in the four other genes, indicating an early role of TP53 in the radio-sarcomagenesis. RB1 and TP53 genes were biallelically coinactivated in all sarcomas developing in the context of the predisposition, indicating that both genes played a major role in the formation of these sarcomas. In the absence of predisposition, TP53 was biallelically inactivated in one-third of the sarcomas, whereas at least one allele of RB1 was wild type. In both genetic contexts, the TP53 pathway was inactivated by genetic lesions and not by the activation of the ARF/MDM2/MDMX pathway, as recently shown in retinoblastomas. Together, these findings highlight the intricate tissue- and aetiology-specific relationships between TP53 and RB1 pathways in tumorigenesis.
Asunto(s)
Genes de Retinoblastoma/efectos de la radiación , Genes p53/efectos de la radiación , Proteína de Retinoblastoma/fisiología , Sarcoma/etiología , Proteína p53 Supresora de Tumor/fisiología , Genes Supresores de Tumor/efectos de la radiación , Humanos , Neoplasias Inducidas por Radiación/genética , Proteína de Retinoblastoma/efectos de la radiación , Sarcoma/genética , Proteína p53 Supresora de Tumor/efectos de la radiaciónAsunto(s)
Genes de Retinoblastoma , Mutación Puntual , Sitios de Empalme de ARN , Proteína de Retinoblastoma/genética , Retinoblastoma/genética , Preescolar , Exones , Femenino , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Humanos , Lactante , Masculino , Linaje , Penetrancia , Nucleótidos de Pirimidina/genética , ARN Neoplásico/análisis , Retinoblastoma/metabolismoRESUMEN
One difficulty in studying molecular changes of tumours has been the inability to isolate DNA and RNA from a homogeneous cell population. The combination of several new technologies should help overcome these hurdles. Microdissection is a technique for rapid and easy procurement of a pure cellular subpopulation away from its complex tissue milieu. Laser-assisted microdissection has recently been identified as a quick, simple and effective method by which microdissection of complex tissue specimens can be routinely performed for molecular analysis. With the advent of laser microdissection, cDNA libraries can be developed from pure cells obtained directly from stained neoplastic tissue, and microarrays of thousands of genes can now be used to examine gene expression in microdissected tumour tissue samples. This review will concentrate on the application of different microdissection techniques in the area of cancer research.
Asunto(s)
Separación Celular/métodos , Citogenética/métodos , Disección/métodos , Perfilación de la Expresión Génica , Micromanipulación/métodos , Neoplasias/patología , Células Madre Neoplásicas/química , Citogenética/instrumentación , ADN Complementario/genética , ADN de Neoplasias/análisis , Progresión de la Enfermedad , Disección/instrumentación , Humanos , Micromanipulación/instrumentación , Proteínas de Neoplasias/análisis , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Neoplásico/análisisRESUMEN
Genome alterations of seven secondary tumors (five osteosarcomas, one malignant peripheral sheath nerve tumor, one leiomyosarcoma) occurring in the field of irradiation of patients treated for bilateral retinoblastoma have been studied. These patients were predisposed to develop radiation-induced tumors because of the presence of a germ line mutation in the retinoblastoma gene (RB1). Tumor cells were characterized by a high chromosome instability whereas microsatellites and minisatellites were found to be stable. In all tumors, the normal RB1 allele was lost with the corresponding chromosome 13, whereas the germ line mutated allele was retained. The two alleles of TP53 were inactivated, one by deletion of the short arm of chromosome 17, the other by mutation. As compared with non-radiation-induced tumors, the observed panel of TP53 mutations was uncommon with sites not recurrently found otherwise and a high rate of deletions (3/7). In these predisposed patients, the loss of the single normal allele of RB1 is rather due to the radiation-induced chromosome instability than a direct effect of ionizing radiation.
Asunto(s)
Neoplasias Inducidas por Radiación/genética , Retinoblastoma/radioterapia , Análisis Citogenético , Genes de Retinoblastoma , Genes p53 , Predisposición Genética a la Enfermedad , Humanos , Hibridación Fluorescente in Situ , Lactante , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Repeticiones de Minisatélite , Mutación , Radiación IonizanteRESUMEN
In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus-transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2-rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3-rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.
