RESUMO
BACKGROUND: Hepatitis C virus (HCV) effectively establishes persistent infection in human livers. The non-structural (NS) 3/4A complex participates in this process by cleavage of interferon beta (IFN beta) promoter stimulator-1 (IPS-1; also termed Cardif/MAVS/VISA), which inhibits responses to double stranded (ds) RNA. However, it is not known whether this effect extends beyond innate responses. AIMS: To test if HCV NS3/4A affects innate and adaptive immune responses in vivo. METHODS: NS3 levels were semi-quantified in human liver biopsies, transfected cells, and in transgenic (Tg) mouse livers by western blot. The effect of NS3/4A on dsRNA-mediated signalling and on the integrity of IPS-1 was analysed using in vitro translation, transfected cells and Tg mice. Cytotoxic T cell (CTL)-mediated clearance of transient firefly luciferase (FLuc)- and/or NS3/4A-Tg hepatocytes was determined using in vivo imaging and western blot. RESULTS: NS3 protein levels were in a comparable range (0.1-49 microg/g tissue) in infected human livers and Tg mouse livers. Importantly, these levels of NS3/4A reduced murine innate responses to synthetic dsRNA in vivo, supporting the possibility that this occurs also in infected humans. The likely explanation for this was the NS3/4A-mediated cleavage of mouse IPS-1, albeit less efficiently than human IPS-1. Despite this, FLuc- and/or NS3/4A-expressing murine hepatocytes were effectively eliminated by hepatic CTLs, utilising the classical molecules for virus-infected cell lysis, including CD8, IFN gamma, perforin and FasL. CONCLUSIONS: Although HCV NS3/4A inhibits the innate immunity, this does not prevent CTL-mediated clearance of NS3/4A-expressing hepatocytes in vivo. Thus, other HCV proteins are most likely responsible for interfering with the adaptive immunity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Hepacivirus , Hepatite C Crônica/imunologia , Hepatócitos/virologia , Linfócitos T/imunologia , Proteínas não Estruturais Virais/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepatite C Crônica/virologia , Hepatócitos/imunologia , Humanos , Imunidade Inata , Interferon beta/imunologia , Fígado/metabolismo , Neoplasias Hepáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , NF-kappa B/metabolismo , RNA de Cadeia Dupla/imunologia , Especificidade da Espécie , Células Tumorais CultivadasRESUMO
Several recent findings have indicated that the promyelocytic leukemia gene product (PML) oncogenic domains (PODs) are involved in proteasome-mediated degradation of ubiquitinated proteins. We wanted to examine the intracellular distribution of PML protein in the presence of a proteasome inhibitor. We used high-resolution microscopy to study the distribution of PML protein and other POD-associated proteins along with the proteasomes themselves under normal conditions and in cells treated with the proteasome inhibitor, MG132. Inhibition of the proteasomes in MCF-7, HeLa, and IB-4 cell lines resulted in a radical redistribution of the POD-associated proteins PML, Sp100, and SUMO-1. After 6-10 h of MG132 treatment, PML, Sp100, and SUMO-1 were no longer detectable in the PODs and accumulated mainly in the nucleolus. Moreover, MG132 treatment changed the cellular distribution of the proteasomes. Interestingly, this included the accumulation in euchromatin areas of the nucleus and within the nucleoli. Several non-POD-associated proteins did not change their cellular distribution under the same conditions. The accumulation of POD-associated proteins and proteasomes in the nucleoli of MG132-treated cells indicates that these proteins may target the nucleoli under normal conditions and that the nucleolus may have a function in the regulation of proteasomal protein degradation.
