RESUMEN
DNA copy number analysis was performed, using single-nucleotide polymorphism mapping arrays, to fine map genomic imbalances in human malignant mesothelioma (MM) cell lines derived from primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. All 22 cell lines examined showed homozygous deletions of 9p21.3, centering at the CDKN2A/ARF and CDKN2B loci. Other commonly underrepresented segments included 1p36, 1p22, 3p21-22, 4q13, 4q34, 11q23, 13q12-13, 14q32, 15q15, 18q12, and 22q12, each observed in 55-90% of cell lines. Focal deletions of 11q23 encompassed the transcriptional repressor gene promyelocytic leukemia zinc finger (PLZF), which was validated by analysis of genomic DNA using real-time polymerase chain reaction (PCR). Semi-quantitative RT-PCR and immunoblot analysis revealed that PLZF is greatly downregulated in MM cell lines compared with non-malignant mesothelial cells. Ectopic expression of PLZF in PLZF-deficient MM cells resulted in decreased cell viability, reduced colony formation, as well as increased apoptosis, the latter based on results of various cell death assays and the observation of increased cleavage of caspase 3, PARP, and Mcl-1. These data indicate that deletions of PLZF are a common occurrence in MM and that downregulation of PLZF may contribute to MM pathogenesis by promoting cell survival.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 9/genética , Regulación hacia Abajo , Factores de Transcripción de Tipo Kruppel/genética , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Caspasa 3/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
BACKGROUND: The recently identified TMPRSS2: ERG fusion gene is a candidate oncogene for prostate cancer (PCa). SUBJECTS AND METHODS: We have tested for the presence of this gene in tumor samples from 84 patients who had radical prostatectomy in 1998-2000. Sixty patients (group A) had surgery only; 24 patients (group B) received androgen ablation therapy for 3 months before surgery. The occurrence of the rearrangement was evaluated by RT-PCR and by fluorescent in situ hybridization analysis. RESULTS: A TMPRSS2:ERG fusion gene was present and expressed, as demonstrated by RT-PCR, in 84% of patients in group A and in 54% of patients in group B (p=0.01). The presence of TMPRSS2:ERG transcripts and the levels of ERG RNA, measured by quantitative Real Time-PCR, did not correlate significantly with clinical and pathologic characteristics of the tumors. In patients of group A, but not in those of group B, ERG expression showed a negative correlation with the Gleason score (p=0.0001). Histochemical analysis showed that ERG expression is limited to tumor cells, and in group A patients (but not in group B patients) it is limited to those glands that express TMPRSS2:ERG. CONCLUSION: The lower proportion of patients expressing TMPRSS2: ERG in group B suggests that androgen ablation inhibits the expression of TMPRSS2:ERG. Moreover, in group B, but not in group A, patients with expression of the fusion gene had earlier prostate specific antigen recurrence (p=0.007). Although preliminary, the data indicate that tumors in which pre-surgery androgen ablation fails to suppress expression of the fusion gene have a higher risk of recurrence.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Proteínas de Fusión Oncogénica/genética , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/prevención & control , Neoplasias de la Próstata/terapia , Técnicas de Ablación , Anciano , Animales , Fusión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/metabolismo , Prostatectomía , Neoplasias de la Próstata/sangre , Recurrencia , Resultado del TratamientoRESUMEN
We analyzed a cohort of 61 follicular lymphomas (FL) with an abnormal G-banded karyotype by spectral karyotyping (SKY) to better define the chromosome instability associated with the t(14;18)(q32;q21) positive and negative subsets of FL and histologic grade. In more than 70% of the patients, SKY provided additional cytogenetic information and up to 40% of the structural abnormalities were revised. The six most frequent breakpoints in both SKY and G-banding analyses were 14q32, 18q21, 3q27, 1q11-q21, 6q11-q15 and 1p36 (15-77%). SKY detected nine additional sites (1p11-p13, 2p11-p13, 6q21, 8q24, 6q21, 9p13, 10q22-q24, 12q11-q13 and 17q11-q21) at an incidence of >10%. In addition to the known recurring translocations, t(14;18)(q32;q21) [70%], t(3;14)(q27;q32) [10%], t(1;14)(q21;q32) [5%] and t(8;14)(q24;q32) [2%] and their variants, 125 non-IG gene translocations were identified of which four were recurrent within this series. In contrast to G-banding analysis, SKY revealed a greater degree of karyotypic instability in the t(14;18) (q32;q21) negative subset compared to the t(14;18)(q32;q21) positive subset. Translocations of 3q27 and gains of chromosome 1 were significantly more frequent in the former subset. SKY also allowed a better definition of chromosomal imbalances, thus 37% of the deletions detected by G-banding were shown to be unbalanced translocations leading to gain of genetic material. The majority of recurring (>10%) imbalances were detected at a greater (2-3 fold) incidence by SKY and several regions were narrowed down, notably at gain 2p13-p21, 2q11-q21, 2q31-q37, 12q12-q15, 17q21-q25 and 18q21. Chromosomal abnormalities among the different histologic grades were consistent with an evolution from low to high grade disease and breaks at 6q11-q15 and 8q24 and gain of 7/7q and 8/8q associated significantly with histologic progression. This study also indicates that in addition to gains and losses, non-IG gene translocations involving 1p11-p13, 1p36, 1q11-q21, 8q24, 9p13, and 17q11-q21 play an important role in the histologic progression of FL with t(14;18)(q32;q21) and t(3q27).