Asunto(s)
Transformación Celular Neoplásica/genética , Aberraciones Cromosómicas/genética , Metilación de ADN , Linfocitos/metabolismo , Linfocitos/patología , Ataxia Telangiectasia/genética , Southern Blotting , Línea Celular Transformada , Bandeo Cromosómico , Células Clonales , Metilasas de Modificación del ADN/genética , ADN Satélite/genética , Herpesvirus Humano 4/fisiología , Heterocromatina/genética , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Oxidorreductasas O-Demetilantes/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telómero/genética , Factores de Tiempo , Cromosoma Y/genéticaRESUMEN
We show that in a series of eight breast cancer cell lines, a direct relationship exists between the overall DNA demethylation and the percentage of rearranged chromosomes, except for cell lines with a highly rearranged genome which can be weakly demethylated. A real time fluorescent detection method was used to quantify by reverse transcription-PCR the expression of the DNA methyltransferase 1 and of the newly discovered DNA demethylase. The overall DNA methylation status seems to result from a complex interplay between the expression of these two genes. Our results suggest that in these tumor cells, the overall DNA demethylation is implicated in one of the mechanisms at the origin of the genome instability and that besides the role of the DNA methyltransferase 1, that of the DNA demethylase may be essential in the control of DNA methylation.
Asunto(s)
Neoplasias de la Mama/genética , Aberraciones Cromosómicas , Metilación de ADN , Bandeo Cromosómico , Regulación Neoplásica de la Expresión Génica , Humanos , Cariotipificación , Células Tumorales CultivadasRESUMEN
Detailed studies of chromosome rearrangements within solid tumors require karyotype analysis after cell culturing. However, different cell subpopulations with various growth capacities within one tumor may introduce biases in karyotype analysis, known as the in vitro selection. In our laboratory, 22% of karyotypes from breast cancers established after short-term culture were normal. Using interphase fluorescence in situ hybridization (FISH) for the determination of chromosome 1 arm imbalances and flow cytometry measurements of ploidy, we demonstrated that at least 2/3 of these tumors were mainly composed of aneuploid cell populations. Thus, the incidence of normal or balanced karyotypes among breast cancers is probably below 7%. This is the first direct proof for the existence of an in vitro selection within breast cancer cultures, suggesting cautious interpretation of cytogenetic data.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Citometría de Flujo , Adulto , Anciano , Anciano de 80 o más Años , Aneuploidia , Artefactos , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Cromosomas Humanos Par 1/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Persona de Mediana Edad , Ploidias , Selección Genética , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The diagnosis of lung cancer is quite often hampered by the existence of various cell types within samples such as biopsies or pleural effusions. We have established a new marker for image cytometry of interphase tumor cells of the lung by using the most recurrent and early cytogenetic event in lung cancer, the loss of the short arm of chromosome 3. The method is based on the detection of the imbalance between the long and the short arms of chromosome 3 by performing two-color fluorescence in situ hybridization on both arms. Fourteen tumors were analyzed after short-term culture and compared with the corresponding cytogenetic data obtained from metaphase analysis. Results on interphase nuclei and control experiments on metaphases were the same, with imbalance ratios ranging from 1.0 to 2.0 (mean value 1.6, median 1.5). To assess the clinical significance of this approach, three pleural effusions were analyzed. Data showed that normal cells within the sample could have been distinguished from the tumor cells based on different imbalance values between the long and the short arms. Thus, our method allows refined detection of lung tumor cells within samples containing heterogeneous cell populations.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/diagnóstico , Carcinoma de Células Pequeñas/genética , Cromosomas Humanos Par 3/genética , Humanos , Interfase/genética , Neoplasias Pulmonares/patología , Metafase/genética , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologíaRESUMEN
In situ alterations of DNA methylation were studied between 14 d postcoitum and 4 d postpartum in Sertoli cells and germ cells from mouse testis, using anti-5-methylcytosine antibodies. Compared to cultured fibroblasts, Sertoli cells display strongly methylated juxtacentromeric heterochromatin, but hypomethylated chromatids. Germ cells always possess hypomethylated heterochromatin, whereas their euchromatin passes from a demethylated to a strongly methylated status between days 16 and 17 postcoitum. This hypermethylation occurs in the absence of DNA replication, germ cells being blocked in the G(0)-G(1) phase from day 15 postcoitum to birth. The DNA hypermethylation of germ cells is maintained until birth and could be visualized on both chromatids of metaphase chromosomes at the first postpartum cell division. Subsequently, the DNA hypermethylation is lost semiconservatively, being replaced by a methylation pattern recalling the typical fibroblast pattern. These alterations of DNA methylation follow a strict chronology, are chromosome structure and cell-type dependent, and may underlie profound changes of genome function.