Assuntos
Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias da Mama , Nucléolo Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Feminino , Células HeLa , Humanos , Leucemia Promielocítica Aguda/genética , Proteína da Leucemia Promielocítica , Complexo de Endopeptidases do Proteassoma , Transporte Proteico , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Ubiquitinas/metabolismoRESUMO
OBJECTIVES: To understand the mechanism for the refractoriness of B-chronic lymphocytic leukemia (B-CLL) cells for Epstein-Barr virus (EBV)-induced immortalization. STUDY DESIGN/METHODS: Cultures were initiated with EBV-infected tonsillar B and B-CLL cells. Expression of EBNA-2 and some of the key players regulating G1/S phase transition such as c-myc expression, phosphorylation of Rb protein, expression of G1 cyclins, and the cyclin-dependent kinase inhibitor p27 were followed. RESULTS: In line with earlier studies, EBV infection induced c-myc expression, pRb phosphorylation, D2 and D3 expression, and disappearance of p27 in normal B cells. In contrast, EBV-infected B-CLL cells remained resting and they did not express c-myc; cyclin D2, ppRb and cyclin D3 were seen only in occasional cells. Importantly, p27 expression was maintained. CONCLUSIONS: In B-CLL cells, the expression of the EBV-encoded nuclear proteins EBNAs is not followed by entrance to the cell cycle. Thus, the difference in the interaction of EBV-normal B cells and EBV-B-CLL cells is already apparent early after infection.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ciclo Celular/biossíntese , Herpesvirus Humano 4/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Animais , Linfócitos B/citologia , Células Cultivadas , Ciclina D2 , Ciclina D3 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/biossíntese , Antígenos Nucleares do Vírus Epstein-Barr/biossíntese , Herpesvirus Humano 4/fisiologia , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Fosforilação , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteína do Retinoblastoma/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/biossíntese , Proteínas ViraisRESUMO
EBNA-3 (also called EBNA-3A) is one of the EBV encoded nuclear antigens that are necessary for B-cell transformation. EBNA-3 is known to target RBPs, nuclear proteins that also interacts with EBNA-2, EBNA-4 and EBNA-6. In order to identify additional EBNA-3 targets, an EBV-transformed human lymphocyte cDNA library was screened in the yeast two-hybrid system with N-terminus truncated EBNA-3 that cannot interact with RBP-Jkappa. A clone, encoding Xap-2 protein, a cellular partner of Hepatitis B virus X-antigen was isolated. This protein is also known as the p38 subunit of the aryl hydrocarbon receptor complex (ARA9). The specific binding to EBNA-3 was confirmed by showing that the GST-Xap-2 precipitated EBNA-3 from CV1 cells that were infected with recombinant vaccinia virus expressing EBNA-3. Deletion of the C-terminus of Xap-2 eliminated the binding. Fusion with green fluorescent protein showed that Xap-2 is preferentially cytoplasmic but translocates to the nucleus upon expression of EBNA-3.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Antígenos da Hepatite B/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Linhagem Celular Transformada , DNA Complementar , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Proteínas/genética , Receptores de Hidrocarboneto Arílico/genética , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais Reguladoras e AcessóriasRESUMO
The D-type cyclins, involved in the regulation of G1 progression of the cell cycle, are expressed in a lineage-specific manner. Normal hematopoietic cells express cyclin D2 and/or D3. In order to determine whether their expression pattern changes in lymphoid tumors, we examined cyclin D2 and D3 expression in non-neoplastic and neoplastic lymphoid lesions, using a sensitive immunohistochemical amplification method. Centroblasts in lymphoid follicles of reactive lymph nodes expressed exclusively cyclin D3 and no D2. Interfollicular areas contained scattered cyclin D3 and D2 positive cells. By double staining, cyclin D3 was detected in CD79a positive B cells, CD3 positive T cells and CD68 positive macrophages. Cyclin D2 was present only in CD3 positive T cells. Neoplastic lymphoid lesions included 33 B cell lymphomas, 9 T cell lymphomas and 12 Hodgkin's lymphomas. The B cell lymphomas comprised 9 follicular lymphomas (FL), 1 Burkitt lymphoma (BL), 22 diffuse large cell lymphomas (DL) and 1 chronic lymphocytic leukemia (CLL). All 9 FLs and the single BL expressed exclusively cyclin D3, similarly to germinal center B cells, that represent their cells of origin. Six DLs expressed both cyclin D2 and D3, while 6 expressed only D3. Among the 9 pleomorphic T cell lymphomas, medium and large cell type, 5 expressed cyclin D2. Cyclin D3 was also detected in scattered cells in 4 of 9 cases and was highly expressed in 2 of 9 T cell lymphomas. The majority of Hodgkin's lymphomas expressed both cyclin D2 and D3 in Hodgkin/Reed-Sternberg (HRS) cells. The high frequency of positive cells indicates that both cyclins were expressed in the same cells.
