RESUMEN
In an effort to elucidate the biological role played by cyclin D1, a candidate BCL-1 oncogene, in human B-cell tumours carrying the t(11;14) translocation, we have studied the properties of this cyclin protein in a series of human lymphoid lines with rearrangements in the BCL-1 locus. The BCL-1/cyclin D1 protein was easily detectable in both immunocytochemistry and immunoblotting, its abundance grossly correlating with the mRNA levels. The cyclin D1 protein was localised predominantly to nuclei and there was a striking variation of staining intensity among the exponentially growing cells, reflecting the maximum level reached in mid/late G1 and the lowest level in S-phase. This characteristic mode of cell cycle-dependent oscillation was confirmed by three independent approaches, demonstrating that even upon rearrangement, the expression of cyclin D1 is regulated in a cyclical manner. Antibody-mediated and anti-sense oligonucleotide 'knockout' experiments revealed that the aberrantly expressed BCL-1/cyclin D1 protein is required for G1 phase progression of all four B-cell tumours with the BCL-1 rearrangement. Consistent with the proposed oncogenic role of this cyclin, our data demonstrate that the BCL-1 deregulation caused by chromosomal rearrangement leads to expression of a functionally active cyclin D1 protein which subverts the G1 phase control in the human B-cell tumours carrying the t(11;14) translocation.
Asunto(s)
Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Ciclinas/fisiología , Leucemia de Células B/genética , Linfoma de Células B/genética , Proteínas Oncogénicas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Translocación Genética , Secuencia de Bases , Ciclina D1 , Fase G1 , Humanos , Leucemia de Células B/patología , Linfoma de Células B/patología , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
We report the molecular cytogenetic analysis of a case of Philadelphia (Ph)-negative, BCR-positive chronic myeloid leukemia (CML) which appeared by conventional cytogenetics to have a t(6;9)(p23;q34) as the sole cytogenetic abnormality. Neither conventional nor pulse-field Southern blots detected any rearrangement of the DEK or CAN genes which are often fused in acute myeloid leukemia (AML) with t(6;9)(p23;q34). However, rearrangements of both BCR and ABL genes were detected. The breakpoint in BCR was located in the major translocation cluster region between exons b1 and b3. ABL rearrangements were detected with an ABL exon 1B probe and with a probe located 5' of the entire ABL gene. Comigration between the rearranged fragments obtained with M-bcr-5' and ABL exon 1B probes was observed, implying that the entire ABL gene was fused to the 5' part of the BCR gene. Fluorescence in situ hybridization (FISH) analyses using BCR and ABL probes showed that in 20% of metaphases BCR and ABL signals were present on one chromosome 6 at 6p23, whilst in 80% of metaphases BCR and ABL signals were identified on both copies of chromosome 6. Furthermore, FISH analysis with a whole-chromosome 22 paint demonstrated that chromosome 22 material was present on both copies of chromosome 6. These data indicate a complex Philadelphia translocation involving chromosome band 6p23 and duplication of the whole aberrant chromosome. The nature of the gene locus on 6p23, involved in this rearrangement, remains unknown. A similar translocation has been previously reported in a case of CML, which also lacked DEK and CAN gene rearrangements implying that abnormalities of 6p23 involving genes other than DEK may be a recurrent abnormality in CML.
Asunto(s)
Cromosomas Humanos Par 6 , Cromosomas Humanos Par 9 , Reordenamiento Génico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Oncogenes , Translocación Genética , Southern Blotting , Mapeo Cromosómico , Citogenética , Exones , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
Chromosome 11q23 is frequently a site of chromosomal translocation in both acute leukemias and chronic lymphoproliferative disorders. In the former, an 8 kb region within the MLL gene is consistently involved, whereas in the latter breakpoints appear to be heterogeneous. In a B cell acute leukemia cell line with t(14;18)(q32.3;q21.3) we have previously demonstrated a reciprocal translocation between the LAZ3/BCL6 gene at 3q27 and the B cell specific transcriptional coactivator gene BOB-1 at 11q23.1, implicating BOB-1 as a potential proto-oncogene. To confirm the chromosomal localization of BOB-1 we have mapped it by FISH to 11q23.1. It lay immediately telomeric of the ATM gene. We have also investigated the frequency of BOB-1 rearrangements in a panel of 32 cell lines and 71 patient samples. In one case of T cell prolymphocytic leukemia-a disease where 11q23 abnormalities are observed-a chromosomal rearrangement was identified 3.3-0.9 kb centromeric of the 3' end of the gene. Thus, there is a heterogeneity of breakpoints associated with BOB-1 while the frequency of the gene's involvement in lymphoproliferative diseases is low.
