RESUMEN
Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.
Asunto(s)
División Celular/fisiología , Daño del ADN , Proteínas Inmediatas-Precoces , Proteínas/genética , Proteína p53 Supresora de Tumor/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Clonación Molecular , Regulación de la Expresión Génica , Genes Supresores de Tumor , Humanos , Células Híbridas , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas/fisiología , Homología de Secuencia de Aminoácido , Proteínas Supresoras de TumorRESUMEN
The tumour suppressor gene PTEN (also called MMAC1 or TEP1) is somatically mutated in a variety of cancer types [1] [2] [3] [4]. In addition, germline mutation of PTEN is responsible for two dominantly inherited, related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome [4]. PTEN encodes a dual-specificity phosphatase that inhibits cell spreading and migration partly by inhibiting integrin-mediated signalling [5] [6] [7]. Furthermore, PTEN regulates the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring [8]. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase [9]. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signalling cascade.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/crecimiento & desarrollo , Genes de Helminto , Genes Supresores de Tumor , Proteínas del Helminto/fisiología , Fosfatidilinositol 3-Quinasas , Monoéster Fosfórico Hidrolasas/genética , Proteínas Supresoras de Tumor , Animales , Caenorhabditis elegans/genética , Catálisis , ADN Complementario/genética , Proteínas del Helminto/genética , Humanos , Larva/crecimiento & desarrollo , Longevidad/genética , Lípidos de la Membrana/metabolismo , Familia de Multigenes , Fosfohidrolasa PTEN , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , Receptor de Insulina/genética , Receptor de Insulina/fisiologíaRESUMEN
Here we report the specific regulation of gadd45 expression in human melanoma cell lines following UVB radiation. This solar wavelength is likely to be involved in melanoma aetiology. We have previously shown that gadd45 expression is strongly enhanced in a p53-independent manner following UVB irradiation, unlike the other p53 target genes studied. Furthermore, gadd45 is specifically activated in melanocytes since its induction in response to UVB, is not observed in other skin cells such as keratinocytes or fibroblasts. To investigate this particular regulation of gadd45, we analysed the UVB-induced response of different gadd45 promoter regions. Thus, a minimal promoter region of 50 bp length, responsible for gadd45 activation in melanoma cell lines following UVB irradiation, was determined. In electrophoretic mobility shift assays (EMSAs), we showed that this region (-106/-56) of the gadd45 promoter which contains two identical octamers, binds the POU family gene products oct-1 and N-oct3. Given the specific expression pattern of N-oct3 in melanocyte, we invalidated the expression of this transcription factor in melanoma cells: such an abrogation of N-oct3 protein expression in melanoma cells impeded gadd45 UVB-response. Thus the response of melanocyte to UVB may use an original and previously undescribed pathway.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Melanocitos/efectos de la radiación , Melanoma/patología , Proteínas de Neoplasias/fisiología , Biosíntesis de Proteínas , Neoplasias Cutáneas/patología , Factores de Transcripción/fisiología , Rayos Ultravioleta , Sitios de Unión/genética , Células Cultivadas/metabolismo , Células Cultivadas/efectos de la radiación , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Marcación de Gen , Genes Reporteros , Proteínas de Homeodominio , Factor C1 de la Célula Huésped , Humanos , Péptidos y Proteínas de Señalización Intracelular , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Melanocitos/metabolismo , Melanoma/genética , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Factor 1 de Transcripción de Unión a Octámeros , Factores del Dominio POU , Regiones Promotoras Genéticas , Proteínas/genética , Eliminación de Secuencia , Neoplasias Cutáneas/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Proteinas GADD45RESUMEN
We have previously shown that BTG1 stimulates myoblast differentiation. In addition, this protein displays a major nuclear localization in confluent myoblasts, decreasing during the early steps of differentiation, and is essentially detected in the cytoplasm of mature myotubes. To identify the domains involved in the cellular trafficking of BTG1, we observed the localization of several BTG1 sequences fused to betaGalactosidase. The highly conserved B box among all members of the BTG family induces a significant nuclear localization of the betaGal moiety, enhanced by presence of the BTG1 carboxy-terminal sequence. In addition, a functional Nuclear Export Signal (NES) overlaps the B box. Moreover, presence of the first 43 NH(2)-terminal amino acids reduced the nuclear localization of each chimeric protein tested. Last, the BTG1 amino-terminal domain bears an LxxLL motif favouring nuclear accumulation, and another region encompassing the A box inhibiting nuclear localization. In contrast to a BTG1 mutant exclusively localized in the cytoplasm, transient expression of a mutant displaying a nuclear localization enhanced myoblasts withdrawal from the cell cycle and terminal differentiation, thus mimicking the myogenic influence of BTG1. In conclusion, several regions of BTG1 are implicated in its cellular localization, and BTG1 myogenic activity is induced at the nuclear level.