Asunto(s)
Cromosomas/metabolismo , Islas de CpG/genética , Metilación de ADN , Células de Sertoli/metabolismo , Espermatozoides/metabolismo , Animales , Núcleo Celular/genética , Células Cultivadas , Centrómero/genética , Centrómero/metabolismo , Cromátides/genética , Cromátides/metabolismo , Bandeo Cromosómico , Cromosomas/genética , Replicación del ADN/genética , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Heterocromatina/genética , Heterocromatina/metabolismo , Interfase , Masculino , Metafase , Ratones , Células de Sertoli/citología , Espermatozoides/citología , Testículo/crecimiento & desarrollo , Factores de TiempoRESUMEN
Angiogenesis is a complex phenomenon likely to be under the strict control of a group of transcription factor(s). Homeobox (HOX)-containing proteins have been identified as regulators controlling the coordinated expression of genes involved in organ development and tissue differentiation. In this study, we have demonstrated that human umbilical vein endothelial cells (HUVEC) express 8 of the 10 HOX genes contained in cluster B. Treatment of HUVEC with tissue plasminogen activator (TPA), an agent known to induce morphologic changes in endothelial cells, or vascular endothelium growth factor (VEGF), a proliferative and angiogenesis inducer, results in a specific time-dependent modulation of the eight HOX genes identified. Interestingly, neither basic fibroblast growth factor, an endothelial proliferative agent, nor TNP-470, a fumagillin derivative with potent antiendothelial cell proliferation properties, affected expression of these HOX genes. Specific modulation of HOX genes by differentiating agents but not by proliferative or antiproliferative molecules suggests that they could be involved in the control of the genetic program that coordinates the construction of new blood vessels.
Asunto(s)
Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/fisiología , Genes Homeobox , Familia de Multigenes , Secuencia de Bases , División Celular/efectos de los fármacos , Ciclohexanos , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Linfocinas/farmacología , Datos de Secuencia Molecular , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , O-(Cloroacetilcarbamoil) Fumagilol , Sesquiterpenos/farmacología , Acetato de Tetradecanoilforbol/farmacología , Activador de Tejido Plasminógeno/farmacología , Transcripción Genética/efectos de los fármacos , Cordón Umbilical , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: Automated image cytometry can allow concurrent quantification of several parameters in each individual cell within a population, opening new possibilities for diagnosis and prognosis. In this study, the authors investigated the capacity of this method for performing a bivariate analysis of DNA ploidy and synthesis in fine-needle samplings obtained without aspiration from breast tumors. METHODS: Samplings from 25 unselected cases of ductal infiltrative breast adenocarcinoma and 2 cases of fibroadenoma were analyzed. For each case, 3-5 slides (containing approximately 1000 cells each) were quantified to assess experimental precision. Ploidy was determined by fluorescent staining of DNA using 4,6-diamidino-2-phenylindole (DAPI). Contaminating lymphocytes were taken as internal controls to calculate DNA indices. DNA synthesis was analyzed by immunofluorescent detection of 5-bromodeoxyuridine (BrdU) incorporation. Measurements were compared with flow cytometric data obtained from the same patients. RESULTS: Relative error in determination of DNA indices was generally below 5%. Determination of proliferation indices were more variable, with a mean relative error of 25%. Two different populations of BrdU positive cells were detected systematically, one in the diploid and another in the aneuploid fraction. For both cytometric methods, DNA indices were similar in all 27 cases, whereas BrdU labeling indices showed no significant correlation in 13 cases. The remaining cases were not comparable due to lack of flow cytometric data. Labeling indices obtained by image cytometry did not reveal any significant correlation with Scarff-Bloom-Richardson grading or clinical staging. CONCLUSIONS: Automated image cytometry allows concurrent measurement of ploidy and cell proliferation within individual breast carcinoma cells. Statistical reliability can be reached with a relative small number of cells (1000), which is crucial for samples in which the cell number is too low for flow cytometry analysis. Visual control for artifact elimination and better characterization of cell populations makes this a powerful tool for tumor cell investigation. Automated image cytometry allows the obtainment of valuable prognostic parameters of traditional flow cytometry with the relatively small number of cells obtained in aspiration procedures.