Asunto(s)
Trastornos Linfoproliferativos/genética , Transactivadores/genética , Secuencia de Bases , Cromosomas Humanos Par 11 , Sondas de ADN , ADN de Neoplasias/genética , Exones , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Leucemia Prolinfocítica/genética , Leucemia-Linfoma de Células T del Adulto/genética , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Células Tumorales CultivadasRESUMEN
Abnormalities of several cell-cycle regulatory genes including cyclin D1, p16CDKN2 and p15CDKN2B have been described in B cell non-Hodgkin's lymphoma (B-NHL). We describe a new B-NHL cell line (Granta 519), with concurrent abnormalities of the cyclin D1, pl6CDKN2 and pl5CDKN2B genes. An independent clinical case of mantle cell NHL (Mc-NHL) with concomitant overexpression of cyclin D1, and deletion of the p16CDKN2 gene was also identified, suggesting that this combination of oncogenic aberration is a pathophysiologic contribution to a subset of NHL cases. More in-depth functional studies of this concept were facilitated by the availability of the cell line Granta 519 which was derived from a case of high-grade NHL and has a mature B cell immunophenotype. Cytogenetic analysis identified translocation t(11;14)(q13;q32) and complex rearrangements involving chromosomes 9p22, 13p21, 17pl1, and 18q21. Molecular analysis identified overexpression of cyclin D1 mRNA and biallelic deletion of the p16CDKN2 and p15CDKN2B genes. To elucidate the effect of these genetic abnormalities on the G1 control of Granta 519 cells, the level and function of the major components of the cyclinD/retinoblastoma (RB) pathway were investigated. Cyclin D1 was dominant among the D-type cyclins, formed abundant complexes with cyclin-dependent kinase (Cdk) Cdk4 rather than Cdk6, and the immunoprecipitated cyclin D1/Cdk4 holoenzyme was active as a pRB kinase. Electroporation of wild-type pl6CDKN2 arrested the Granta 519 cells in G1, consistent with the p16CDKN2 loss as a biologically relevant event during multistep evolution of the tumor, and with the expression of functional pRB. Direct cooperation of these distinct abnormalities to cell-cycle, deregulation in NHL cells was suggested by G1 acceleration upon inducible overexpression of cyclin D1 in a control breast cancer cell line lacking p16CDKN2, an effect which could be prevented by ectopic expression of p16CDKN2. Taken together, these data suggest that concurrent overexpression of cyclin D1 and functional elimination of p16CDKN2 and p15CDKN2B may characterize certain cases of mantle cell NHL, and that cooperation of the abnormalities is likely to provide a growth advantage of the tumour cells through more efficient inactivation of the RB tumor suppressor. Further clinicopathologic studies of this possibility are warranted.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Ciclinas/metabolismo , Eliminación de Gen , Linfoma de Células B/genética , Proteínas de Neoplasias/metabolismo , Proteínas Oncogénicas/metabolismo , Translocación Genética/genética , Proteínas Supresoras de Tumor , Anciano , Anciano de 80 o más Años , Proteínas Portadoras/genética , Ciclina D1 , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Ciclinas/genética , Genes Supresores de Tumor , Humanos , Inmunofenotipificación , Cariotipificación , Proteínas de Neoplasias/genética , Proteínas Oncogénicas/genética , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
The BCL7A gene, which maps to human chromosome 12q24.13, was cloned through its direct involvement with MYC and IGH in a three-way translocation in a Burkitt lymphoma cell line. Here, we describe the identification of two related human genes, BCL7B and BCL7C, which share 90% identity to the amino-terminal 51 amino acids of human BCL7A, as well as 41% identity in the same region to Drosophila melanogaster, Caenorhabditis elegans, and Brugia malayi EST sequences. This degree of relatedness in the amino-terminal domain suggests we have defined a new gene family of unknown function. There was little sequence conservation between the family members outside this conserved domain and no identified protein motifs could be deduced. Human BCL7B and BCL7C mapped to chromosome 7q11.23, and 16p11, respectively. No chromosomal rearrangements affecting BCL7B or BCL7C were detected in lymphoid malignancies. BCL7B did, however, map within the region of 7q11.23 which is commonly deleted in the congenital disorder, Williams syndrome.