Asunto(s)
Inestabilidad Cromosómica , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 18 , Linfoma Folicular/genética , Translocación Genética , Adulto , Anciano , Anciano de 80 o más Años , Bandeo Cromosómico , Progresión de la Enfermedad , Femenino , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Linfoma Folicular/patología , Masculino , Persona de Mediana EdadRESUMEN
The neurofibromatosis type 2 NF2 gene product, merlin, is a tumor suppressor frequently inactivated in malignant mesothelioma (MM). To investigate a possible correlation between merlin inactivation and MM invasiveness, we restored merlin expression in NF2-deficient MM cells. Re-expression of merlin markedly inhibited cell motility, spreading and invasiveness, properties connected with the malignant phenotype of MM cells. To test directly whether merlin inactivation promotes invasion in a nonmalignant system, we used small interfering RNA to silence Nf2 in mouse embryonic fibroblasts (MEFs) and found that downregulation of merlin resulted in enhanced cell spreading and invasion. To delineate signaling events connected with this phenotype, we investigated the effect of merlin expression on focal adhesion kinase (FAK), a key component of cellular pathways affecting migration and invasion. Expression of merlin attenuated FAK phosphorylation at the critical phosphorylation site Tyr397 and disrupted the interaction of FAK with its binding partners Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase. In addition, NF2-null MM cells stably overexpressing FAK showed increased invasiveness, which decreased significantly when merlin expression was restored. Collectively, these findings suggest that merlin inactivation is a critical step in MM pathogenesis and is related, at least in part, with upregulation of FAK activity.
Asunto(s)
Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Mesotelioma/patología , Mesotelioma/prevención & control , Neurofibromina 2/biosíntesis , Neurofibromina 2/genética , Animales , Línea Celular Tumoral , Inhibición de Migración Celular , Células Cultivadas , Regulación hacia Abajo/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Humanos , Mesotelioma/enzimología , Ratones , Invasividad Neoplásica/prevención & control , Neurofibromina 2/fisiología , Transducción de Señal/genéticaRESUMEN
With the advent of array-based comparative genomic hybridization technology, the analog cytogenetic analysis that has been used for the past 100 years could be replaced by the quantitative, microarray-based molecular analysis. Major advantages of the new array-based cytogenetic technologies are the high resolution and the high throughput. This technology is the first to offer an autonomous whole-chromosome analysis in one hybridization reaction for the detection of submicroscopic gains/losses. However, as with any new technology, it needs to be validated with regard to its performance in various applications (e.g. clinical genetic testing and cancer applications), comparative cost, and the data interpretation.