Assuntos
Ciclinas/análise , Doença de Hodgkin/patologia , Linfonodos/patologia , Doenças Linfáticas/patologia , Linfoma de Células B/patologia , Linfoma de Células T/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Northern Blotting , Ciclo Celular , Criança , Ciclina D2 , Ciclina D3 , Ciclinas/genética , Feminino , Doença de Hodgkin/genética , Humanos , Hiperplasia , Imuno-Histoquímica , Linfonodos/metabolismo , Doenças Linfáticas/genética , Linfoma de Células B/genética , Linfoma de Células T/genética , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To find cellular proteins that associate with EBNA-3 (also called EBNA-3A), one of the Epstein-Barr virus (EBV)-encoded growth transformation-associated nuclear proteins. METHODS: Screening human cDNA libraries in the yeast two-hybrid system and performing an analysis of interaction in vitro as well as in cell lysates. RESULTS: EBNA-3 binds to the epsilon subunit of the chaperonin containing T-complex protein 1 (epsilon-TCP-1) in the yeast two-hybrid system. The cDNA clone isolated from a human lymphocyte library was found to encode the middle and C-terminal part of epsilon-TCP-1. The interaction was confirmed by showing that a GST fusion protein specifically precipitated EBNA-3 from CV1 cells infected with recombinant vaccinia virus expressing EBNA-3. The interacting region was mapped to the putative apical domain of epsilon-TCP-1. CONCLUSIONS: This study shows that large, virus-encoded transforming proteins such as EBNA-3 may receive help for their initial folding by chaperonin complexes. The recognition of the chaperonin complex likely occurs through specific interaction with one of the subunits. We suggest that nascent EBNA-3 may recognize the TCP-1 complex by interacting with the apical region of the epsilon subunit.
Assuntos
Chaperoninas/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Chaperonina com TCP-1 , Chaperoninas/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Infection of human B lymphocytes with Epstein-Barr virus (EBV) in vitro induces a G0 to G1 transition followed by DNA synthesis and cell division. The virus activation of the cell cycle closely mimics the antigen-dependent normal B cell activation pathway. Infected B cells undergo blast transformation followed by the emergence of immortalized lymphoblastoid cell lines. Numerous cellular proteins are switched on in the infected cells, including p53. In view of the frequent association of wild-type p53 (wtp53) expression with growth arrest and apoptosis, p53 expression, cell viability (absence of apoptosis) and cell cycle progression at the single cell level during the first week after EBV infection were assessed. The rate of EBV infection was scored by EBNA-5 staining between 20 and 72 h after infection and varied between 20 and 25% of the cell population. All EBNA-5-positive blasts were p53-positive as well. Double staining for p53 and for DNA ends (TUNEL) revealed that p53-positivity and apoptosis were mutually exclusive. Quantification of the DNA content by Hoechst staining and computer-assisted image analysis showed that a fraction of the p53-positive blasts had a DNA content higher than 2N, indicating entry into the S/G2 phases. Double p53 and BrdU staining of the cells, pulse-labelled with BrdU, revealed that 65% of the p53-positive blasts were in S phase 3 days after infection. Similarly, B cell activation by CD40L and IL-4 induced p53 expression without any adverse effect on cell cycle progression. Therefore, the phenomenon is not EBV-specific but correlates with immunoblast activation.