Asunto(s)
Sondas de ADN/genética , Proteínas de Neoplasias , Proteínas , Síndrome de Williams/genética , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Brugia/química , Brugia/genética , Mapeo Cromosómico , Cromosomas Humanos Par 16/genética , Cromosomas Humanos Par 7/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Expresión Génica , Genes/genética , Humanos , Hibridación Fluorescente in Situ , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
The translocation (11;14)(q13;q32) and its molecular counterpart the BCL-1 rearrangement are features observed in mantle cell lymphoma (MCL) and less commonly in other B-cell disorders. This rearrangement leads to cyclin D1 overexpression, which may be the main pathogenic event in these tumours and is therefore recognised as a diagnostic marker. We developed a flow cytometry method to detect cyclin D1 overexpression using the monoclonal antibody (MoAb) 5D4, and characterised its frequency in 93 B-cell malignancies. The competitive reverse transcriptase polymerase chain reaction (RT-PCR) for cyclin D1, D2 and D3 was then performed on 40 of these cases to assess the validity of the flow cytometry method. Fluorescence in situ hybridisation (FISH) to detect t(11;14)(q13;q32) was carried out on 31 cases and results were compared with cyclin D1 expression by flow cytometry. Twenty five cases showed cyclin D1 expression using 5D4, including MCL (12/13, 92%), chronic lymphocytic leukaemia (CLL) (4/30), B-prolymphocytic leukaemia (B-PLL) (1/4), splenic lymphoma with villous lymphocytes (SLVL) (4/13), hairy cell leukaemia (HCL) (1/7) and other B-non Hodgkins Lymphoma (B-NHL) (3/15). There was a good correlation between flow cytometry results and RT-PCR in 36/40 cases (90%), and with FISH for t(11;14) in 25/31 cases (80%). We concluded that the detection of cyclin D1 expression by flow cytometry in cell suspensions could be applied routinely to the study of B-lymphoproliferative disorders and may be of value for their diagnosis and management.
Asunto(s)
Biomarcadores de Tumor/análisis , Ciclina D1/análisis , Leucemia de Células B/diagnóstico , Linfoma de Células B/diagnóstico , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Leucemia de Células B/genética , Linfoma de Células B/genética , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Translocación GenéticaRESUMEN
UNLABELLED: Indacaterol is a novel, inhaled, long-acting ß(2)-agonist providing 24-h bronchodilation with once-daily (o.d.) dosing in patients with COPD. In this double-blind, incomplete block crossover study, patients with moderate-to-severe COPD were randomised to receive three treatment cycles from: indacaterol 300 µg o.d. dosed PM or AM, salmeterol 50 µg twice daily or placebo, each for 14 days. Trough FEV(1) was measured 24 h after indacaterol, and 12 h after salmeterol. Ninety-six patients (mean age: 64 years; post-bronchodilator FEV(1) 57% predicted, FEV(1)/FVC 55%) were randomised; 83 completed. After 14 days, the difference vs. placebo in trough FEV(1) for PM indacaterol was 200 mL (p < 0.001 [primary analysis]) and for AM indacaterol was 200 mL (p < 0.001). Compared with salmeterol, trough FEV(1) for PM indacaterol was 110 mL higher (p < 0.001), and for AM indacaterol was 50 mL higher (p = NS). Over 14 days, vs. placebo, both PM and AM indacaterol improved the % of nights with no awakenings (by 11.9 and 8.1 points; p < 0.01); the % of days with no daytime symptoms (by 6.7 and 5.5 points; p < 0.05); and the % of days able to perform usual activities (by 6.7 and 7.8 points; p < 0.05). Indacaterol provided 24-h bronchodilation and improvement in symptoms regardless of whether taken regularly in the morning or evening. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT00615030.
Asunto(s)
Agonistas Adrenérgicos beta/administración & dosificación , Indanos/administración & dosificación , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Quinolonas/administración & dosificación , Administración por Inhalación , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Volumen Espiratorio Forzado/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Factores de TiempoRESUMEN
We have investigated an extended pedigree with three cousins affected by Duchenne muscular dystrophy with apparent transmission through the male line. However, molecular studies have shown that one boy has a de novo duplication, another has a deletion, and the molecular mutation has yet to be defined in the third boy. All three X chromosomes in the affected boys appear to have a different origin. We speculate on the mechanisms by which the Duchenne locus may be particularly prone to mutation in this family and the possible involvement of transposons is discussed. Whatever the mechanism involved, the occurrence of three different mutations in one pedigree is a rare event.
