RESUMO
The MLL gene at chromosome band 11q23 is specifically cleaved at a unique site within its breakpoint cluster region (bcr) during the higher order chromatin fragmentation associated with apoptosis. We now show that the same specific DNA cleavage event can be detected in an exogenous MLL bcr fragment that is integrated into the genome outside of its normal chromosomal context, as well as in an extrachromosomal episome containing an MLL bcr fragment. We also show that episomal or randomly integrated copies of the MLL bcr behave similar to the endogenous MLL bcr when tested in a scaffold-associated region (SAR) assay. Furthermore, an episomal murine MLL bcr introduced into human cells is cleaved at the same site as the endogenous murine MLL bcr; this episomal murine MLL bcr also functions as a SAR in human cells. We conclude that both nuclear DNA scaffold attachment as well as site-specific DNA cleavage can be directed by sequences contained within the MLL bcr, and that it is feasible to study these events using episomal shuttle vectors.
Assuntos
Proteínas de Ligação a DNA/genética , Rearranjo Gênico , Proto-Oncogenes , Fatores de Transcrição , Animais , Apoptose/genética , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Quebra Cromossômica/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Vetores Genéticos/genética , Histona-Lisina N-Metiltransferase , Humanos , Células Jurkat , Camundongos , Proteína de Leucina Linfoide-Mieloide , Matriz Nuclear/genética , Matriz Nuclear/fisiologia , Plasmídeos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Tumorais CultivadasRESUMO
We have cloned the genomic breakpoints for a balanced t(14;21)(q11. 2;q22) chromosomal translocation associated with T-cell acute lymphoblastic leukemia. Sequence analysis of the genomic breakpoints indicated that the translocation had been mediated by an illegitimate V(D)J recombination event that disrupted the T-cell receptor (TCR) alpha locus and placed the TCR alpha locus enhancer on the derivative 21 chromosome. We identified a previously unreported transcript, designated BHLHB1 (for basic domain, helix-loop-helix protein, class B, 1) that had been activated by the translocation. BHLHB1 mapped to the region of chromosome 21 that has been proposed to be responsible, at least in part, for the learning deficits seen in children with Down's syndrome. Although BHLHB1 expression normally is restricted to neural tissues, T-cell lymphoblasts with the t(14;21)(q11.2;q22) also expressed high levels of BHLHB1 mRNA. Expression of BHLHB1 dramatically inhibited E2A-mediated transcription activation in NIH 3T3 fibroblasts and Jurkat T cells. This observation suggests that BHLHB1, similar to SCL/TAL1, may exert a leukemogenic effect through a functional inactivation of E2A or related proteins.
Assuntos
Cromossomos Humanos , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Leucemia-Linfoma de Células T do Adulto/genética , Fatores de Transcrição/genética , Translocação Genética , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Criança , Feminino , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
A novel chromosomal translocation, t(2;11)(q31;p15), was identified in a patient with therapy-related acute myelogenous leukemia (t-AML). Fluorescence in situ hybridization experiments mapped the breakpoint near NUP98; Southern blot analysis demonstrated that the nucleoporin gene NUP98 was disrupted by this translocation. We used rapid amplification of cDNA ends to identify a chimeric mRNA. An in-frame, chimeric mRNA that fused NUP98 sequences to the homeobox gene HOXD13 was cloned; the predicted fusion protein contains both the GLFG repeats from NUP98 as well as the homeodomain from HOXD13. The NUP98-HOXD13 fusion is structurally similar to the NUP98-HOXA9 fusion previously identified in patients with AML, leading to the speculation that NUP98-homeobox gene fusions may be oncogenic. Moreover, this report, along with a recent study that demonstrated NUP98-DDX10 fusions in patients with t-AML, raises the possibility that NUP98 may be a previously unsuspected target for chromosomal translocations in patients with t-AML.