Asunto(s)
Aberraciones Cromosómicas , Citogenética/métodos , Hibridación de Ácido Nucleico , HumanosRESUMEN
We report here the molecular cloning and characterization of a t(1;14)(q21;q32) in a follicular lymphoma (FL) with an unusual BCL2 aberration. Fluorescence in situ hybridization (FISH) and Southern blot analysis of tumor cells identified the translocation breakpoint within the 5' switch region of IGHG (Sgamma). We cloned the chimeric breakpoint region approximately 1.5 kbp downstream from the HindIII site of 5'Sgamma2 on chromosome 14q32 and identified a 360-bp novel segment with homology to the CpG island clone 11h8. Two BAC clones containing this sequence were isolated and mapped to 1q21 by FISH. BAC 342/P13 contained sequences homologous to Fcgamma receptors 2A, 3A, 2B, 3B, and a heat shock protein gene HSP70B. The translocation brought the Sgamma2 region of a productive IGH allele 20 approximately 30 kbp upstream of FCGR2B. As a result of the translocation, the b2 isoform of FCGR2B was overexpressed in the tumor. Screening of a panel of 76 B-cell lymphomas with 1q21-23 cytogenetic aberrations by Southern blot analysis using breakpoint probes identified an additional FL with a t(14;18)(q32;q21) and a breakpoint in the FCGR2B region. These results suggest that FCGR2B may be deregulated by 1q21 aberration in BCL2 rearranged FLs and possibly play a role in their progression.
Asunto(s)
Antígenos CD/genética , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 1 , Regulación de la Expresión Génica , Linfoma Folicular/genética , Receptores de IgG/genética , Translocación Genética , Adulto , Southern Blotting/métodos , Aberraciones Cromosómicas , Clonación Molecular , Femenino , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ/métodos , Linfoma no Hodgkin/genética , Mutagénesis Insercional , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Previous comparative genomic hybridization and allelic loss analyses demonstrated frequent deletions from 15q11.1-15 in malignant mesothelioma. Recurrent losses of 15q11-22 have also been reported in several other tumor types such as breast and colorectal cancers. To more precisely map the commonly deleted region, we have performed a high density loss of heterozygosity analysis of 46 malignant mesotheliomas, using 26 polymorphic microsatellite markers spanning the entire long arm of chromosome 15. Allelic loss from 15q was observed in 22 of 46 (48%) cases. These analyses have defined a minimally deleted region of approximately 3-cM, which was confirmed to reside at 15q15 by fluorescence in situ hybridization analysis with yeast artificial chromosome probes. No tumor suppressor genes have been reported to map to this site. The minimally deleted region identified in this investigation overlaps those observed in other kinds of cancer, and is the smallest site of recurrent 15q loss identified to date in human tumors. The identification of this commonly deleted site implicates a putative tumor suppressor gene(s) at 15q15 involved in diverse forms of human neoplasia.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 15 , Pérdida de Heterocigocidad , Mesotelioma/genética , Alelos , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Heterocigoto , Humanos , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite , Modelos Genéticos , Polimorfismo Genético , Células Tumorales CultivadasRESUMEN
Four new cell lines were established from the primary tumors of patients with untreated colorectal adenocarcinoma. Drug sensitivity and characterization of these cell lines was performed. Three of the four cell lines formed colonies in soft agar and all were tumorigenic in nude mice. The cell lines were morphologically similar but had differences in growth characteristics. Two of the cell lines, C18 (CCCL-4) and C29 (CCCL-6), had a longer doubling time compared with C85 (CCCL-1) and C86 (CCCL-2). The C18 and C29 cell lines had chromosome 17 abnormalities and evidence by immunohistochemistry of a mutant p53 and had decreased levels of thymidylate synthase and dihydrofolate reductase proteins, associated with decreased thymidylate synthase catalytic activity in C18 and no detectable activity in C29. Raltitrexed and GW1843U89 showed potent cytotoxic activity and all four cell lines displayed similar cytotoxicity to these folate thymidylate synthase inhibitors. The C18 and C29 cell lines were in general resistant to the other agents tested (methotrexate, 5-fluorouracil, nolatrexed) when compared with the C85 and C86 cell lines. These new cell lines may be useful for the study of colorectal adenocarcinoma and for evaluating new drugs or treatment schedules.