Asunto(s)
Distrofias Musculares/genética , Mutación/genética , Adolescente , Niño , Preescolar , Deleción Cromosómica , ADN , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Masculino , Familia de Multigenes , Distrofias Musculares/etiología , Distrofias Musculares/fisiopatología , Linaje , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The translocation t(11;14)(q13;q32) occurs in about 15% of patients with splenic lymphoma with villous lymphocytes (SLVL) or the closely related disorder lymphoplasmacytic lymphoma (LPL). To characterize the nature and frequency of rearrangements of the BCL-1 locus in SLVL/LPL and to document the effect of these genetic alterations on the expression of the cyclin D1 gene, we analyzed 22 cases of SLVL/LPL with defined cytogenetic abnormalities by both conventional electrophoresis (CE) and pulse-field gel electrophoresis (PFGE) and by Northern blotting. Four SLVL/LPL cases showed rearrangement of the BCL-1 locus. In two cases with t(11;14)(q13;q32), different breakpoints were identified; one mapped adjacent to the major translocation cluster (MTC) and the other within a 28-kb region telomeric of it. In a third case of SLVL with no cytogenetic abnormality of 11q13, a novel breakpoint approximately 100 kb centromeric of MTC was detected by PFGE. The fourth case, which had a normal karyotype, demonstrated rearrangement with a BCL-1 probe immediately telomeric of MTC. This case may have had a small deletion of 0.5 kb from within the BCL-1 locus. No rearrangement of the BCL-1 locus or within the cyclin D1 gene was detected by CE or PFGE in any of the remaining 18 SLVL/LPL samples. Northern blot analysis showed expression of a normal-sized cyclin D1 transcript in the two SLVL/LPL cases with t(11;14)(q13;q32). In cases that lacked a cytogenetically demonstrable t(11;14) translocation, no cyclin D1 transcript was detected. Analysis of the BCL-1 locus was also performed in three other cases of B-cell disorders with t(11;14)(q13;q32) detected cytogenetically. Two cases were analyzed by Southern blot and showed rearrangement of the BCL-1 locus. Expression of high-level normal-sized and/or truncated cyclin D1 transcript was also detected in these cases. These data show the importance of PFGE in the detection of rearrangements in the BCL-1 locus and show further the complexity of rearrangements in this locus.
Asunto(s)
Ciclinas/genética , Reordenamiento Génico , Linfoma/genética , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Neoplasias del Bazo/genética , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Ciclina D1 , Genes de Inmunoglobulinas , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Linfocitos/patología , Linfoma/patología , Neoplasias del Bazo/patología , Translocación GenéticaRESUMEN
BACKGROUND: The role of apoptosis (programmed cell death) in the development and progression of breast cancer is unknown. Recently the bcl-2 gene has been shown to block apoptosis and thus may promote tumour development. BCL-2 is localized to the luminal cells of the normal breast, which are considered to be the origin of malignant breast disease. PATIENTS AND METHODS: Immunocytochemistry using anti bcl-2- antibody was performed on 107 breast cancer specimens belonging to node-positive patients from the Ludwig Breast Cancer Studies I-IV and the results were correlated with survival, tumour grade, S-phase, oestrogen and progesterone receptor status and c-erb B-2 expression. Western and Southern blotting together with immunofluorescence were performed on the breast cancer cell lines BT-20, BT-474, MDA-MB-361, T47-D and MCF-7. RESULTS: In the breast cancer derived cell line MCF-7 BCL-2 is expressed to a level similar to that of the B-lymphoma cell line Karpas 231 with t(14;18)(q32.3;q21.3), but no evidence of a rearrangement or gene amplification was identified. In a study of 107 breast cancers from the International Breast Cancer Study Group Trials I-IV we have demonstrated a very significant inverse correlation of BCL-2 with c-erbB-2 expression (p = 0.002), and a positive correlation with oestrogen receptors (p = 0.001) and progesterone receptors (p = 0.05). In this study there was no correlation of expression with S-phase fraction in the tumours or with any stage in the cell cycle as assessed in MCF-7 cells. CONCLUSION: We conclude that BCL-2 might contribute to the malignant phenotype of breast cancer by modulation of biological behaviour of cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Expresión Génica , Proteínas Proto-Oncogénicas/metabolismo , Proto-Oncogenes , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Southern Blotting , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Receptores ErbB/metabolismo , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Fenotipo , Pronóstico , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Receptor ErbB-2 , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