Asunto(s)
Proteínas de Neoplasias/fisiología , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/metabolismo , Secuencia Conservada , Humanos , Datos de Secuencia Molecular , Músculos/citología , Músculos/metabolismo , Músculos/fisiología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estructura Terciaria de Proteína , Codorniz , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
It is well known that loss of tumor suppressor genes and more generally of antiproliferative genes plays a key role in the development of most tumors. We report here the cloning of the mouse BTG3 gene and show that its human counterpart maps on chromosome 21. This evolutionarily conserved gene codes for a 30 kDa protein and is expressed in most adult murine and human tissues analyzed. However, we demonstrate that its expression is cell cycle dependent and peaks at the end of the G1 phase. This gene is homologous to the human BTG1, BTG2 and TOB genes which were demonstrated to act as inhibitors of cell proliferation. Its description allowed us to define better this seven gene family (the BTG gene family) at the structural level and to speculate about its physiological role in normal and tumoral cells. This family is mainly characterized by the presence of two conserved domains (BTG boxes A and B) of as yet undetermined function which are separated by a non-conserved 20-25 amino acid sequence.
Asunto(s)
ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Genes Supresores de Tumor , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Ciclo Celular/fisiología , Cromosomas Humanos Par 21 , Clonación Molecular , Humanos , Ratones , Datos de Secuencia Molecular , FilogeniaRESUMEN
The human BTG1 gene (expressing an anti-proliferative function) is an evolutionarily conserved gene homologous to the murine PC3/TIS21 genes. Here, we report the cloning and sequencing of the murine BTG1 coding region and chicken BTG1 cDNA. The putative human and mouse BTG1 proteins are 100% identical; the chicken BTG1 cDNA contains an open reading frame of 170 amino acids with a 91% identity to its human and murine counterparts. The 3'-untranslated region of BTG1 is also highly conserved (82% homology between human and chicken), suggesting that it plays a key role in the regulation of BTG1 expression. These data confirm that BTG1 is phylogenetically highly conserved and that BTG1 and PC3/TIS21 may constitute the first members of a new family of functionally related genes.
Asunto(s)
Secuencia Conservada , Genes Reguladores , Proteínas de Neoplasias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Apoptosis of human B cells and murine T and B cells was analyzed by DNA agarose gel electrophoresis, clamped homogeneous electric field, measurement of cell DNA content by flow cytometry, transmission electron microscopy and by UV microscopy. Apoptosis was induced by etoposide (an inhibitor of topoisomerase II), by the calcium ionophore ionomycin or by cross-linking of membrane immunoglobulins (Ig) with anti-Ig-antibodies. Two types of apoptosis could be defined. Apoptosis resulting in small DNA fragments (180-200 base pairs and multiples thereof) was associated with a typical 'ladder' in agarose gel electrophoresis and a decrease in cell DNA content assessed by flow cytometry. Conversely apoptosis with large DNA fragments (100-150 kilobase pairs) was only demonstrated by clamped homogeneous electric field but was not associated with decreased cell DNA content or the observation of DNA ladders. Nuclear condensation without fragmentation was more frequent when apoptosis generated large DNA fragments. The type of apoptosis appears to be an intrinsic property of each cell type.