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antimetabolitos Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Antagonistas del Ácido Fólico/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Células Tumorales Cultivadas , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Adenocarcinoma/patología , Aneuploidia , Animales , Antimetabolitos Antineoplásicos/metabolismo , Western Blotting , División Celular/efectos de los fármacos , Bandeo Cromosómico , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Resistencia a Antineoplásicos , Antagonistas del Ácido Fólico/metabolismo , Humanos , Inmunohistoquímica , Cariotipificación , Cinética , Ratones , Ratones Desnudos , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismo , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute megakaryocytic leukemia is a rare subtype of AML that is often difficult to diagnose; it is most commonly associated with Down syndrome in children. To identify chromosomal imbalances and rearrangements associated with acute megakaryocytic leukemia, we used G-banding, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) on a variety of primary patients' samples and leukemia cell lines. The most common abnormality was gain of chromosome 19 or arm 19q, which was detected by CGH in four of 12 (33.3%) primary samples and nine of 11 (81.8%) cell lines. In none of the primary samples was this abnormality detected by G-banding analysis. WCP was used to define further the nature of the chromosome 19 gain in the cell lines, which was found to be due to the presence of additional 19q material on marker chromosomes or to cryptic translocations involving 19q. The most common chromosomal loss--detected only in the cell lines--was deletion of chromosomal band 13q14, which was seen in six of 11 (54.5%) cell lines. Other recurrent changes included gains of 1p, 6p, 8q, 11q, 15q, 17q, and 21q and losses of 2, 4q, 5q, 7q, 9p, and 11p. Combining conventional and molecular cytogenetic analyses defined recurrent clonal chromosomal abnormalities, which will aid in the identification of critical genes that are abnormal in acute megakaryocytic leukemia cells.
Asunto(s)
Cromosomas Humanos Par 19/genética , Amplificación de Genes/genética , Leucemia Megacarioblástica Aguda/genética , Adulto , Anciano , Preescolar , Aberraciones Cromosómicas , Pintura Cromosómica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Hibridación de Ácido NucleicoRESUMEN
Deletions of the long arm of chromosome 20 have been reported in a wide range of myeloid disorders and may reflect loss of critical tumor suppressor gene(s). To identify such candidate genes, 65 human myeloid cell line DNAs were screened by polymerase chain reaction (PCR) for evidence of allelic loss at 39 highly polymorphic loci on the long arm of chromosome 20. A mono-allelic pattern was present in eight cell lines at multiple adjacent loci spanning the common deleted regions (CDRs) previously defined in primary hematological samples, suggesting loss of heterozygosity (LOH) at 20q. Fluorescence in situ hybridization (FISH) was then performed using a series of yeast artificial chromosomes (YACs) ordered in the CDR, and in five of eight cell lines, the deletions resulted from cytogenetically detectable whole chromosomal loss or large interstitial deletion, whereas in another cell line deletion was associated with an unbalanced translocation. LOH in the CMK megakaryocytic cell line, which has a hypotetraploid karyotype, was associated with a der(20)t(1;20)(q32;q12)x2 leading to complete deletion of the CDR. Three additional unbalanced translocations were found within the CDR and all three breakpoints mapped to a single YAC. We then used a series of P1 artificial chromosomes (PACs) spanning this YAC clone, and two PACs produced 'split' signals suggesting that they each span one of these breakpoints. Exon trapping using PACs that overlap the breakpoint regions yielded portions of six genes and evaluation of these genes as candidate tumor suppressor genes is underway. The limited information available about these genes suggests that the h-l(3)mbt gene is the most attractive candidate.
Asunto(s)
Fragilidad Cromosómica , Cromosomas Humanos Par 20 , Leucemia Mieloide/genética , Translocación Genética , Secuencia de Bases , Southern Blotting , Cromosomas Artificiales de Levadura , Cartilla de ADN , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Leucemia Mieloide/patología , Pérdida de Heterocigocidad , Repeticiones de Microsatélite/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
The MYCN gene is often amplified but rarely rearranged in neuroblastoma. We report, for the first time, a rearrangement within the MYCN coding region in a metastatic neuroblastoma in a 3-year-old boy with MYCN amplification in his primary tumor. The rearrangement occurred 46 nucleotides downstream from the ATG codon in exon 2 of MYCN. The amplification level of the rearranged copies of the MYCN gene was lower than that of the unrearranged copies of MYCN. These results indicate that the rearrangement occurred after initial MYCN gene amplification. Monochromosomal somatic cell hybrid mapping of the novel region fused to exon 2 of MYCN localized it to chromosome 2, suggesting that this rearrangement resulted from an interstitial deletion, presumably within the MYCN amplicon itself.