A B-cell non-Hodgkin's lymphoma (B-NHL) cell line (Karpas 1106) with an unusual three-way translocation involving 18q21.3 has been derived from a patient with mediastinal lymphoblastic B-NHL. Although conventional cytogenetics showed a derivative 18q-identical to that seen in cases with t(14;18)(q32.3;q21.3), no translocations of either chromosome 14 could be detected. Instead fluorescent in situ hybridization analysis using a chromosome-18 paint showed that the segment 18q21.3-18qter had become sandwiched on a derivative chromosome X between segments Xqter-c-Xq28 and 13q12-qter, with the centrometric site of 18q21.3 subband juxtaposed to the X sequences. Pulsed-field DNA blots failed to detect rearrangement of the BCL2 gene. Conventional DNA blots using a variety of restriction digests and both 5' and 3' BCL2 and FVT 1 probes also failed to detect rearrangement in Karpas 1106. A rearranged fragment seen only in HindIII digests with 5' BLC2 probes may represent a local microalteration, which is either a mutation or small deletion involving the HindIII site as seen in other cases of B-NHL. Neither BCL2 RNA nor BCL2 protein expression were detected. These and other data suggest that genes at 18q21.3, other than BCL2 and FVT1, may be targets for translocation in certain subgroups of B-NHL.
Asunto(s)
Cromosomas Humanos Par 18 , Expresión Génica , Reordenamiento Génico , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas/genética , Translocación Genética , Adulto , Alelos , Antígenos CD/análisis , Antígenos CD/biosíntesis , Northern Blotting , Línea Celular , Deleción Cromosómica , Mapeo Cromosómico , Femenino , Citometría de Flujo , Proteínas de Unión al GTP/genética , Genotipo , Humanos , Inmunoglobulinas/análisis , Inmunoglobulinas/biosíntesis , Inmunofenotipificación , Hibridación Fluorescente in Situ , Linfoma de Células B/inmunología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2 , ARN Mensajero/análisis , Mapeo Restrictivo , Células Tumorales CultivadasRESUMEN
Von Recklinghausen neurofibromatosis (NF-1) is a common autosomal dominant disorder. The estimated new mutation rate (1 x 10(-4] is one of the highest for a human disorder. Here we report that in 12 of 14 families we have analysed, the new mutation is of paternal origin. This result is similar to that recently obtained for retinoblastoma. In other genetic disorders that show a bias towards paternal origin of new mutations, there is a marked increase in the incidence of mutations with paternal age, consistent with the mutations arising from replication errors in mitosis of spermatogonial stem cells. In retinoblastoma and NF-1, however, such paternal age effects are slight or absent. The mechanism or timing of germline mutation could therefore be different in the two cases.
Asunto(s)
Mutación , Neurofibromatosis 1/genética , Adulto , ADN/genética , Humanos , Masculino , Mitosis , Linaje , Espermatozoides/ultraestructuraRESUMEN
Clonal rearrangements of the Ig heavy chain (IGH) locus consisting of either intrachromosomal (VDJ) rearrangements or interchromosomal translocations are a consistent feature of all B-cell malignancies and may be used both diagnostically and to monitor response to therapy. Many of these rearrangements are targeted to the IGHJ segments, but only some can be amplified with regular polymerase chain reaction (PCR) techniques. To permit PCR amplification of potentially all IGHJ rearrangements, we have devised a method incorporating self-ligation of restriction endonuclease-digested DNA fragments with long-distance PCR (long-distance, inverse PCR [LDI-PCR]). We show here, using only 4 nested oligonucleotide primers, the successful amplification and DNA sequencing of all IGHJ rearrangements up to 5.4 kb in length from a panel of 13 cases and cell lines of various types of B-cell malignancy. In all cases, both VDJ and DJ IGH rearrangements and translocation breakpoints were amplified. Six cases exhibited t(14;18)(q32;q21). All translocation breakpoints were cloned and sequenced. Three cases exhibited a rearrangement to the BCL2 major breakpoint region (MBR). However, 2 other cases exhibited rearrangements between the MBR and the minor cluster region (mcr). These 2 cases broke within 44 bp of each other, confirming the presence of an additional 3' BCL2 breakpoint cluster region. The final case fell immediately 3' of the 3' UTR of the BCL2 gene adjacent to an Alu repeat. No other BCL2 breakpoints within this region have been reported. Four cases exhibited t(11;14)(q13;q32). All 3 cases with translocations targeted to the IGHJ segments were successfully amplified and sequenced, including 1 case in which the BCL1 translocation could not be detected by DNA blot using the currently available probes. All three translocation breakpointsfell outside the BCL1 major translocation cluster between 20 and 40 kb telomeric and showed no clustering. Two of the three fell within or adjacent to Alu repeat regions. LDI-PCR is a simple and robust technique that allows PCR amplification of nearly all IGHJ rearrangements.