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 2 , Rearranjo Gênico , Proteínas de Homeodomínio/genética , Leucemia Mieloide Aguda/induzido quimicamente , Leucemia Mieloide Aguda/genética , Proteínas de Membrana/genética , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Fatores de Transcrição , Translocação Genética , Fusão Gênica Artificial , Células da Medula Óssea/patologia , Criança , Mapeamento Cromossômico , Proteínas de Homeodomínio/biossíntese , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Proteínas de Membrana/biossíntese , Segunda Neoplasia Primária/induzido quimicamente , Segunda Neoplasia Primária/genética , Proteínas Nucleares/biossíntese , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Segmental jumping translocations are chromosomal abnormalities in treatment-related leukemias characterized by multiple copies of the ABL and/or MLL oncogenes dispersed throughout the genome and extrachromosomally. Because gene amplification potential accompanies loss of wild-type p53, we examined the p53 gene in a case of treatment-related acute myeloid leukemia (t-AML) with MLL segmental jumping translocation. The child was diagnosed with ganglioneuroma and embryonal rhabdomyosarcoma (ERMS) at 2 years of age. Therapy for ERMS included alkylating agents, DNA topoisomerase I and DNA topoisomerase II inhibitors, and local radiation. t-AML was diagnosed at 4 years of age. The complex karyotype of the t-AML showed structural and numerical abnormalities. Fluorescence in situ hybridization analysis showed multiple copies of the MLL gene, consistent with segmental jumping translocation. A genomic region including CD3, MLL, and a segment of band 11q24 was unrearranged and amplified by Southern blot analysis. There was no family history of a cancer predisposing syndrome, but single-strand conformation polymorphism (SSCP) analysis detected identical band shifts in the leukemia, ganglioneuroma, ERMS, and normal tissues, consistent with a germline p53 mutation, and there was loss of heterozygosity in the ERMS and the t-AML. Sequencing showed a CGA-->TGA nonsense mutation at codon 306 in exon 8. The results of this analysis indicate that loss of wild-type p53 may be associated with genomic instability after DNA-damaging chemotherapy and radiation, manifest as a complex karyotype and gene amplification in some cases of t-AML.
Assuntos
Cromossomos Humanos Par 11 , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Leucemia Mieloide/genética , Proto-Oncogenes , Fatores de Transcrição , Translocação Genética , Proteína Supressora de Tumor p53/genética , Doença Aguda , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Pré-Escolar , Histona-Lisina N-Metiltransferase , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide/tratamento farmacológico , Masculino , Proteína de Leucina Linfoide-MieloideRESUMO
Type I procollagen COOH-terminal proteinase (C-proteinase) enhancer, a glycoprotein that binds to the COOH-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity, was purified from mouse fibroblast culture media. Partial amino acid sequences obtained from proteolytic fragments were found to have identity with the deduced amino acid sequence of a cDNA clone of unknown function, previously isolated from a mouse astrocyte library. Sequences of mouse enhancer cDNA, obtained in the present study, predict a approximately 50-kDa, 468-amino acid protein that differs from the 43-kDa, 402-amino acid protein predicted by the previously reported astrocyte-derived clone. Human cDNAs encode an enhancer of 449 amino acids. Previous biochemical studies have found the mouse enhancer as a 55-kDa form, which is readily processed to 36- and 34-kDa forms, retaining full C-proteinase enhancing activity and the ability to bind the COOH-terminal propeptide. Data presented here show the 36-kDa form to correspond to the amino-terminal portion of the 55-kDa protein. This is the most conserved region between mouse and human enhancers, comprising two domains with homology to domains found in a number of proteases and proteins with developmental functions. Such domains are thought to mediate interactions between proteins. Mouse enhancer RNA is shown to be at highest levels in collagen-rich tissues, especially tendon. The human enhancer gene, PCOLCE, is localized to 7q21.3-->q22, the same chromosomal region containing the type I collagen alpha 2 chain gene, COL1A2.