Asunto(s)
Apoptosis/fisiología , Linfocitos B/citología , ADN/metabolismo , Linfocitos T/citología , Linfocitos B/metabolismo , Electroforesis en Gel de Agar , Etopósido/farmacología , Humanos , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
The neuropeptide bombesin stimulates tumour cell proliferation in vitro. Through pharmacological testing, 20-40% of human colorectal tumours have been shown to be equipped with bombesin/gastrin releasing peptide receptor (GRP-R). The aim of the present study was to test whether GRP-R expression is correlated with tumour characteristics and usual prognostic factors in colorectal adenocarcinomas. A sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) method was validated by studying GRP-R mRNA in separated layers of normal colonic wall, and GRP-R mRNA levels (in parallel with binding studies) in colon cancer cell lines LoVo and Caco-2. GRP-R mRNA levels were then determined in 29 surgical tumour specimens and the results compared with tumour histology and, using histochemistry, with the accumulation of p53 protein and a Ki-67 cell proliferation index. The mRNA was not detected in normal colonic epithelium, whereas a distinct signal was observed after amplification in 27/29 (93%) tumour specimens. Estimates of mRNA levels in the 27 positive tumours ranged from 52 to 8000 amol/0.25 microgram total RNA, and were significantly higher in poorly/moderately differentiated tumours (P < 0.05) and in tumours with lymphatic vessel invasion (P < 0.01). There was no relationship with p53 accumulation or to the proliferation index. Our results show that GRP-R mRNA can be detected in most colorectal tumour specimens, and suggest a link between high mRNA levels and both tumour dedifferentiation and lymph vessel invasion, but not proliferation.
Asunto(s)
Neoplasias del Colon/metabolismo , Péptido Liberador de Gastrina/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Mensajero/metabolismo , Anciano , Transformación Celular Neoplásica , Neoplasias del Colon/patología , Femenino , Humanos , Inmunohistoquímica , Enfermedades Linfáticas/patología , Invasividad NeoplásicaRESUMEN
We have recently reported that transformation of murine NIH 3T3 cells by v-fos oncogene interfered with Hsp70 and Hsp25 accumulation after heat shock. Here, we have investigated the effect mediated by other oncogenes on the accumulation of these stress proteins. We report that T-antigen transformation of NIH 3T3 cells delayed and reduced the accumulation of Hsp25 after heat shock and decreased the heat-mediated phosphorylation of this protein. This decreased level of Hsp25 correlated with a reduced accumulation of the corresponding mRNA and was related to T-antigen level. In contrast, T-antigen had no effect on the expression of the major stress protein Hsp70 nor did it interfere with the level of Hsp90 or Hsp60. We report also that v-src or Ha-ras oncogenes delayed Hsp25 accumulation after heat shock but that only v-src reduced the heat-induced phosphorylation of this protein. v-src, but not Ha-ras, interfered with Hsp70 expression and none of these oncogenes had an effect on Hsp60 or Hsp90 levels. Taken together, these observations suggest that an altered accumulation of Hsp25 after heat shock is a common characteristic of NIH 3T3 fibroblasts transformed by different oncogenes.
Asunto(s)
Células 3T3/metabolismo , Antígenos Virales de Tumores/fisiología , Transformación Celular Viral/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Antígenos Virales de Tumores/genética , Genes ras/fisiología , Genes src/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Ratones , Chaperonas Moleculares , ARN Mensajero/metabolismoRESUMEN
The t(14;18) chromosomal translocation is widely recognized as a cytogenetic abnormality associated with follicular lymphomas, but estimates of its frequency in this type of lymphoma vary from less than 40% to almost 90% according to the geographic origin of the patients. Using two human genomic probes for major and minor breakpoint cluster regions mapping at chromosome 18q21, we have analysed 131 cases of B non-Hodgkin's lymphomas obtained from France, by the Southern blot technique. The genotypic study was complemented in most cases by immunophenotypic and cell kinetic analyses. The BCL2 gene located at 18q21 band was rearranged in 39 of 56 (70%) follicular lymphomas and in 9 of 74 (12%) diffuse lymphomas; probes for major and minor breakpoint regions detected two thirds and one third of the rearrangements respectively. Regarding the morphologic subtypes of follicular and diffuse lymphomas, no significant differences were observed irrespective of the probe used. Review of the literature showed that comparable results have been obtained previously using both cytogenetic and molecular approaches and our results support the view that the global incidence of the t(14;18)(q32;q21) translocation in follicular lymphomas is about 70% with wide geographic variations. The immunological study provides evidence for a significant correlation of BCL2 rearrangement with surface immunoglobulin gamma isotype expression and with the lack of reactivity of the malignant cells with an antibody against the CD5 cluster. In the cases where cell kinetics was analysed, we did not find any significant difference between the rate of proliferation and BCL2 rearrangement. These data should be compared with previously reported observations made in humans or in transgenic mice and enable us to propose a model accounting for the role of BCL2 in B cell tumorigenesis.