Asunto(s)
Aberraciones Cromosómicas/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Reacción en Cadena de la Polimerasa/métodos , Translocación Genética , Secuencia de Bases , Trastornos de los Cromosomas , Clonación Molecular , Ciclina D1 , Genes bcl-2 , Humanos , Cariotipificación , Leucemia de Células B/genética , Linfoma no Hodgkin/genética , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/genética , Mapeo Restrictivo , Células Tumorales CultivadasRESUMEN
Although translocations of the BCL2 gene are frequent in B-cell non-Hodgkin's lymphomas (B-NHL) the incidence, nature, and prognostic significance of similar translocations in the phenotypically related chronic leukemias of mature B cells are unknown. Therefore, we examined 170 cases of B-cell chronic lymphocytic leukemia (B-CLL), 7 cases of B-cell prolymphocytic leukemia (B-PLL), 25 cases of hairy cell leukemia (HCL) and 22 cases of splenic lymphoma with villous lymphocytes (SLVL) with defined cytogenetic abnormalities by DNA blot using both 5' and 3' BCL2 probes to search for rearrangement of the BCL2 locus. Translocation t(14;18) (q32.3;q21.3) was detected cytogenetically in 3 cases of B-CLL. All had breakpoints in the 3' region of BCL2, mapping between the major breakpoint region (MBR) and the minor cluster region (mcr), the breakpoint clusters commonly detected in B-NHL. In 2 of the 3 cases, the breakpoint within BCL2 was mapped to a 1.0-kb EcoRI-HindIII fragment indicating a clustering of breakpoints. Two cases of B-CLL had cytogenetically detectable t(2;18)(p11;q21.3) or t(18;22)(q21.3;q11). Both had rearranged the 5' region of the BCL2 gene to the corresponding lg light-chain gene. Molecular cloning of the t(18;22)(q21.3;q11) showed that the translocation disrupted the BCL2 promoter region and the first untranslated BCL2 exon. Nevertheless, high levels of BCL2 protein were seen in this case. Only 2 other cases in whom cytogenetic analysis was not successful showed rearrangement of the 5' region of BCL2, an overall incidence of 2.3%. No cases of B-PLL, HCL, or SLVL showed either 5' or 3' BCL2 rearrangement. These data confirm the cytogenetic observations that translocations involving the BCL2 locus in all forms of leukemia of mature B cells are rare, and limited to a minor subset of B-CLL. BCL2 translocations in B-CLL involve hot spots of recombination of both the 5' and 3' regions of the BCL2 gene, which are distinct from those commonly seen in B-NHL, suggesting distinct pathogenic mechanisms.
Asunto(s)
Leucemia de Células B/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Translocación Genética , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 18 , Cromosomas Humanos Par 2 , Femenino , Humanos , Linfoma/genética , Masculino , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
Chromosome 12q24.1 is a recurrent breakpoint in high-grade B-cell non-Hodgkin lymphoma (B-NHL). To identify the genes involved at 12q24.1, molecular cloning of a three-way translocation t(8;14;12)(q24.1;q32.3;q24.1) in a Burkitt lymphoma cell line (Wien 133) was performed; all four translocation breakpoints were cloned and sequenced. Analysis of clones encompassing the der(12)(12;14)(q24.1;q32.3) breakpoint showed a CpG island from chromosome 12q24.1 juxtaposed in a tail-to-tail configuration with a productively rearranged Ig VH4-DH-JH5 gene. A total of 4.5 kb of genomic DNA including the CpG island was sequenced and analyzed using gene-identification programs; all three programs identified a potential 92-bp exon within the centromeric boundary of the CpG island. Using this as a probe, an RNA transcript of 3.8 kb, expressed at low levels in a wide variety of normal tissues, was detected. Overlapping cDNA clones were isolated and sequenced. The longest open-reading frame predicted a serine-rich protein of 231 amino acids. This protein, termed BCL7A, exhibited no recognizable protein motifs but showed homology with the actin-binding protein, caldesmon. In Wien 133, the BCL7A breakpoint occurred within the first intron and resulted in a MYC-BCL7A fusion transcript, with exon I of BCL7A being replaced by MYC exon I. The normal, untranslocated allele of BCL7A was also expressed without mutation. One of the 11 other B-NHL cell lines examined with 12q24.1 cytogenetic abnormalities, a mediastinal B-NHL cell line (Karpas 1106), showed biallelic rearrangement within the first intron of BCL7A, which was adjacent to the breakpoint observed in Wien 133. Disruption of the amino-terminus of BCL7A defines a new mechanism in the pathogenesis of a subset of high-grade B-NHL.