Asunto(s)
Neoplasias Abdominales/genética , Reordenamiento Génico , Genes myc/genética , Neuroblastoma/genética , Neoplasias Abdominales/patología , Neoplasias Abdominales/terapia , Secuencia de Bases , Southern Blotting , Preescolar , Fragilidad Cromosómica , Terapia Combinada , ADN de Neoplasias/análisis , Resultado Fatal , Amplificación de Genes , Humanos , Hibridación Fluorescente in Situ , Masculino , Datos de Secuencia Molecular , Neuroblastoma/secundario , Neuroblastoma/terapia , Reacción en Cadena de la PolimerasaRESUMEN
Cytogenetic abnormalities at chromosome 1q21 are among the most common second genetic events observed in Non-Hodgkin's Lymphomas and have prognostic significance. Recently, BCL9 has been cloned from a pre-B-cell lymphoblastic leukemia cell line, which carried a t(1:14)(q21;q32). However, among a panel of 39 B-cell malignancies with 1q21 translocation, only two cases showed rearrangement for the BCL9 gene. We report the establishment of a new lymphoma cell line from a patient with relapsed diffuse large cell lymphoma. This cell line SKI-DLCL-1 showed cell surface antigens identical to the original tumor and demonstrated the profile of a mature B-cell phenotype: CD19 and CD20 positive, CD5 and C10 negative. It carried a t(1;14)(q21;q32) translocation identical to the original tumor. Although the clinical presentation was an isolated effusion lymphoma, studies for HIV-1, HHV8 and EBV were all negative. Southern blot analysis demonstrated that BCL9 was not rearranged in the SKI-DLCL-1 cell line. In addition, the BCL9 gene was not over-expressed in SKI-DLCL-1 cell line. The identification of a new locus at 1q21 will help clarify the pathogenesis of B-cell malignancies with a translocation involving this locus.
Asunto(s)
Cromosomas Humanos Par 14 , Cromosomas Humanos Par 1 , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Translocación Genética/genética , Células Tumorales Cultivadas/citología , Anciano , Ascitis/genética , Ascitis/patología , Análisis Citogenético , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Masculino , Células Tumorales Cultivadas/metabolismoRESUMEN
The BCL10 gene, located at 1p22, has been implicated in a number of human malignancies, including malignant mesotheliomas (MMs) and colorectal carcinomas. Subsequent reports, however, have revealed an absence of BCL10 mutations in genomic DNA from such tumors. It has been proposed that some abnormalities of this gene may be found only in RNA and not in genomic DNA, suggesting that BCL10 may be mutated post-transcriptionally, rather than at the genomic level. To explore this possibility, we performed SSCP mutation analysis and direct sequencing of cDNA from 17 MM cell lines displaying LOH in 1p22, 12 MM tumor specimens, and 11 colon carcinoma cell lines. SSCP revealed several different band shifts in these samples. The nucleotide changes observed in the cDNA samples were also seen in matched genomic DNA and corresponded to known polymorphisms in the general population. Thus, we conclude the BCL10 mutations are absent at the cDNA level, and that this gene does not undergo "molecular misreading." Since BCL10 also does not possess mutations at the genomic DNA level, it can be ruled out as a gene involved in the pathogenesis of MM and colorectal cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Neoplasias Colorrectales/genética , Mesotelioma/genética , Mutación , Proteínas de Neoplasias/genética , Procesamiento Postranscripcional del ARN/genética , Proteína 10 de la LLC-Linfoma de Células B , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , Análisis Mutacional de ADN , Humanos , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , ARN Neoplásico/genética , Eliminación de Secuencia , Células Tumorales CultivadasRESUMEN
A multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay for both major forms of BCR-ABL was compared with fluorescence in situ hybridization (FISH), karyotyping, and Southern blotting for disease monitoring in 37 follow-up bone marrow samples from 32 patients with Ph1-positive leukemia. Of these 37 samples, 33 were from patients with chronic myeloid leukemia (CML) (26 post allogeneic bone marrow transplantation [AlloBMT] and seven during interferon-alpha therapy) and 4 from Ph1-positive acute lymphoblastic leukemia (ALL) patients (1 post AlloBMT and 3 post high dose chemotherapy). For the 27 samples studied after AlloBMT (26 CML and 1 Ph1-positive ALL) the time after transplantation ranged from 1 to 107 months (median 47.5 months). In 8 (22%) of the 37 samples there were discrepant results among methods. The discrepancy rates relative to other techniques were: karyotyping 17% (5 of 29), Southern blotting 18% (6 of 33), multiplex RT-PCR 8% (3 of 37), and FISH 8% (3 of 37). Therefore, the relative accuracy of each method for disease monitoring in Ph1-positive leukemia was: 83% (24 of 29) for karyotyping, 82% (27 of 33) for Southern blotting, 92% (34 of 37) for FISH, and 92% (34 of 37) for multiplex RT-PCR. This multiplex RT-PCR assay appears equivalent to FISH in terms of accuracy, simplicity, and turnaround time and both are superior to Southern blot and conventional cytogenetics in the laboratory monitoring of Ph1-positive leukemias.