Asunto(s)
Neoplasias de la Mama/genética , ADN de Neoplasias/análisis , Citometría de Imagen/métodos , Ploidias , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Neoplasias de la Mama/patología , Femenino , Fluorescencia , Humanos , Persona de Mediana EdadRESUMEN
Cytogenetic analyses were performed on 223 breast carcinomas, of which 60% contained homogeneously staining regions (hsr), an intrachromosomal cytogenetic feature of gene amplification. The precise hsr localization could be determined for 123 hsr from 72 cases. The juxtacentromeric region of chromosome 8, band 11q13, and the whole of chromosome 17 were frequently involved. For 28 cases, the origin of the DNA sequences forming HSR could be investigated by chromosome painting, comparative genomic hybridization, and/or Southern blotting. Sequences from chromosomes 11 and 17 were mostly found within hsr located on chromosomes 11 and 17, respectively. In contrast, sequences from chromosome 8 were rarely found within hsr localized on chromosome 8. These observations suggest that different mechanisms lead to hsr formation in breast cancer. Band 11 q13 and the 17p chromosome arm may correspond to sites of in situ amplification driven by deletions distal to the amplification target genes. hsr in the region 17q2, which is also a frequent site of in situ amplification, takes place without the occurrence of a distal deletion. The short arm of chromosome 8 is often deleted, but frequently becomes the site of hsr formed elsewhere in the genome.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Recurrencia Local de Neoplasia/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/química , Carcinoma/química , Rotura Cromosómica , Cromosomas Humanos Par 11/química , Cromosomas Humanos Par 17/química , Cromosomas Humanos Par 8/química , Femenino , Genoma Humano , Humanos , Cariotipificación , Persona de Mediana Edad , Recurrencia Local de Neoplasia/química , Coloración y EtiquetadoRESUMEN
We have used two-dimensional electrophoresis of enzyme-digested genomic DNA to identify a novel gene GAC1, which maps at 1q32.1 and which is overexpressed in malignant gliomas in which it is amplified. GAC1 encodes a protein which belongs to the leucine-rich repeat superfamily. Amplification and overexpression of GAC1 was demonstrated in two of eight tumors where amplifications were previously evidenced by comparative genomic hybridization (one glioblastoma multiforme and one anaplastic astrocytoma), and in one of eight unselected glioblastomas multiforme. GAC1 exhibits sequence homology with other proteins which function as cell-adhesion molecules or as signal transduction receptor and is a likely candidate for the target gene in the 1q32.1 amplicon in malignant gliomas.
Asunto(s)
Moléculas de Adhesión Celular Neuronal , Cromosomas Humanos Par 1 , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Proteínas de la Membrana , Proteínas de Neoplasias/genética , Secuencia de Aminoácidos , Astrocitoma/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Amplificación de Genes , Glioblastoma/genética , Humanos , Leucina , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Oligodendroglioma/genética , Renina/genética , Transcripción GenéticaRESUMEN
Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.
Asunto(s)
Neoplasias de la Mama/genética , Núcleo Celular/patología , Aberraciones Cromosómicas , Cromosomas Humanos Par 1 , Reordenamiento Génico , Adulto , Anciano , Neoplasias de la Mama/patología , Sondas de ADN , Femenino , Citometría de Flujo/métodos , Humanos , Hibridación Fluorescente in Situ/métodos , Interfase , Persona de Mediana Edad , Estadificación de Neoplasias , Células Tumorales CultivadasRESUMEN
The expression patterns of the class I homeogenes HOXB and HOXC clusters in the presence of retinoic acid (RA) were studied in two human small-cell lung cancer (SCLC) cell lines and compared to that of NT2/D1 embryonal carcinoma cells. Contrasting with the sequential 3'-5' induction of the HOX genes observed after RA treatment of embryonic NT2/D1 cells, in the SCLC cells the responding genes (induced or down-regulated) were interspersed with insensitive genes (expressed or unexpressed), while no genomic alteration affected the corresponding clusters. These findings imply that HOX gene regulatory mechanisms are altered in non-embryonic SCLC cells, perhaps reflecting their ability to respond to more diversified stimuli, in relation with their origin from adult tissues.
