RESUMEN

The mechanism that generates the extreme aneuploidy that characterizes osteosarcoma (OS) is poorly understood. In this study, interphase fluorescence in situ hybridization (FISH) analysis was used to enumerate cell-to-cell variation of several different chromosomes. We also investigated whether there was an association between TP53 mutation and centrosome aberrations in the generation of chromosomal aneuploidy in OS in four OS cell lines (HOS, SAOS2, U2OS, and MG63) and in a subset of seven tumors. Our analysis showed that there was a wide range of numerical changes affecting multiple chromosomes in OS cell lines and tumors. These data suggest that chromosomal instability (CIN) could be responsible for the extensive aneuploidy associated with this tumor. The results also showed an increased frequency of atypical mitotic figures in three OS cell lines with defective TP53, function and significantly, a more marked CIN phenotype was present in these lines. Furthermore, numerical aberrations of centrosomes were also present in these three OS cell lines with TP53 mutations. In two of three OS patients' tumors there was a large increase in the percentage of abnormal centrosome numbers. We conclude that CIN is a consistent feature of OS and that an intrinsic disturbance of the chromosomal segregation mechanisms is likely associated with centrosome aberrations.

Asunto(s)

Neoplasias Óseas/genética , Centrosoma/patología , Aberraciones Cromosómicas , Osteosarcoma/genética , Adolescente , Adulto , Niño , Femenino , Genes p53 , Humanos , Hibridación Fluorescente in Situ , Masculino , Mitosis , Mutación , Células Tumorales Cultivadas

RESUMEN

BACKGROUND: Glial tumors are the most common tumors of the central nervous system, affecting individuals of all ages. Conventional cytogenetics have been unable to identify a consistent chromosomal translocation or rearrangement in this group of tumors; thus, more advanced molecular cytogenetic approaches are required. METHODS AND RESULTS: In this study, 16 glial tumors, including two recurrences and six glioma cell lines, were analyzed by spectral karyotyping (SKY) and comparative genomic hybridization (CGH). From 169 rearrangements detected by SKY, chromosomes 1 and 10 were the most frequently affected by translocation (18 of 169 and 16 of 169 rearrangements, respectively). Other frequently altered chromosomes included chromosomes 3 (13 of 169 rearrangements), 5 (ten of 169 rearrangements), 7 (ten of 169 rearrangements ), and 11 (ten of 169 rearrangements). A clustering of centromeric breakpoints was detected in chromosomes 3, 5, 10, 11, 16, 17, and 20. CGH analysis identified consistent gain of part or all of chromosome 7 among the 10 astrocytic tumors (five of ten specimens) in the study group. Analysis of the three gangliogliomas and one ependymoma identified a much simpler pattern of primarily numerical change. CONCLUSION: Application of improved cytogenetic methods can increase our abilities to progress toward effective strategies of molecular diagnosis and classification of glial tumors.

Asunto(s)

Neoplasias del Sistema Nervioso Central/diagnóstico , Neoplasias del Sistema Nervioso Central/genética , Cariotipificación/métodos , Neuroglía/patología , Hibridación de Ácido Nucleico/métodos , Adulto , Astrocitoma/diagnóstico , Astrocitoma/genética , Centrómero , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 10 , Citogenética/métodos , Ependimoma/diagnóstico , Ependimoma/genética , Femenino , Ganglioglioma/diagnóstico , Ganglioglioma/genética , Glioblastoma/diagnóstico , Glioblastoma/genética , Humanos , Hibridación Fluorescente in Situ , Masculino , Translocación Genética , Células Tumorales Cultivadas

RESUMEN

The object of the study was to investigate the share of the polymorphisms I/D ACE, endothelin 1 4127G/A and TNF-beta NcoI in the susceptibility to proliferative diabetic retinopathy (PDR) in non-insulin-dependent diabetes mellitus (NIDDM). Genotypes were detected by polymerase chain reactions and determined in a set of 246 Caucasian NIDDM subjects with defined PDR status. The relevance of genotypes and clinical characteristics to the PDR occurrence was tested using multiple linear regression models and discrimination analysis. The best predictive value for PDR was given by a combination of two parameters - NIDDM duration and the TNF-beta genotype (p < 1.10(-6) and p = 1.10(-2), respectively) with a correct retrograde prediction of 82.6%. A comparison of the TNF-beta NcoI allele frequencies revealed no difference between NIDDM and nondiabetic subjects (n = 176), but a statistically significant difference was found between PDR and non-PDR NIDDM subjects (after a correction for the number of comparisons p = 0.03), allele beta2 being associated with PDR. Our results identified the allele variant TNF-beta2 being associated with PDR in NIDDM. Diabetes duration and the TNF-beta NcoI genotype were proven to significantly predict PDR occurrence. The TNF-beta2 allele could be regarded as a separate genetic risk factor that increases the relative incidence of PDR in patients with NIDDM.