RESUMEN
We have studied the regulation of ATAD2 gene expression by androgens in prostate cells. ATAD2 is a coactivator of the androgen receptor (AR) and the MYC protein. We showed that ATAD2 expression is directly regulated by AR via an AR binding sequence (ARBS) located in the distal enhancer of its regulatory region. The gene is also regulated by the E2F1 transcription factor. Using knockdown and chromatin immunoprecipitation technique approaches, we could demonstrate that AR and E2F1 functionally collaborate and physically interact between each other. From the analysis of chromatin conformation, we conclude that this cooperation results from a chromatin looping over the ATAD2 promoter region between the ARBS and E2F1 binding site in an androgen-dependent manner. Furthermore, we could show that several genes overexpressed in prostate cancer and potentially involved in several aspects of tumor development have an ARBS and an E2F1 binding site in their regulatory regions and exhibit the same mechanism of regulation by both transcription factors as ATAD2.
Asunto(s)
Factor de Transcripción E2F1/metabolismo , Próstata/metabolismo , Receptores Androgénicos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Factor de Transcripción E2F1/genética , Ensayo de Cambio de Movilidad Electroforética , Humanos , Inmunoprecipitación , Masculino , Regiones Promotoras Genéticas/genética , Unión Proteica , Receptores Androgénicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
miR-16, a miRNA involved in cell proliferation and apoptosis regulation, may interfere with either oncogenic or tumor-suppressor pathways and is implicated in leukemogenesis. We then explored its expression in 93 childhood acute lymphoblastic leukemia (ALL) cases. A high miR-16 expression was associated with hyperleukocytosis and poor cytogenetic groups. In the whole group and in B-cell ALLs, disease-free survival (DFS) was significantly shorter for miR-16 above quartile 75. In T-cell ALLs, for both DFS and overall survival, a significant trend was found with a survival shortening from the lowest to the highest miR-16 levels. miR-16 expression neither significantly correlated with normal and malignant lymphocyte proliferation nor varied according to lymphocyte differentiation. The prognostic value of miR-16 in childhood ALL highlighted the complexity of miR-16 functions.