Asunto(s)
Southern Blotting , Análisis Citogenético , Genes abl/genética , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adolescente , Adulto , Niño , Femenino , Humanos , Células K562 , Masculino , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
Multifocal presentation, defined as the presence of tumor at two or more anatomically separate sites, before the manifestation of disease in sites where sarcomas usually metastasize (e.g., lungs) occurs in about 1% of extremity soft tissue sarcomas (STSs). Debate still persists whether multifocal STSs represent an unusual pattern of metastasis or multiple separate primary tumors. Among STSs with multifocal presentation, myxoid liposarcoma is the predominant histological type. This subtype of liposarcoma contains the specific t(12;16) chromosomal translocation, which results in rearrangement of the TLS and CHOP genes that is clone specific at the DNA level. We, therefore, sought to address the question of clonality by molecular analysis in six patients who presented with either synchronous or metachronous multifocal myxoid liposarcoma. In all six cases, adequate frozen tumor was available for DNA extraction from at least two distinct anatomical sites. Southern blot analysis using CHOP, TLS, and EWS cDNA probes was performed on genomic DNA. Five cases contained a TLS-CHOP rearrangement, and one case had the variant EWS-CHOP fusion (seen in <5% of cases). The size of the rearranged CHOP fragment differed among the six patients, as expected, but was identical in all anatomically separate tumor samples from each patient. Likewise, the sizes of the rearranged bands observed with either the TLS or EWS probes supported the monoclonality of all cases. Our results confirm the monoclonal origin of multifocal myxoid liposarcoma, establishing the metastatic nature of distant soft tissue lesions in these cases. It remains unclear whether this unusual pattern of metastasis represents an intrinsic property of this subset of myxoid liposarcoma or merely a rare chance occurrence. The clinical outcomes observed in this small series suggest that the prognosis of multifocal myxoid liposarcoma is poor, regardless of its often bland or "low-grade" histological appearance.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Reordenamiento Génico , Liposarcoma/genética , Neoplasias Primarias Múltiples/genética , Ribonucleoproteínas/genética , Factores de Transcripción/genética , Adulto , Anciano , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 16 , Células Clonales , Daño del ADN , ADN de Neoplasias/aislamiento & purificación , Femenino , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Liposarcoma/química , Liposarcoma/patología , Masculino , Persona de Mediana Edad , Neoplasias Primarias Múltiples/química , Neoplasias Primarias Múltiples/patología , Proteína EWS de Unión a ARN , Proteína FUS de Unión a ARN , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción CHOP , Transcripción Genética , Translocación GenéticaRESUMEN
Cytogenetic investigations of malignant mesothelioma (MM) have revealed frequent losses in chromosomes 13 and 14, suggesting that inactivation of tumor suppressor genes (TSGs) residing in these chromosomes may contribute to mesothelial cell tumorigenesis. To define the shortest region of overlap (SRO) of deletions from these chromosomes, we performed loss of heterozygosity (LOH) analyses on 30 MMs using 25 microsatellite markers in 13q and 21 markers in 14q. Twenty of the 30 MMs (67%) showed allelic loss of at least one marker in 13q. The SRO of deletions was delineated as an approximately 7 centiMorgan region, flanked by markers D13S1253 and D13S291, located at 13q13.3-14.2. Thirteen of the 30 MMs (43%) displayed allelic losses from 14q, with at least three distinct regions of LOH located at segments q11.2-13.2, q22.3-24.3, and q32. 12. These data highlight a single region of chromosomal loss in 13q in many MMs, implicating the involvement of a TSG that is critical to the pathogenesis of this malignancy. In contrast, the lower incidence and diffuse pattern of allelic losses in 14q suggest that several TSGs in this chromosome arm may contribute to tumorigenic progression in a subset of MMs. Genes Chromosomes Cancer 28:337-341, 2000.
