RESUMEN

The nucleolar Arf protein has been shown to regulate cell cycle through both p53-dependent and -independent pathways. In addition to the well-characterized Arf-mdm2-p53 pathway, several partners of Arf have recently been described that could participate in alternative regulation process. Among those is the nucleolar protein B23/NPM, involved in the sequential maturation of rRNA. p19ARF can interact with B23/NPM in high molecular complexes and partially inhibit the cleavage of the 32S rRNA, whereas the human p14ARF protein has been shown to participate in the degradation of NPM/B23 by the proteasome. These data led to define Arf as a negative regulator of ribosomal RNA maturation. Our recent finding that the human p14ARF protein was able to specifically interact with the rRNA promoter in a p53-independent context, led us to analyse in vitro and in vivo the consequences of this interaction. Luciferase assay and pulse-chase experiments demonstrated that the rRNA transcription was strongly reduced upon p14ARF overexpression. Investigations on potential interactions between p14ARF and the transcription machinery proteins demonstrated that the upstream binding factor (UBF), required for the initiation of the transcriptional complex, was a new partner of the p14ARF protein. We next examined the phosphorylation status of UBF as UBF phosphorylation is required to recruit on the promoter factors involved in the transcriptional complex. Upon p14ARF overexpression, UBF was found hypophosphorylated, thus unable to efficiently recruit the transcription complex. Taken together, these data define a new p53-independent pathway that could regulate cell cycle through the negative control of rRNA transcription.

Asunto(s)

Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , ARN Ribosómico/genética , Transcripción Genética , Proteína p14ARF Supresora de Tumor/metabolismo , Animales , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Factor de Transcripción E2F1/metabolismo , Humanos , Fosforilación , Regiones Promotoras Genéticas , Transfección

RESUMEN

The ARF gene (p19(ARF) in mouse and p14(ARF) in man) has become a central actor of the cell cycle regulation process as it participates to the ARF-MDM2-p53 pathway and the Rb-E2F-1 pathway. By use of immunoprecipitation and Western blotting (IP/WB), we now show that ARF physically associates with topoisomerase I (Topo I). ARF-Topo I immune complexes were detected in SF9 insect cells infected with recombinant baculoviruses encoding the two genes as well as in 293 cells that express endogenously these proteins. Preparations of a GST-ARF recombinant protein stimulated the DNA relaxation activity of Topo I but, in contrast, had no effect on the decatenation activity of Topo II. The Topo I stimulation was also detected in cell extracts of SF9 cells expressing both proteins. A confocal microscopy study indicated that part of ARF and Topo I colocalized in the granular component structure of the nucleolus. As a whole, our data indicate that Topo I is a new partner of ARF and suggest that ARF is involved in cell reactions that require Topo I.

Asunto(s)

Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas/metabolismo , Animales , Baculoviridae/genética , Compartimento Celular , Nucléolo Celular/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Activación Enzimática , Humanos , Conformación de Ácido Nucleico , Unión Proteica , Proteínas/genética , Proteínas Recombinantes/metabolismo , Spodoptera/citología , Proteína p14ARF Supresora de Tumor

RESUMEN

The INK4a/ARF locus which is frequently inactivated in human tumours encodes two different tumour suppressive proteins, p16(INK4a) and ARF. p16(INK4a) is a major component of the RB pathway. ARF is part of an ARF-mdm2-p53 network that exerts a negative control on hyperproliferative signals emanating from oncogenic stimuli. Among these is the transcription factor E2F1, a final effector of the RB pathway, that induces ARF expression. Recent data suggest that ARF function is not restricted to the p53 pathway. However, ARF target(s) implicated in this p53-independent function remains to be identified. We show that ARF is able to inhibit the proliferation of human cell lines independently of their p53 status. In this context, we demonstrate that ARF interacts physically with E2F1 and inhibits its transcriptional activity. Moreover, we show that mdm2 is required for the modulation of E2F1 activity by ARF. Beside the well-known p53 and mdm2 partners, these results identify E2F1 as a new ARF target. Thus, ARF can be viewed as a dual-acting tumour suppressor protein in both the p53 and RB pathways, further emphasizing its role in tumour surveillance.