Assuntos
Proteínas Morfogenéticas Ósseas , Mapeamento Cromossômico , Endopeptidases/genética , Elementos Facilitadores Genéticos , Metaloendopeptidases , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 1 , DNA Complementar/isolamento & purificação , Humanos , Camundongos , Dados de Sequência Molecular , RNA/análise , Homologia de Sequência de AminoácidosRESUMO
A targeted yeast artificial chromosome (YAC) library for chromosome 11 has been constructed from the J1 cell line that carries a single human chromosome 11 within a hamster DNA background. Interspecies chimeric clones generated during construction of the library were detected during the screening process and eliminated from the library. Contig assembly becomes much less difficult using such a library as the complexity is decreased and the ends of the clone inserts can be rescued for walking to neighboring clones. The library contains > 1824 clones with an average insert length of 337 kb. This represents a fourfold coverage of chromosome 11 or a > 95% chance of recovering a unique single-copy sequence from the library. Two hundred YAC clones were localized by fluorescence in situ hybridization and found to be randomly distributed along the chromosome. The library has been screened with probes for the chromosome 11 markers HBB, GLUR4, H19, and D11S193. Corresponding YAC clones have been isolated for each locus. This analysis has indicated that the library is unbiased, that cognate YAC clones can be recovered with chromosome 11 markers, and that extensive contig assembly should be feasible.
Assuntos
Cromossomos Fúngicos , Cromossomos Humanos Par 11 , Biblioteca Gênica , Genoma Humano , Animais , Southern Blotting , Linhagem Celular , Bandeamento Cromossômico , Clonagem Molecular , Cricetinae , Humanos , Hibridização In SituRESUMO
Translocations involving 11q23 have been shown to be a consistent finding in human hematopoietic malignancies and in some constitutional abnormalities. The identification of a gene, MLL (myeloid/lymphoid or mixed-lineage leukemia), that spans the breakpoints in four different recurrent 11q23 translocations was recently reported. We describe a rare (11;12)(q23;p13) translocation, observed in leukemic cells from a patient with acute lymphoblastic leukemia, which also disrupts this gene.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 12 , Genes , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Southern Blotting , Medula Óssea/patologia , Pré-Escolar , Bandeamento Cromossômico , Mapeamento Cromossômico , Humanos , Células Híbridas , Cariotipagem , Masculino , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Mapeamento por RestriçãoRESUMO
The human beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1) (SiaT-1) gene is localized to human chromosome 3 (q21-q28) by Southern analysis of somatic cell hybrids and by in situ hybridization of metaphase chromosomes. Comparative analysis between the human and the previously reported rat SiaT-1 genomic sequences demonstrates precise conservation of the intron/exon boundaries throughout the coding domains. Furthermore, there is extensive inter-species sequence similarity in some of the exons that contain information only for the 5'-leader regions. Human genomic sequences were also analyzed to reconcile reported differences in the 5'-untranslated region in SiaT-1 mRNAs. In cultured cell lines of the B-lineage, Reh, Nalm-6, Jok-1, Ball-1, Daudi, and Louckes, the study demonstrates that three upstream exons, Exons(Y+Z) and Exon(X), are mutually exclusively utilized, resulting in at least two distinct populations of SiaT-1 mRNA being synthesized. None of these exons is present in the SiaT-1 mRNA isotype expressed in HepG2 human hepatoma cells. In all B-lymphoblastoid cell lines examined, the basal level SiaT-1 mRNA is maintained by the expression of an isotype containing the Exons(Y+Z) sequence. The slightly smaller SiaT-1 mRNA, which contains the Exon(X) sequence but not Exons(Y+Z) sequence, is synthesized at a high level and found only in Jok-1, Daudi, and Louckes, the cell lines with mature B-cell phenotype. The study also provides further evidence that induced SiaT-1 expression accompanies the appearance of CDw75, a putatively sialylated cell surface epitope and a marker of human mature B-lymphocytes. The SiaT-1 induction is the result of the appearance of a novel form of SiaT-1 mRNA isotype.
Assuntos
Linfócitos B/metabolismo , Cromossomos Humanos Par 3 , Éxons , Sialiltransferases/genética , Animais , Antígenos CD/genética , Sequência de Bases , Células Cultivadas , Mapeamento Cromossômico , DNA , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
It has been established that nonrandom chromosome rearrangements are characteristic of specific types of neoplasia. We present six new cases of sarcoma that had in common the same chromosome abnormality, i.e., a balanced translocation between chromosomes X and 18, t(X;18)(p11.2;q11.2), and evaluate the 15 cases with this translocation in the literature. The histological diagnosis was synovial sarcoma in 19 cases and malignant fibrous histiocytoma and fibrosarcoma in the remaining two tumors, respectively. The translocation was found in tumors of both the biphasic and monophasic types, as well as in poorly differentiated synovial sarcoma. The two nonsynovial sarcomas with the t(X;18) were described as spindle cell tumors but failed to show the presence of cytokeratins by immunohistochemical stains. Even with the numerous variabilities on which this test depends, the cytogenetic analysis holds great promise as a tool for the diagnosis of synovial sarcoma.