Asunto(s)
Proliferación Celular , Linfocitos/citología , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Northern Blotting , Línea Celular Tumoral , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Humanos , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , PronósticoRESUMEN
Previous studies have shown that the BCL-2 protooncogene encodes a mitochondrial protein that promotes cell survival by blocking programmed cell death. Bcl-2 protein has been detected in normal immature myeloid cells and in acute myeloid leukemia (AML) cells. To assess its functional role in normal and leukemic hematopoiesis, we performed serum-free cultures of CD34+ normal marrow cells, of bcl-2-positive myeloid lines, and of AML cells in the presence of bcl-2 sense, nonsense, and antisense phosphorothioate oligodeoxynucleotides. In all antisense-treated cultures, we observed (1) an inhibition of bcl-2 protein expression by day 4 to 6 of culture; (2) a decrease in cell survival duration; and (3) a decrease in the number of clonogenic cells present in the culture. Moreover, exposure to chemotherapeutic drugs resulted in more effective killing of AML cells in the presence of antisense oligomers. We conclude that bcl-2 protein is necessary for the survival of myeloid cells in culture, and that it may be implicated in the resistance of AML cells to chemotherapy.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Leucemia/patología , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas/fisiología , Secuencia de Bases , División Celular , Supervivencia Celular , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
Group I and Epstein-Barr virus-negative Burkitt's lymphoma cell lines and the B104 lymphoma cell line which expresses a phenotype of immature B cells undergo apoptosis after cross-linking of their surface Ig receptors or after exposure to a calcium ionophore. We show here that tumor necrosis factor (TNF)-alpha protects these B cell lines against Ca(2+)-dependent apoptosis. Protection was associated with up-regulation of bcl-2 mRNA and protein expression. The increase of Bcl-2 expression induced by TNF-alpha was inhibited by chelerythrine, a specific inhibitor of protein kinase C (PKC), suggesting that Bcl-2 expression was dependent on PKC activation. Furthermore, we show that phorbol esters and cyclosporin A (CsA), which prevent Ca(2+)-dependent apoptosis, up-regulated Bcl-2 expression. The effect of CsA on Bcl-2 expression is controlled by calcineurin since we have shown that FK506 but not rapamycin had the same effect on Bcl-2 expression, whereas okadaic acid, an inhibitor of phosphatases 1, 2A and 2C, was ineffective. These data provide direct evidence that TNF-alpha prevents Ca(2+)-dependent apoptosis by a Bcl-2-dependent mechanism mediated by PKC.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos B/metabolismo , Calcio/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Calcineurina , Calcio/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/farmacología , Línea Celular , Ciclosporina/farmacología , Activación Enzimática , Humanos , Fosfoproteínas Fosfatasas/farmacología , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2 , Acetato de Tetradecanoilforbol/farmacología , Regulación hacia ArribaRESUMEN
The effect of v-fos transformation on the cellular response to heat shock has been investigated. NIH 3T3 fibroblasts were transfected with the FBR p75gag-fos gene fusion under the control of the long terminal repeat (LTR) promoter of Finkel-Biskin-Reilly (FBR) murine sarcoma virus and with the gene encoding hygromycin resistance. Several hygromycin-resistant clone isolates, that expressed various levels of p75gag-fos oncoprotein, were analyzed as they displayed properties of transformed cells, such as altered morphology, shorter doubling time, serum-independent growth and foci formation in soft agar. The thermal response of these clones was compared to that of the control cells expressing the hygromycin-resistance gene only. Here, we report that the v-fos-transformed clones displayed an enhanced thermosensitivity which resulted in a reduced tolerance to thermal stress. Heat-treated v-fos-transformed cells displayed a decreased expression and accumulation of the major stress proteins Hsp68 (68-kDa heat-shock protein) and Hsp25 which probably resulted of a reduced accumulation of the corresponding mRNAs. This effect was particularly intense at the level of Hsp25. These alterations in cell survival and stress-protein expression appeared correlated to the level of p75gag-fos. At least for Hsp68, the transcription of this gene was not found altered by v-fos expression suggesting that this oncogene increases the turn-over of Hsp68 mRNA. After the heat-shock treatment, v-fos transformation also reduced the time period during which the constitutively expressed stress protein Hsc70 redistributes inside the nucleus. Since Hsp68 and Hsp25 are molecular chaperones that in vivo protect cells against the deleterious effects of heat shock, it is conceivable that their reduced accumulation and altered cellular distribution following heat shock may contribute, at least in part, to the thermosensitivity of v-fos-transformed NIH 3T3 fibroblasts.
Asunto(s)
Proteínas de Choque Térmico/biosíntesis , Proteínas Oncogénicas v-fos/genética , Células 3T3 , Animales , División Celular , Línea Celular Transformada/fisiología , Técnicas de Transferencia de Gen , Calor , Ratones , ARN Mensajero/biosíntesisRESUMEN
Post-transplant lymphoproliferative disorders (PTLD) are well-known complications of iatrogenic immune deficiency and are thought to result from the proliferation of B cells infected by the Epstein-Barr virus (EBV). Some large cell lymphomas occurring in the general population carry a rearrangement of the Bcl6/Laz3 zinc-finger-encoding gene. 15 EBV-associated PTLD were tested for the presence of Bcl6/Laz3 rearrangements by Southern blot analysis using two specific probes (F370, F372). One out of 15 cases displayed a rearranged band independent of the germline one. In contrast, 10 lymphoblastoid cell lines and one lymphoblastoid cell line passaged in an SCID mouse carried only germline alleles of Bcl6/Laz3 after Southern blot hybridization. This indicates that genetic abnormalities may also play an important role in the development of some PTLD.