Asunto(s)
Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 14/genética , Pérdida de Heterocigocidad/genética , Mesotelioma/genética , Neoplasias Pleurales/genética , Alelos , Humanos , Cariotipificación , Mesotelioma/epidemiología , Neoplasias Pleurales/epidemiología , Células Tumorales CultivadasRESUMEN
The development of an in vivo model of retinoblastoma could be important for studying its biological behaviour and developing novel therapeutic strategies. We examined the ability of patient-derived retinoblastoma cells to grow and disseminate in severe combined immunodeficiency CB-17-SCID mice after subcutaneous (s.c.) inoculation without conditioning treatment. 24/30 (80%) of patient-derived tumours engrafted and grew as s.c. nodules in SCID mice. Whilst most xenografted tumours appeared to be localised, by PCR assay a positive DNA band of human minisatellite region (YNZ.22) was determined in the bone marrow of 19/25 (76%), in the spleen of 14/25 (56%) and in the liver of 16/25 (64%) mice, respectively, indicating dissemination to distant organs. Cytogenetic analysis demonstrated i(6p) in 5/12 (42%) and trisomy 1 or 1q abnormalities in 8/12 (67%) of the xenografted tumour samples studied, respectively, suggesting that retinoblastoma tumour cells maintain their cytogenetic abnormalities following adoptive growth in SCID mice. In this report we demonstrate the ability to propagate human primary retinoblastoma cells in SCID mice after s.c. inoculation and suggest the possibility of using the SCID mouse model to study the intrinsic biological behaviour of human retinoblastoma and to develop novel therapeutic strategies in the treatment of this disease.
Asunto(s)
Neoplasias de la Retina/patología , Retinoblastoma/patología , Animales , Niño , Preescolar , Aberraciones Cromosómicas/genética , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 16/genética , Cromosomas Humanos Par 6/genética , Femenino , Humanos , Lactante , Cariotipificación , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasias de la Retina/genética , Retinoblastoma/genéticaRESUMEN
Gene expression changes in rat asbestos-induced malignant mesothelioma (MM) cells were investigated by differential mRNA display. A mRNA transcript identified by this approach was abundant in normal rat mesothelial cells but not expressed in rat MM cell lines. Northern blot analysis confirmed that this transcript is uniformly silenced in rat MM cell lines and primary tumors. Nucleotide sequence analysis revealed that this transcript is encoded by the rat glypican 3 gene (GPC3), whose human homolog is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. Allelic loss at the GPC3 locus was infrequent (6.9%) in MM cell lines, and no mutations were found. GPC3 transcript levels were markedly decreased in 16 of 18 primary tumors and 17 of 22 human MM cell lines. Most of the cell lines were shown to have aberrant methylation of the GPC3 promoter region. In two of four human MM cell lines tested, GPC3 expression was restored after 2-deoxy 5-azacytidine (DAC)-mediated demethylation of its promoter region. Ectopic expression of GPC3 inhibited in vitro colony formation of human MM cells. Collectively, these data suggest that down-regulation of GPC3 is a common occurrence in MM and that GPC3, an X-linked recessive overgrowth gene, may encode a negative regulator of mesothelial cell growth.