Asunto(s)
Linfoma de Burkitt/genética , Proteínas de Unión a Calmodulina/genética , Cromosomas Humanos Par 12/ultraestructura , Cromosomas Humanos Par 14/ultraestructura , Cromosomas Humanos Par 8/ultraestructura , Genes , Proteínas de Microfilamentos/genética , Proteínas Oncogénicas , Secuencia de Aminoácidos , Linfocitos B/patología , Secuencia de Bases , Linfoma de Burkitt/patología , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 8/genética , Clonación Molecular , ADN Complementario/genética , ADN de Neoplasias/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes myc , Humanos , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/patología , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/patología , Proteínas de Fusión Oncogénica/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Nine markers from the pericentromeric region of chromosome 17 were typed in 16 British and five South African families with neurofibromatosis type 1 (NF1). The markers--p17H8, pHHH202, and EW204--were linked to NF1 at recombination fractions less than 1%. No evidence of locus heterogeneity was detected. Inspection of recombinant events in families informative for several markers suggests that the NF1 gene is located between the markers EW301 (cen-p11.2) and EW206 (cen-q12) and possibly distal to pHHH202 (q11.2-q12).
Asunto(s)
Cromosomas Humanos Par 17 , Ligamiento Genético , Marcadores Genéticos , Neurofibromatosis 1/genética , Femenino , Humanos , Masculino , Sudáfrica , Reino UnidoRESUMEN
Recurrent abnormalities of the short arm of chromosome 9, including translocations and interstitial deletions, have been reported in both leukemia and lymphoma. The pathologic consequences of these abnormalities remain unknown. The cyclin-dependent kinase 4 inhibitor (CDKN2) gene, which maps to 9p21, has been implicated by the finding of a high frequency of biallelic deletions in leukemic cell lines. We have determined the incidence of structural abnormalities affecting CDKN2 by DNA blot in a panel of 231 cases of leukemia and lymphoma and 66 cell lines derived from patients with lymphoid malignancies with defined cytogenetic abnormalities. Structural alterations of CDKN2 were seen in 20 (8.3%) of all fresh cases and 10 (15.1%) of all cell lines. Biallelic CDKN2 deletions were seen in 11 of 53 (21%) cases of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There was no association with any particular cytogenetic abnormality. Biallelic deletions were also found in high-grade and transformed non-Hodgkin's lymphoma (NHL) of both B- and T-cell lineages. In two cases of transformed NHL, analysis of sequential samples showed loss of CDKN2 with transformation. Neither deletions nor rearrangements of the CDKN2 gene were seen in any of the 119 leukemias of mature B or T cells analyzed. Biallelic deletions of CDKN2 were observed in 6 of 13 NHL cell lines. Three of the 6 cases had undergone transformation from low- to high-grade disease: in 2 of these cases it was possible to show that the CDKN2 deletions were present in fresh material from the patient and were therefore not an artifact of in vitro culture. Rearrangements of CDKN2 were seen in 2 cases (4%) of BCP-ALL, in 1 case of B-NHL, and in 1 Burkitt's lymphoma cell line and suggest the presence of a "hot spot" for recombination in the vicinity of the CDKN2 gene. These data indicate that the loss of CDKN2 expression may be involved in the pathogenesis of a subset of BCP-ALL, some high-grade NHL, and in the transformation of NHL from low- to high-grade disease. CDKN2 deletions and rearrangements occurred in the absence of detectable cytogenetic changes of chromosome 9p in 25 of 30 (83%) cases. Finally, of 10 cases of BCP-ALL that produced overt, transplantable leukemia in mice with severe combined immunodeficiency (SCID), seven showed biallelic CDKN2 deletions. In contrast, none of 11 cases that failed to engraft showed biallelic CDKN2 deletions. BCP-ALL cases that lack CDKN2 expression may have a particular propensity to grow in SCID mice.