Asunto(s)

Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/farmacología , Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteínas Nucleares , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/metabolismo , Western Blotting , División Celular/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Ensayo de Unidades Formadoras de Colonias , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Exones/fisiología , Eliminación de Gen , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Humanos , Luciferasas/metabolismo , Mutagénesis/fisiología , Osteosarcoma/genética , Osteosarcoma/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Transfección , Proteína p53 Supresora de Tumor/análisis

RESUMEN

The IPC-81 myeloid leukaemia cells undergo apoptosis rapidly after cAMP stimulation (6 h) and cell death is prevented by early over-expression of the cAMP-inducible transcription repressor ICER, that blocks cAMP-dependent nuclear signalling. Therefore, the expression of specific genes controlled by CRE-containing promoters is likely to determine cell fate. We now show that cAMP-induced cell death also is abrogated by the over-expression of the anti-apoptotic gene, Bcl-2. Contrary to ICER, Bcl-2 does not affect cAMP-signalling and allows the analysis of cAMP responses in death rescued cells. The Bcl-2 transfected cells treated with 8-CPT-cAMP were growth-arrested and thereafter cells embarked in granulocytic differentiation, with no additional stimulation. Neutrophilic polynuclear granulocytes benefited from a long life span in G0-G1 and remained functional (phagocytosis). This work demonstrates that, using anti-apoptosis regulators, 'death signals' could be exploited to trigger distinct biological responses. Indeed, cAMP signal can trigger several simultaneously developing biological programs, in the same cell, i.e., growth regulation, apoptosis and differentiation. This cell system should prove useful to determine how a tumour cell can be re-programmed for either apoptosis or functional maturation by physiological signals.

Asunto(s)

Apoptosis , Diferenciación Celular , Núcleo Celular/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Granulocitos/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Ciclo Celular/fisiología , Fragmentación del ADN , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Regulación de la Expresión Génica , Granulocitos/citología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ratas , Transducción de Señal , Tionucleótidos/farmacología , Transfección , Células Tumorales Cultivadas

RESUMEN

Numerous alternative implants have been proposed for cervical fusion, including bovine bone. We report a case of a two level cervical interbody fusion for degenerative disc disease using bovine dowels of Unilab Surgibonetrade mark. We describe the clinical evolution and histological examination after failure of the procedure due to non-union of the Surgibone implants. Alternative grafting substitutes are discussed with respect to their immunological, cytotoxic and osteoinductive aspects. Based on a literature review we conclude that allografts have a significantly better cytological biocompatibility than Surgibonetrade mark and allow a higher fusion rate. The ideal graft remains autologous bone.

RESUMEN

The cAMP pathway plays a central role in the response to hormonal signals for cell proliferation, differentiation and apoptosis. In IPC-81 leukaemia cells, activation of the cAMP pathway by prostaglandin E1 treatment, or other cAMP-elevating agents, induces apoptosis within 4-6 h. Inhibition of mRNA or protein synthesis during the first 2 h of cAMP induction protects cells from apoptosis, suggesting a requirement for early gene expression. cAMP-dependent protein kinase phosphorylates a class of nuclear factors and thereby regulates the transcription of a specific set of genes. Here we show that CREM (cAMP Responsive Element Modulator) expression is induced rapidly upon prostaglandin E1 treatment of IPC-81 cells. The induced transcripts correspond to the early product ICER (Inducible cAMP Early Repressor). ICER expression remains elevated until the burst of cell death. Protein synthesis inhibitors which prevent cAMP-induced apoptosis also block de novo ICER synthesis. Transfected IPC-81 cell lines, constitutively expressing high level of ICER are resistant to cAMP-induced cell death. In these transfected cells, cAMP fails to upregulate the ICER transcripts demonstrating that ICER exerts strongly its repressor function on CRE-containing genes. That an early expression of ICER blocks apoptosis, suggests that gene repression by endogenous ICER in IPC-81 is insufficient or occurs too late to protect cells against death. ICER transfected cells rescued from cAMP-induced apoptosis are growth arrested. It shows for the first time that CREM activation directly participates to the decision of the cell to die. ICER, by sequentially repressing distinct sets of CRE-containing genes could modulate cell fate.