Assuntos
Cromossomos Humanos Par 18 , Sarcoma Sinovial/genética , Neoplasias de Tecidos Moles/genética , Translocação Genética/genética , Cromossomo X , Adulto , Feminino , Humanos , Queratinas/análise , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Sarcoma Sinovial/patologia , Neoplasias de Tecidos Moles/patologiaRESUMO
We have used panels of somatic cell hybrids and fluorescent in situ hybridization to determine the chromosomal localization of the novel nontransmembrane tyrosine phosphatase PTPN6 (protein tyrosine phosphatase, nonreceptor type 6), which contains two SH2 domains. PTPN6 maps to 12p13, a region commonly involved in leukemia-associated chromosomal abnormalities. Since PTPN6 is expressed at high levels in hematopoietic cells of all lineages and its expression is induced early in hematopoietic differentiation, altered expression and/or structure of PTPN6 may play a role in leukemogenesis.
Assuntos
Cromossomos Humanos Par 12 , Proteínas Tirosina Fosfatases/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA/genética , Humanos , Células Híbridas , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Proteínas Tirosina Fosfatases/química , Compostos de Sulfidrila/químicaRESUMO
Type V collagen is a fibrillar collagen that is widely distributed in tissues as a minor component of extracellular matrix and is usually composed of one pro alpha 2 (V) and two pro alpha 1 (V) chains. In this report, recently isolated cDNA and genomic clones, which encode the pro alpha 1 (V) chain, are used as probes for hybridization to filter-bound DNA from a panel of human-mouse hybrid cell lines and for in situ hybridization to metaphase chromosomes. These studies establish the chromosomal location of the COL5A1 gene, which encodes the pro alpha 1 (V) chain, within segment 9q34.2----q34.3. These findings add to the previously characterized dispersion of collagen genes in the human genome, as this is the first example of a collagen locus on chromosome 9. In addition, these studies place COL5A1 near the locus for the genetic disorder, nail-patella syndrome (hereditary osteo-onychodysplasia), which also maps to 9q34.
Assuntos
Cromossomos Humanos Par 9 , Colágeno/genética , Síndrome da Unha-Patela/genética , Mapeamento Cromossômico , DNA/genética , Sondas de DNA , HumanosRESUMO
KDR (kinase insert domain receptor), a new type III receptor tyrosine kinase gene, maps to human chromosome 4q31.2----q32 by fluorescence in situ hybridization. This differs from the chromosomal locations of other members of this gene family.
Assuntos
Cromossomos Humanos Par 4 , Proteínas Tirosina Quinases/genética , Receptores de Superfície Celular/genética , Mapeamento Cromossômico , Fluorescência , Substâncias de Crescimento , Humanos , Hibridização de Ácido NucleicoRESUMO
The q13 to q15 region of human chromosome 12 is frequently and consistently rearranged in malignant and benign adipose tissue tumors as well as benign tumors of smooth muscle and salivary glands. A reciprocal translocation, (12;16) (q13;p11), is characteristic of the myxoid subtype of liposarcoma, whereas translocations within 12q13-14 are frequently observed in benign lipomas. We are using pulsed-field gel electrophoresis to study the 12q13-q14 region in order to detect and clone the respective translocation breakpoints in these tumors. The locus GLI, which encodes a zinc-finger protein, has been mapped to the same region as the myxoid liposarcoma breakpoint. Pulsed-field analysis of myxoid liposarcoma and lipoma DNA has allowed us to construct a 600-kilobase physical map surrounding the GLI locus, which shows that breakpoints in both types of tumor are outside this region. However, myxoid liposarcoma DNA samples contained altered restriction fragments detectable with GLI probes that were highly specific and reproducible from case to case. These altered fragments are due to highly specific and reproducible methylation differences that are unique to myxoid liposarcoma DNA. These methylation changes may prove to be useful clinically as a diagnostic tool to differentiate subtypes of liposarcoma.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 16 , Genes , Lipossarcoma/genética , Translocação Genética , Dedos de Zinco , Linhagem Celular , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Humanos , Metilação , Mapeamento por RestriçãoRESUMO