Asunto(s)

Diabetes Mellitus Tipo 2/genética , Retinopatía Diabética/genética , Linfotoxina-alfa/genética , Polimorfismo Genético , Alelos , Cartilla de ADN/química , Endotelina-1/genética , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Peptidil-Dipeptidasa A/genética , Reacción en Cadena de la Polimerasa , Factores de Tiempo

RESUMEN

BCR/ABL fluorescent in situ hybridization study of chronic myeloid leukemia (CML) and Philadelphia(+) (Ph(+)) acute lymphoid leukemia (ALL) indicated that approximately 9% of patients exhibited an atypical hybridization pattern consistent with a submicroscopic deletion of the 5' region of ABL and the 3' region of the BCR genes on the 9q(+) chromosome. The CML patients with deletions had a shorter survival time and a high relapse rate following bone marrow transplant. Since deletions are associated with both Ph(+) CML and ALL, it seemed probable that other leukemia-associated genomic rearrangements may also have submicroscopic deletions. This hypothesis was confirmed by the detection of deletions of the 3' regions of the CBFB and the MLL genes in AML M4 patients with inv(16) and in patients with ALL and AML associated with MLL gene translocations, respectively. In contrast, analysis of the AML M3 group of patients and AML M2 showed that similar large deletions were not frequently associated with the t(15;17) or t(8;21) translocations. Analysis of sequence data from each of the breakpoint regions suggested that large submicroscopic deletions occur in regions with a high overall density of Alu sequence repeats. These findings are the first to show that the process of deletion formation is not disease specific in leukemia and also implicate that the presence of repetitive DNA in the vicinity of breakpoint regions may facilitate the generation of submicroscopic deletions. Such deletions could lead to the loss of one or more genes, and the associated haploinsufficiency may result in the observed differences in clinical behavior. (Blood. 2001;97:3581-3588)

Asunto(s)

Aberraciones Cromosómicas , Eliminación de Gen , Leucemia/genética , Adolescente , Adulto , Trasplante de Médula Ósea , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 22 , Cromosomas Humanos Par 9 , Subunidad beta del Factor de Unión al Sitio Principal , Análisis Citogenético , Proteínas de Unión al ADN/genética , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Hibridación Fluorescente in Situ , Leucemia/mortalidad , Leucemia/terapia , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Persona de Mediana Edad , Cadenas Pesadas de Miosina/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pronóstico , Recurrencia , Análisis de Secuencia de ADN , Tasa de Supervivencia , Factor de Transcripción AP-2 , Factores de Transcripción/genética , Translocación Genética

RESUMEN

Activation of the p21-ras signaling pathway from aberrantly expressed receptors promotes the growth of malignant human astrocytomas. We developed a transgenic mouse astrocytoma model using the glial fibrillary acidic protein (GFAP) promoter to express oncogenic V(12)Ha-ras, specifically in astrocytes. The development of GFAP-immunoreactive astrocytomas was directly proportional to the level of V(12)Ha-ras transgene expression. Chimeras expressing high levels of V(12)Ha-ras in astrocytes died from multifocal malignant astrocytomas within 2 weeks, whereas those with moderate levels went to germ-line transmission. Ninety-five percent of these mice died from solitary or multifocal low- and high-grade astrocytomas within 2-6 months. These transgenic astrocytomas are pathologically similar to human astrocytomas, with a high mitotic index, nuclear pleomorphism, infiltration, necrosis, and increased vascularity. Derivative astrocytoma cells are tumorigenic upon inoculation in another host. The transgenic astrocytomas exhibit additional molecular alterations associated with human astrocytomas, including a decreased or absent expression of p16, p19, and PTEN as well as overexpression of EGFR, MDM2, and CDK4. Cytogenetic analysis revealed consistent clonal aneuploidies of chromosomal regions syntenic with comparable loci altered in human astrocytomas. Therefore, this transgenic mouse astrocytoma model recapitulates many of the molecular histopathological and growth characteristics of human malignant astrocytomas in a reproducible, germ-line-transmitted, and high-penetrance manner.