Asunto(s)
Proteínas Portadoras/genética , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Cromosomas Humanos Par 9 , Eliminación de Gen , Reordenamiento Génico , Leucemia/genética , Linfoma/genética , Inhibidores de Proteínas Quinasas , Adolescente , Adulto , Anciano , Animales , Proteínas Portadoras/biosíntesis , Línea Celular , Niño , Preescolar , Mapeo Cromosómico , Cromosomas Humanos Par 8 , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Exones , Femenino , Humanos , Leucemia/enzimología , Linfoma/enzimología , Masculino , Ratones , Ratones SCID , Mapeo Restrictivo , Translocación Genética , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Abnormalities of chromosome 1q21 are common in B-cell malignancies and have been associated with a poor response to therapy. The nature of the involved gene(s) on chromosome 1q21 remains unknown. A cell line (CEMO-1) has recently been established from a patient with precursor-B-cell acute lymphoblastic leukemia (ALL), which exhibited a t(1;14)(q21;q32). To identify the gene involved in this translocation, we have cloned both rearranged IGHJ alleles using long-distance inverse polymerase chain reaction (LDI-PCR). Two IGHJ fragments were amplified from CEMO-1 DNA and sequenced. One allele showed novel sequences upstream of JH5 with no homology to either IGH or any other sequences on the databases. Using a single-copy Xho I fragment immediately 5' of JH5, PAC clones were isolated and mapped to chromosome 1q21 on normal metaphases by fluorescence in situ hybridization (FISH), confirming that this allele represented the t(1;14)(q21;q32) breakpoint. Sequence analysis of the 1q21 Xho I fragment showed identity with an expressed sequence tag (EST), and this probe was therefore used to probe Northern blots. Two transcripts of 6.3 kb and 4.2 kb expressed at low level in mRNA from all tissues were detected: a third transcript of 1.6 kb was expressed only in thymus, spleen, and small intestine. Full-length BCL9 cDNA clones were obtained from a normal human fetal brain cDNA library supplemented by 5' and 3' RACE. Sequence analysis predicted a protein of 1394 amino acids containing 18% proline, 11% glycine, 11% serine, and 6% methionine, but no recognizable protein motifs or significant homologies to any other known proteins. The CEMO-1 1q21 breakpoint fell within the 3' UTR of the BCL9 gene. Low-level expression of BCL9 was detected in Epstein-Barr virus-transformed normal B cells by Northern blot; in contrast, abundant BCL9 expression was observed in CEMO-1, indicating that deregulated expression of this gene was one pathological consequence of the translocation. Screening of a panel of 39 B-cell malignancies with 1q abnormalities by Southern blot showed one additional case with a breakpoint in the 3' UTR of BCL9, indicating that this was a recurrent breakpoint. FISH analysis using an 850-kb YAC spanning BCL9 identified a further case with t(1;22)(q21;q11) causing juxtaposition of BCL9 to the IGlambda locus. Other breakpoints were heterogeneous, falling both centromeric (10 cases) and telomeric (10 cases) of the BCL9 gene. These data suggest that BCL9 may be the target of translocation in some B-cell malignancies with abnormalities of 1q21 and that deregulated BCL9 expression may be important in their pathogenesis.
Asunto(s)
Cromosomas Humanos Par 14/ultraestructura , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 1/ultraestructura , Genes , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Translocación Genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Línea Celular Transformada , Mapeo Cromosómico , Clonación Molecular , ADN de Neoplasias/genética , Regulación Leucémica de la Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Especificidad de Órganos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Factores de TranscripciónRESUMEN
MALT B cell lymphomas with t(1;14)(p22;q32) showed a recurrent breakpoint upstream of the promoter of a novel gene, Bcl10. Bcl10 is a cellular homolog of the equine herpesvirus-2 E10 gene: both contain an amino-terminal caspase recruitment domain (CARD) homologous to that found in several apoptotic molecules. Bcl10 and E10 activated NF-kappaB but caused apoptosis of 293 cells. Bcl10 expressed in a MALT lymphoma exhibited a frameshift mutation resulting in truncation distal to the CARD. Truncated Bcl10 activated NF-kappaB but did not induce apoptosis. Wild-type Bcl10 suppressed transformation, whereas mutant forms had lost this activity and displayed gain-of-function transforming activity. Similar mutations were detected in other tumor types, indicating that Bcl10 may be commonly involved in the pathogenesis of human malignancy.