Asunto(s)
Carcinoma de Células Pequeñas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Homeobox , Proteínas de Homeodominio/genética , Neoplasias de Células Germinales y Embrionarias/genética , Tretinoina/farmacología , Cartilla de ADN , Electroforesis en Gel de Agar , Proteínas de Homeodominio/biosíntesis , Humanos , Reacción en Cadena de la Polimerasa , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Células Tumorales CultivadasRESUMEN
The global DNA methylation status was investigated on a series of 59 breast cancers by Southern blotting, using methylation sensitive restriction enzymes. By comparison to control DNA, almost all tumor DNAs were found globally hypomethylated. However, the demethylation was variable from tumor to tumor. Compared to other biological parameters, the methylation did not correlate with chromosome alterations, steroid hormone receptor status, or histopathological grading. Tumors which appeared to be the most evolved for other parameters were only mildly hypomethylated, whereas tumors with strongly hypomethylated DNA corresponded to those with slight alterations of the other parameters. Thus, DNA hypomethylation is a consistent characteristic of breast cancer, but its variations may not correlate with tumor progression of most breast cancers.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Metilación de ADN , ADN de Neoplasias/metabolismo , Factores de Edad , Mama/metabolismo , Neoplasias de la Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , ADN/metabolismo , Femenino , Humanos , Metástasis Linfática , Menstruación , Ploidias , Pronóstico , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismoRESUMEN
The most prominent class of centromeric DNA sequences belongs to the alpha satellite family of tandemly repeated DNA. The human chromosome 7 has been shown to contain two distinct alpha satellite arrays: D7Z1 and D7Z2, separated by 1 Mb. The order of these arrays was analyzed in normal blood cells and in the melanoma cell line IPC182 with two approaches using in situ hybridization: (1) Relative mapping on high-resolution chromosomes in fluorescence and electron microscopy (EM); and (2) simultaneous visualization of the two sequences using fluorochromes of different colors or gold particles of different sizes. The location within the centromeric area of chromosome 7, on the side of the short arm for D7Z2 and near the long arm for D7Z1 is confirmed. In addition, the hybridization signal of D7Z2 is confined to two small areas of the centromeric region in external positions, whereas the D7Z1 signal covers the entire width of the primary constriction. In situ hybridization with D7Z1 and D7Z2, performed on the melanoma cell line IPC 182, allowed characterization of two isochromosomes, i(7)(q10) and idic(7)(q11), as well as the der(7)t(7;12) observed in this cell line. The three-derived chromosomes appeared to result from different breakpoints, but only D7Z1 was conserved in all cases, suggesting the importance of this sequence for the centromeric function.
Asunto(s)
Cromosomas Humanos Par 7/genética , ADN Satélite/genética , Melanoma/genética , Centrómero , Humanos , Hibridación Fluorescente in Situ , Linfocitos , Microscopía Electrónica , Microscopía Fluorescente , Células Tumorales CultivadasRESUMEN
Using chromosome painting, a study of chromosomal abnormalities has been performed in two prostatic carcinoma cell lines, PC-3 and DU145. In PC-3, this analysis revealed a highly rearranged hypotriploid karyotype with 54 to 61 chromosomes and numerous rearrangements of chromosomes 1, 3, 5, 8, 10, and 14. At passage 73, DU145 had a hypotriploid karyotype with few rearrangements of chromosomes 1, 3, 5, 12, 13, and 20, whereas at passage 153, this cell line showed a near-tetraploid karyotype with a great number of rearrangements involving chromosomes 3, 6, 8, 10, 12, and 17. A single rearrangement was shared by the 2 cell lines, an i(5)(p10). A comparative genomic hybridization study demonstrated a noticeable amplification of bands 10q22.3-q23 and 14q22-q24 in the PC-3 cell line. No amplification signal was detected for DU145.
Asunto(s)
Carcinoma/patología , Aberraciones Cromosómicas/genética , Neoplasias de la Próstata/patología , Carcinoma/genética , Aberraciones Cromosómicas/patología , Trastornos de los Cromosomas , ADN de Neoplasias/genética , Amplificación de Genes , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , Hibridación de Ácido Nucleico , Neoplasias de la Próstata/genética , Eliminación de Secuencia , Células Tumorales CultivadasRESUMEN
The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.