Asunto(s)

Astrocitos/fisiología , Astrocitoma/genética , Modelos Animales de Enfermedad , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Animales , Astrocitos/metabolismo , Astrocitoma/metabolismo , Astrocitoma/patología , Transformación Celular Neoplásica , Femenino , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Plásmidos/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Proteínas Proto-Oncogénicas p21(ras)/genética

RESUMEN

Embryonic stem cells offer unprecedented opportunities for random or targeted genome alterations in the mouse. We present here an efficient strategy to create chromosome-specific loss of heterozygosity in embryonic stem cells. The combination of this method with genome-wide mutagenesis in ES cells (using chemical mutagens or gene-trap vectors) opens up the possibility for in vitro or in vivo functional screening of recessive mutations in the mouse.

Asunto(s)

Pérdida de Heterocigocidad , Células Madre/metabolismo , Animales , Femenino , Marcación de Gen , Técnicas Genéticas , Homocigoto , Kanamicina Quinasa/genética , Masculino , Ratones , Ratones Transgénicos , Mutagénesis

RESUMEN

Immortal epithelial cell lines were previously established after transduction of the HPV16-E6E7 genes into primary cultures of normal pancreatic duct epithelial cells. Single clones were isolated that demonstrated near normal genotype and phenotype. The proliferation of HPDE6-E6E7c7 and c11 cells is anchorage-dependent, and they were nontumorigenic in SCID mice. The cell lines demonstrated many phenotypes of normal pancreatic duct epithelium, including mRNA expression of carbonic anhydrase II, MUC-1, and cytokeratins 7, 8, 18, and 19. These cells have normal Ki-ras, p53, c-myc, and p16(INK4A) genotypes. Cytogenetic studies demonstrated losses of 3p, 10p12, and 13q14, the latter included the Rb1 gene. The wild-type p53 protein was detectable at very low levels consistent with the presence of E6 gene product, and the lack of functional p53 pathway was confirmed by the inability for gamma-irradiation to up-regulate p53 and p21waf1/cip1 protein. The p110/Rb protein level was also not detectable consistent with the expression of E7 protein and haploid loss of Rb1 gene. Despite this, the proliferation of both c7 and c11 cells were markedly inhibited by transforming growth factor-beta1. This was associated with up-regulation of p21cip1/waf1 but not p27kip1. Further studies showed that p130/Rb2 and cyclin D3 were expressed, suggesting that p130/Rb2 may have partially assumed the maintenance of G(1) cell cycle checkpoint regulation. These results indicate that except for the loss of p53 functional pathway, the two clones of HPDE6-E6E7 cells demonstrated a near normal genotype and phenotype of pancreatic duct epithelial cells. These cell lines will be useful for future studies on the molecular basis of pancreatic duct cell carcinogenesis and islet cell differentiation.

Asunto(s)

Conductos Pancreáticos/patología , Células Tumorales Cultivadas , Animales , División Celular , Línea Celular Transformada , Técnicas Citológicas , Células Epiteliales/patología , Células Epiteliales/fisiología , Expresión Génica , Regulación de la Expresión Génica , Genotipo , Humanos , Cariotipificación , Ratones , Ratones SCID , Trasplante de Neoplasias , Conductos Pancreáticos/fisiopatología , Fenotipo , Valores de Referencia , Proteína de Retinoblastoma/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/genética

RESUMEN

BACKGROUND: Currently, prostate cancer (CaP) cytogenetics is not well defined, largely because of technical difficulties in obtaining primary tumor metaphases. METHODS AND RESULTS: We examined three CaP cell lines (LNCaP, DU145, PC-3) using sequential Giemsa banding and spectral karyotyping (SKY) to search for a common structural aberration or translocation breakpoint. No consistent rearrangement common to all three cell lines was detected. A clustering of centromeric translocation breakpoints was detected in chromosomes 4, 5, 6, 8, 11, 12, 14, and 15 in DU145 and PC-3. Both these lines were found to have karyotypes with a greater level of complexity than LNCaP. CONCLUSION: The large number of structural aberrations present in DU145 and PC-3 implicate an underlying chromosomal instability and subsequent accumulation of cytogenetic alterations that confer a selective growth advantage. The high frequency of centromeric rearrangements in these lines indicates a potential role for mitotic irregularities associated with the centromere in CaP tumorigenesis.