Asunto(s)

Alprostadil/farmacología , Apoptosis/efectos de los fármacos , AMP Cíclico/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras , Animales , Apoptosis/genética , AMP Cíclico/antagonistas & inhibidores , Modulador del Elemento de Respuesta al AMP Cíclico , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Cicloheximida/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Ratas , Transfección

RESUMEN

Nine KOX zinc finger genes were localized on four human chromosomes by in situ hybridization of cDNA probes to metaphase chromosomes. KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively. Six other KOX genes were localized on chromosome 19: KOX6 (ZNF14) and KOX13 (ZNF20) to 19p13.3-p13.2, KOX5 (ZNF13) and KOX22 (ZNF27) to 19q13.2-qter, and KOX24 (ZNF28) and KOX28 (ZNF30) to 19q13.4. Pulsed field gel electrophoresis experiments showed that the pairs of KOX genes found on the chromosome bands 12q24.33, 16q22-q23, 19p13.3-p13.2, or 19q13.3-qter lie within 200-300 kb DNA fragments. This suggests the existence of KOX gene clusters on these chromosomal bands.

Asunto(s)

Mapeo Cromosómico , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 19 , Cisteína/genética , Histidina/genética , Familia de Multigenes/genética , Dedos de Zinc/genética , Sondas de ADN , Electroforesis en Gel de Campo Pulsado , Genes , Humanos , Hibridación in Situ , Masculino , Datos de Secuencia Molecular

RESUMEN

Two rearrangements affecting the same allele of the BCL2 gene were characterized by molecular analysis of an untreated follicular lymphoma. The first rearrangement interested the major breakpoint region (mbr) on chromosome 18 and a JH segment on chromosome 14. The other one was located at the 5' end of the BCL2 gene, in the so called variant cluster region (vcr), and consisted of a series of deletions that removed part of a DNA region where initiation of transcription normally occurs. Interestingly, both rearrangements involved the same BCL2 allele. The simultaneous presence of mbr (or mcr) translocations and of minor rearrangements in vcr has been previously suggested by restriction map analysis in a significant number of follicular lymphomas. The significance of these abnormalities on the oncogenic process is discussed.

Asunto(s)

Eliminación de Gen , Reordenamiento Génico , Linfoma Folicular/genética , Oncogenes , Proteínas Proto-Oncogénicas/genética , Translocación Genética , Alelos , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-bcl-2

RESUMEN

We describe a patient with stage IV non-Hodgkin's lymphoma (NHL) and a t(11;18)(q21;q21) translocation. He presented with a gastric small B-cell lymphocytic lymphoma, expressing IgAL immunoglobulins without expression of CD10, CD5, and CD23 antigens. The lymphoma was the final development of a 6-year history of a monoclonal IgAL increase complicated by severe renal failure due to membranoproliferative glomerulonephritis. The clinical, histological, immunologic, and cytogenetic features of this patient are very similar to those observed in the five other patients with t(11;18) reported to date. This translocation therefore seems to delineate a new subtype of diffuse small B-cell lymphoma with involvement of mucosal sites. Involvement of the BCL2 oncogene on 18q21 could not be detected using molecular techniques with 5' as well as 3' BCL2 probes, indicating that other, so far unknown, genes relevant to lymphoid differentiation could be located in 18q21 and 11q21.

Asunto(s)

Cromosomas Humanos Par 11 , Cromosomas Humanos Par 18 , Leucemia Linfocítica Crónica de Células B/genética , Linfoma no Hodgkin/genética , Translocación Genética , Antígenos CD/sangre , Mapeo Cromosómico , Marcadores Genéticos , Humanos , Inmunoglobulina A/sangre , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Linfoma no Hodgkin/sangre , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Mapeo Restrictivo

Asunto(s)

Secuencia de Bases , Cromosomas Humanos Par 18/ultraestructura , Cromosomas Humanos Par 22/ultraestructura , Reordenamiento Génico de Linfocito B , Reordenamiento Génico de Linfocito T , Leucemia-Linfoma de Células T del Adulto/genética , Linfoma Folicular/genética , Translocación Genética , Secuencia de Consenso , ADN Nucleotidiltransferasas/metabolismo , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , VDJ Recombinasas