Asunto(s)
Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/genética , Mesotelioma/genética , Proteoglicanos/genética , Alelos , Animales , Línea Celular , Metilación de ADN , ADN Complementario/análisis , Regulación hacia Abajo , Femenino , Glipicanos , Humanos , Mesotelioma/patología , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas F344RESUMEN
The band 1q21 is among the most common sites affected by chromosomal translocations in lymphoid, myeloid, epithelial, and sarcomatous lesions. In non-Hodgkin's lymphoma (NHL), translocations and duplications affecting this chromosomal site are frequently, but not exclusively, seen in association with primary abnormalities such as the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, suggesting a role for 1q21 rearrangements in tumor progression. We report here the characterization and cloning of breakpoints in a case of extranodal ascitic B-cell lymphoma with a t(1;14)(q21;q32) translocation. The breakpoints on the der(1) and der(14) chromosomes were mapped by fluorescence in situ hybridization and Southern blot analysis and cloned using an IGHG (Cgamma) probe. The translocation linked the IGHG4 switch (Sgamma4) sequences of the productively rearranged allele to chromosome 1 sequences downstream of MUC1, leaving the MUC1 transcriptional unit intact. MUC1 was markedly overexpressed in the tumor at the mRNA and protein levels relative to lymphoma cell lines lacking a 1q21 rearrangement. Presumably, MUC1 transcription is aberrantly regulated by the IGHA (Calpha) 3' enhancer element retained on the same chromosome. Screening of a panel of B-cell lymphomas by Southern blot analysis identified a subset with a 3' MUC1 breakpoint and another with low-level amplification of MUC1. MUC-1 mucin has previously been shown to be frequently overexpressed in human epithelial cancers and to be associated with tumor progression and poor clinical outcome. Thus, MUC1 activation by chromosomal translocation, rearrangement, and amplification, identified here for the first time in NHL, is consistent with its suggested role in tumorigenesis. (Blood. 2000;95:2666-2671)
Asunto(s)
Cromosomas Humanos Par 14 , Cromosomas Humanos Par 1 , Linfoma de Células B/genética , Mucinas/genética , Translocación Genética , Subgrupos de Linfocitos B/patología , Secuencia de Bases , Mapeo Cromosómico , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Marcadores Genéticos , Humanos , Linfoma de Células B/patología , Datos de Secuencia MolecularRESUMEN
Myxoid liposarcoma (LS), the most common subtype of LS, is known to be characterized by the specific t(12;16) resulting in a TLS-CHOP fusion in almost all cases. We wished to address the following questions: (i) Is this genetic hallmark also present in other types of LS with predominant myxoid change? (ii) What is the proportion of cases with the variant EWS-CHOP fusion? (iii) What is the optimal approach for Southern blot detection of TLS breakpoints? We identified 59 LS characterized histologically by >90% myxoid component, in which frozen tissue tumor was available for DNA extraction. These 59 LS with myxoid features were divided into 2 groups: 42 LS with classic myxoid/round cell appearance (myxoid LS) and 17 well-differentiated LS (WDLS) with a predominant (>90%) myxoid component. Within the myxoid LS group, 29 tumors were low grade and 13 high grade (>20% round cell component). Among the 17 predominantly myxoid WDLS, there were 15 low grade and 2 focally high grade tumors. In addition, we selected as control group, 20 LS of other histological types with minimal or no myxoid change (17 WDLS and 3 pleomorphic LS) and 13 myxofibrosarcomas. Southern blot analysis was performed in all cases using a CHOP cDNA probe, and in all CHOP rearranged cases using a TLS cDNA probe. Probe/enzyme combinations for Southern blot analysis were CHOP exon 3-4 cDNA probe with BamHI or SacI, TLS exon 3-6 cDNA probe with BclI. All 42 cases of myxoid LS showed a CHOP rearrangement and 38 of them also had a TLS rearrangement. Among the 4 myxoid LS without Southern blot evidence of TLS rearrangement, 1 showed an EWS-CHOP fusion by Southern blotting and reverse transcriptase-polymerase chain reaction and in another case, reverse transcriptase-polymerase chain reaction detected a TLS-CHOP fusion transcript. None of the predominantly myxoid WDLS and none of the tumors included in the control group showed rearranegements with CHOP probe. In addition, 12 predominantly myxoid WDLS, 10 other LS, and 5 myxofibrosarcoma from the control group were also tested for TLS rearrangement; all were negative. The TLS-CHOP fusion is highly sensitive and specific for the entity of classic myxoid/round cell LS. Other types of LS, even with a predominant myxoid component, lack the TLS-CHOP rearrangement, confirming that they represent a genetically distinct group of LS. The prevalence of the EWS-CHOP variant fusion was approximately 2% in this series. The optimal enzyme for TLS genomic breakpoint detection is BclI.