Asunto(s)

Centrómero , Aberraciones Cromosómicas , Bandeo Cromosómico/métodos , Trastornos de los Cromosomas , Cariotipificación/métodos , Neoplasias de la Próstata/genética , Colorantes Azulados/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/patología , Células Tumorales Cultivadas

RESUMEN

Brca1 (breast cancerl, early onset) deficiency results in early embryonic lethality. As Brca1 is highly expressed in the T cell lineage, a T cell-specific disruption of Brca1 was generated to assess the role of Brca1 in relation to T lymphocyte development. We found that thymocyte development in Brca1-/- mice was impaired not as a result of V(D)J T cell receptor (TCR) recombination but because thymocytes had increased expression of tumor protein p53. Chromosomal damage accumulation and abnormal cell death were observed in mutant cells. We found that cell death inhibitor Bcl-2 overexpression, or p53-/- backgrounds, completely restored survival and development of Brca1-/- thymocytes; peripheral T cell numbers were not totally restored in Brcal-/- p53-/- mice; and that a mutant background for p21 (cyclin-dependent kinase inhibitor 1A) did not restore Brca1-/- thymocyte development, but partially restored peripheral T cell development. Thus, the outcome of Brca1 deficiency was dependent on cellular context, with the major defects being increased apoptosis in thymocytes, and defective proliferation in peripheral T cells.

Asunto(s)

Proteína BRCA1/genética , Proteína BRCA1/inmunología , Reordenamiento Génico de Linfocito T/inmunología , Linfocitos T/inmunología , Animales , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Regulación de la Expresión Génica/inmunología , Ratones , Ratones Noqueados , Linfocitos T/citología

RESUMEN

BACKGROUND: The cytogenetic examination of the bone marrow (BM) depends on the presence of sufficient amount of mitoses on slides prepared for analysis. The quantity and the quality of mitoses found in BM cultivations of patients with MDS is rather low and a new possibilities to increase the proliferative activity of the cells are under research. One promising method is the stimulation of the BM cells with addition of hemopoietic growth factors. The aim of this study was to evaluate in vitro influence of GM-CSF on the proliferative activity and the representation of chromosomally different clones in the patients with MDS. METHODS AND RESULTS: The bone marrow cells of 25 patients (12 males, 13 females) of mean age 52.5 years (range 20-85) were examined. The mitotic index (MI), quality of mitoses and chromosomal complement were evaluated in 24 and 72 h cultivations with 100 and 1000 ng/ml GM-CSF concentrations. No stimulation was used in control cultivations. After 24 h of cultivation 100 ng/ml of GM-CSF increased the mitotic index (112%) but lowered the quality of mitoses (-7.6%). On contrary after 72 h of presence of 100 ng/ml GM-CSF both parameters showed the decrease (MI-30%, quality of mitoses-36%). Similar effect had the addition of 1000 ng/ml for 24 h of cultivation (MI-5%, quality of mitoses-11%). Twelve patients had normal karyotype on all studied cells in every type of cultivation. The ratio of chromosomally abnormal clones was the same in six other cases and only non-significant changes appeared in seven patients after GM-CSF stimulation. CONCLUSIONS: The influence of GM-CSF on the division of the bone marrow cells is strictly individual. GM-CSF in concentration of 100 ng/ml in 24 h cultivation increases the activity of proliferation, but at the same time moderately decreases the quality of mitoses. The presence of GM-CSF has not changed the cytogenetic results.

Asunto(s)

Médula Ósea/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Síndromes Mielodisplásicos/genética , Adulto , Anciano , Anciano de 80 o más Años , División Celular , Células Cultivadas , Femenino , Humanos , Cariotipificación , Masculino , Persona de Mediana Edad , Índice Mitótico , Síndromes Mielodisplásicos/diagnóstico