Asunto(s)
Proteínas de Unión al ADN/genética , Reordenamiento Génico , Terapia de Inmunosupresión/efectos adversos , Trastornos Linfoproliferativos/etiología , Trastornos Linfoproliferativos/genética , Trasplante de Órganos/efectos adversos , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Animales , Southern Blotting , Transformación Celular Viral/genética , Infecciones por Herpesviridae/complicaciones , Herpesvirus Humano 4 , Humanos , Trastornos Linfoproliferativos/virología , Ratones , Ratones SCID , Infecciones Oportunistas/complicaciones , Proteínas Proto-Oncogénicas c-bcl-6 , Células Tumorales Cultivadas , Infecciones Tumorales por Virus/complicaciones , Dedos de ZincRESUMEN
Transplant recipients are at increased risk for the development of post-transplant lymphoproliferative disorders (PTLDs). PTLDs harbor genomes of the Epstein-Barr virus, a herpesvirus that immortalizes B cells in vitro. At least five viral proteins are required for immortalization. Two of them are particularly important. Latent membrane protein (LMP) has transforming activity in fibroblasts, and Epstein-Barr antigen (EBNA)2 transactivates the expression of numerous cellular and viral genes. To determine whether the expression of EBNA2 and LMP is related to the histological and clinical presentation of PTLD, we tested their expression in 14 Epstein-Barr virus-positive cases. Using monoclonal antibodies to EBNA2 and LMP on paraffin sections, we found an expression of both proteins in 2 of 3 polymorphic PTLD and in 7 of 8 cases of monomorphic, large cell PTLD, without plasmacytic differentiation. One polymorphic and one large cell PTLD expressed LMP only. LMP and EBNA2 were found particularly in immunoblasts. The number of positive cells was extremely variable in the different cases as well as within the same biopsy. Three cases of PTLD had morphological and phenotypical features of plasmacytomas and did not stain for EBNA2 or LMP. This suggests that the expression of EBNA2 and LMP is related to the differentiation stage of the infected cells and that other viral or cellular proteins may contribute to tumor growth.
Asunto(s)
Antígenos Virales/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Infecciones por Herpesviridae/patología , Herpesvirus Humano 4/metabolismo , Trastornos Linfoproliferativos/virología , Trasplante/efectos adversos , Infecciones Tumorales por Virus/patología , Proteínas de la Matriz Viral/biosíntesis , Southern Blotting , Antígenos Nucleares del Virus de Epstein-Barr , Infecciones por Herpesviridae/mortalidad , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Trastornos Linfoproliferativos/mortalidad , Trastornos Linfoproliferativos/patología , Infecciones Tumorales por Virus/mortalidadRESUMEN
Antilymphocyte and antithymocyte globulins (ALG) are currently used as immunosuppressive agents in clinical transplantation and for the treatment of severe aplastic anemia. ALG contain a mixture of antibodies that recognize T- and B-cell-specific antigens but mostly nonlineage-specific molecules. We reported previously that ALG could inhibit the proliferation of activated B cells and B cell lines (Bonnefoy-Bérard et al, Blood 79:2164, 1992). We show here that ALG induce apoptosis of several human hematopoietic cell lines, as shown by nuclear condensation and fragmentation in fluorescence and electronic microscopy and by double-strand DNA breaks shown by DNA electrophoresis. Apoptosis was achieved without elevation of intracellular Ca2+ and requirement for mRNA and protein synthesis. Most of the B-cell lines tested (Epstein-Barr virus [EBV]-transformed lymphoblastoid cell lines, EBV-negative and groups I/III EBV-positive Burkitt's lymphoma cell lines, as well as other B-lymphoma cell lines) were susceptible to ALG-induced cytotoxicity. Myelomonocytic and T-cell lines were much less susceptible than B-cell lines. Susceptibility to ALG-induced cytotoxicity was not correlated with intracellular Bcl-2 level. Most cell lines that express high levels of Fas/Apo-1 antigen were susceptible to ALG. However, several lines of evidence support the conclusion that, in addition to Fas/Apo-1, other cell surface molecules can mediate ALG-induced apoptosis. The cytotoxic activity could be fully removed by adsorption on susceptible cell lines but not on a resistant cell line, indicating that it was mediated by antibodies specific for surface antigens expressed only on susceptible cell lines. Apoptosis was triggered by ALG F(ab')2 fragments as well as by intact ALG. This cytotoxic property of ALG may account for their antiproliferative effect and might contribute to some extent to the relatively lower risk of posttransplant lymphoproliferative disorders previously reported in ALG-treated patients.