RESUMEN

Rearrangements of the BCL2 gene and expression of bcl-2 protein were analyzed in a series of 64 cases of follicular lymphomas in order to establish a relationship between the rearrangements and the protein overexpression. Of the 64 cases, 41 showed BCL2 rearrangement involving one of the three breakpoint clusters: 30 in mbr, eight in mcr, and three in vcr. A double rearrangement mbr+vcr was detected in two cases. Twenty cases with bcl-2 protein expression in tumor cells exhibited no apparent rearrangement, suggesting the possible existence of other mechanisms activating the gene. Interestingly, expression of the LMP1 protein, the Epstein-Barr virus (EBV) encoded gene, whose capacity to induce BCL2 has been recently demonstrated, was only found in 2/41 cases in which BCL2 was rearranged. These data suggest that EBV infection is not an important mechanism in the activation of BCL2 in follicular lymphoma.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/fisiología , Linfoma Folicular/genética , Proteínas Proto-Oncogénicas/genética , Antígenos Virales/fisiología , Reordenamiento Génico/fisiología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/fisiología , Humanos , Inmunohistoquímica , Linfoma Folicular/química , Linfoma Folicular/inmunología , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2 , Activación Transcripcional , Translocación Genética/genética , Regulación hacia Arriba/fisiología , Proteínas de la Matriz Viral/fisiología

RESUMEN

Some recent aspects of the molecular biology of chromosome abnormalities that activate the BCL2 gene in B cell malignancies (follicular lymphoma, chronic lymphocytic leukemia) are discussed. We also report data showing that the Epstein-Barr virus (EBV) LMP-1 protein which can induce the expression of BCL2, is found infrequently in follicular lymphomas which exhibit no apparent BCL2 gene rearrangement.

Asunto(s)

Linfoma no Hodgkin/genética , Oncogenes , Antígenos Virales/genética , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Recombinación Genética , Translocación Genética , Proteínas de la Matriz Viral/genética

RESUMEN

We previously reported a 5' rearrangement of the BCL2 locus in a t(18;22) variant translocation found in a lymphocytic lymphoma. Primary structure analysis of both rearranged chromosomes confirmed the localization of the breakpoint in the so-called vcr region (for variant cluster region) that encompasses Z-DNA stretches 5' of the BCL2 locus, and in between J lambda 1 and C lambda 1 segments on the IGL locus. A 1,027 nucleotide segment from chromosome 22 was repeated on both derivative chromosomes 18q+ and 22q-. This segment contained an octanucleotide that was also present in the normal chromosome 18 close to the breakpoint. As a consequence of the translocation, a normal-sized BCL2 transcript was overexpressed in tumor cells.

Asunto(s)

Cromosomas Humanos Par 18 , Cromosomas Humanos Par 22 , ADN de Neoplasias/análisis , Leucemia Linfoide/genética , Translocación Genética , Secuencia de Bases , Northern Blotting , Clonación Molecular , Biblioteca Genómica , Humanos , Datos de Secuencia Molecular , ARN Neoplásico/análisis , Mapeo Restrictivo , Análisis de Secuencia de ADN , Transcripción Genética

RESUMEN

Two members of the human zinc finger Krüppel family, ZNF 12 (KOX 3) and ZNF 26 (KOX 20), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization. The presence of individual human zinc finger genes in mouse-human hybrid DNAs was correlated with the presence of specific human chromosomes or regions of chromosomes in the corresponding cell hybrids. Analysis of such mouse-human hybrid DNAs allowed the assignment of the ZNF 12 (KOX 3) gene to chromosome region 7p. The ZNF 26 (KOX 20) gene segregated with chromosome region 12q13-qter. The zinc finger genes ZNF 12 (KOX 3) and ZNF 26 (KOX 20) were localized by in situ chromosomal hybridization to human chromosome regions 7p22-21 and 12q24.33, respectively. These genes and the previously mapped ZNF 24 (KOX 17) and ZNF 29 (KOX 26) genes, are found near fragile sites.

Asunto(s)

Cromosomas Humanos Par 12 , Cromosomas Humanos Par 7 , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Bandeo Cromosómico , Mapeo Cromosómico , Humanos , Células Híbridas , Ratones , Mitosis , Datos de Secuencia Molecular , Familia de Multigenes , Hibridación de Ácido Nucleico

Asunto(s)

Neoplasias Laríngeas , Melanoma , Humanos , Masculino , Persona de Mediana Edad