To determine the cytogenetic origin of maturing granulocytes in the bone marrow of patients with acute myelogenous leukemia, bone marrow cells were studied using a modified cytogenetic technique, which does not disrupt the cell membrane, in conjunction with periodic acid-Schiff (PAS) staining. In four cases successfully studied, myeloblasts were PAS-negative and granulocytes were PAS-positive. In three cases successfully studied following 0-2 days of culture, metaphase spreads with abnormal karyotypes characteristic of the patients' leukemic clones were seen in five of five, six of nine, and four of four PAS-positive cells successfully studied. These patients' bone marrows were AN, AA, and AA, respectively, by standard cytogenetic study. Therefore, the cytogenetic status of PAS-positive cells did not necessarily correlate with presence or absence of normal metaphases determined by standard cytogenetic study. Bone marrow cells which underwent full and partial granulocytic maturation in suspension culture were studied following 2 weeks of culture. Abnormal karyotypes were seen in five of five and two of two metaphases in PAS-positive cells successfully studied in two patients. Therefore, we have demonstrated that when acute myelogenous leukemia cells undergo myeloid maturation in culture, the mature cells may be definitely proven to derive from leukemic progenitors rather than from normal stem cells.
Assuntos
Células da Medula Óssea , Granulócitos/fisiologia , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Medula Óssea/fisiologia , Células Cultivadas , Aberrações Cromossômicas/patologia , Transtornos Cromossômicos , Feminino , Humanos , Cariotipagem , Masculino , Pessoa de Meia-Idade , Mitose/fisiologia , Reação do Ácido Periódico de Schiff , Coloração e Rotulagem , Fatores de TempoRESUMO
This study was designed to determine if HL-60 cells could undergo one or more cycles of DNA synthesis despite containing 3H-cytosine arabinoside (3HaraC) in their genome. HL-60 cells were incubated with 3HaraC for 2 hours, washed and maintained in a medium containing bromodeoxyuridine (BrdU). At fixed time points, cells were arrested in metaphase and prepared for chromosomal analysis. Treatment of the sample by an immunofluorescent monoclonal anti-BrdU antibody allowed us to determine the differential fluorescent pattern of sister chromatids in metaphase cells that had undergone two or more rounds of DNA synthesis in the presence of BrdU. Processing the samples by autoradiography demonstrated the presence of black grains (3HaraC) overlying the chromosomes. Thus, we were able to examine each metaphase for the presence of 3HaraC as well as the number of cycles it had completed in the presence of BrdU. We showed that despite the presence of 3HaraC in their DNA, some HL-60 cells were able to undergo two or more complete rounds of DNA replication.
Assuntos
Ciclo Celular/efeitos dos fármacos , Citarabina/farmacologia , Troca de Cromátide Irmã/efeitos dos fármacos , Autorradiografia , Linhagem Celular , DNA/biossíntese , Humanos , Cariotipagem , Coloração e RotulagemRESUMO
Trisomy of chromosome #8 is a change commonly associated with acute nonlymphocytic leukemia (ANLL). However, tetrasomy of chromosome #8 in ANLL as a single chromosome change without accompanying abnormalities is extremely rare and, to the best of our knowledge, has not been reported to date. We present here a case of acute monocytic leukemia (AMoL, FAB M5b) with four copies of chromosome #8.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 8 , Leucemia/genética , Doença Aguda , Adulto , Bandeamento Cromossômico , Humanos , Cariotipagem , MasculinoRESUMO
Changes affecting the Y chromosome in neoplasia have been documented. However, this usually occurs as a loss of the Y chromosome in the affected cells accompanied by or preceding other karyotypic changes. Involvement of the Y chromosome in a translocation is extremely rare, there being only 14 cases reported in the literature. Presented here is a case of t(Y;18) in acute myeloblastic leukemia type M2 (FAB).