Asunto(s)
Suero Antilinfocítico/farmacología , Apoptosis/inmunología , Linfocitos B/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Calcio/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Replicación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2 , ARN Mensajero/metabolismo , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
In a previous work, we have shown that T3 induces a potent stimulation of avian myoblast differentiation. In this study, we demonstrated that this hormone did not affect MyoD and myogenin expression. As numerous data suggest that T3 could affect the cAMP pathway, we have studied its involvement in the myogenic activity of triiodothyronine on quail myoblast. In agreement with Zalin and Montagues (Cell 2, 103-108 (1974)), we observed a transient rise in myoblast intracellular cAMP level some hours before the onset of terminal differentiation. Interestingly, this rise occurred earlier in T3-treated than in control myoblasts, and cAMP production was significantly increased by the hormone. Moreover, T3 increased CREB transcriptional activity, thus suggesting that the entire cAMP signaling pathway was stimulated by this hormone. In addition, we observed that addition of an inhibitor of adenylate cyclase activity prior to the cAMP rise dramatically inhibited myoblast differentiation. Last, we showed that cAMP mimicked all T3 actions upon myoblast differentiation: (1) T3 and cAMP reduced myoblast proliferation by increasing the number of postmitotic myoblasts at cell confluence; (2) T3 and cAMP increased BTG1 nuclear accumulation; (3) T3 and cAMP stimulated terminal differentiation only when added during the proliferative phasis. These data strongly suggest that the transient rise in cAMP production could be essential for myoblast terminal differentiation. In addition, it appears that, at least in avian myoblasts, T3 stimulation of terminal differentiation involves the cAMP pathway.
Asunto(s)
AMP Cíclico/metabolismo , Músculos Pectorales/embriología , Codorniz/embriología , Transducción de Señal , Triyodotironina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Fusión Celular , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Iminas/farmacología , Músculos Pectorales/citología , Músculos Pectorales/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Transcripción GenéticaRESUMEN
Post-transplant lymphoproliferative disorders (PTLD) are potentially fatal complications of organ transplants. Impairment of the immune system by immunosuppressive drugs is the assumed cause of PTLD. The Epstein-Barr virus (EBV) is detected in most of the PTLD studied and is considered as the main aetiological agent. The clinical course of PTLD patients remains unpredictable, some lymphoproliferations regress after discontinuation of the immunosuppressive treatment, others behave as true malignant tumours. The mechanism by which a viro-induced lymphoproliferation evolves to an autonomous tumour remains unclear, and little is known about the genetic changes that occur during this process. We report two cases of fatal EBV-associated PTLD in heart transplant recipients. Both tumours were monoclonal and carried numerous chromosomal abnormalities, including a classic t(8;14)(q24;q32) with rearrangement of the MYC proto-oncogene. One tumour demonstrated an amplification of the proto-oncogene N-MYC. The EBNA2 gene was not expressed in tumoral cells, suggesting that the chromosomal abnormalities contributed the function of EBNA2 in these cells. The morphology of the tumours indicated that the cases presented here were not Burkitt's lymphomas. These findings provide some clues with regard to the genetic changes which lead to a B-cell malignancy in some transplant patients.