Assuntos
Apoptose , Linfócitos B/virologia , Herpesvirus Humano 4/fisiologia , Proteínas Nucleares , Proteína Supressora de Tumor p53/biossíntese , Linfócitos B/fisiologia , Ligante de CD40 , Divisão Celular , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/análise , Humanos , Interleucina-4/farmacologia , Ativação Linfocitária , Glicoproteínas de Membrana/farmacologia , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-mdm2 , Fase S , Proteína Supressora de Tumor p53/análiseRESUMO
Simultaneous detection of two antigens by immunostaining usually requires primary antibodies from two different species or a hapten modification of one of the antibodies if they are from the same species. A novel double staining method is described for immunodetection of two independent antigens using two mouse monoclonal antibodies. The principle of the method is the following: The first antigen is detected by a monoclonal antibody that is diluted so extensively that it cannot be recognized with conventional detection systems. A highly sensitive biotin-tyramide amplification system is used to identify this antibody. The second antigen is stained with a monoclonal antibody by dilution and detected by conventional immunostaining. The method was tested for both alkaline-phosphatase staining on paraffin sections and immunofluorescence staining on cultured cells in cytospin preparation. The absence of cross-reaction in the former system was demonstrated by the mutually exclusive detection of T- and B-cells in human lymph nodes or T-cells and carcinoma cells in nasopharyngeal carcinoma biopsies. Similarly, the EBV encoded EBNA2 and ZEBRA proteins showed a mutually exclusive staining by immunofluorescence on B95-8 cells. The method could be used to demonstrate co-expression of two independent antigens in the same cells, such as PCNA and keratin in carcinoma cells in paraffin sections and for EBNA2 and LMP1 EBV proteins in immunofluorescence preparations of B95-8 cells.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/análise , Imunofluorescência , Técnicas Imunoenzimáticas , Fosfatase Alcalina , Animais , Antígenos/imunologia , Antígenos CD/análise , Antígenos Virais/análise , Biotina/análogos & derivados , Reações Cruzadas , Herpesvirus Humano 4/imunologia , Humanos , Queratinas/análise , Queratinas/imunologia , Neoplasias Pulmonares , Tecido Linfoide , Camundongos , Neoplasias Nasofaríngeas , Inclusão em Parafina , Antígeno Nuclear de Célula em Proliferação/análise , Células Tumorais Cultivadas , Tiramina/análogos & derivadosRESUMO
A body cavity lymphoma-derived cell line (BC1), known to carry both Epstein-Barr virus (EBV) and human herpes virus type 8 (HHV-8; or Kaposi's sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth transformation-associated antigens and cellular phenotype by immunofluorescence staining, Western blotting, RT-PCR and flow cytometry. A similar phenotypic analysis was also performed on another body cavity lymphoma line, BCBL1, that is singly infected with HHV-8. Phenotypically, the two lines were closely similar. Although both lines are known to carry rearranged immunoglobulin genes, they were mostly negative for B-cell surface markers. Both expressed the HHV-8-encoded nuclear antigen (LNA1). Similarly to Epstein-Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the chromatin in interphase nuclei and the mitotic chromosomes in metaphase. It accumulated in a few well-circumscribed nuclear bodies that did not co-localize with EBNA1. BC1 cells expressed EBNA1, LMP2A and EBV-encoded small RNAs but not EBNA2-6, LMP1 and LMP2B. They were thus similar to type I Burkitt's lymphoma cells and latently infected peripheral B-cells. Analysis of the splicing pattern of the EBNA1-encoding message by RT-PCR showed that BC1 cells used the QUK but not the YUK splice, indicating that the mRNA was initiated from Qp and not from Cp or Wp.
Assuntos
Linfócitos B/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 8/genética , Linfoma/virologia , Antígenos Virais/biossíntese , Antígenos Virais/genética , Linfócitos B/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/biossíntese , Antígenos Nucleares do Vírus Epstein-Barr/genética , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 8/imunologia , Humanos , Cariotipagem , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Fenótipo , Reação em Cadeia da Polimerase , Splicing de RNA , Células Tumorais Cultivadas , Proteínas da Matriz Viral/biossíntese , Proteínas da Matriz Viral/genéticaRESUMO
In a recent study, we described a group of monoclonal antibodies that identify five high molecular mass proteins which associate with intermediate filaments in the cytoplasm and accumulate in nuclear foci as well. The proteins have been designated pNDCFs, proteins associated with nuclear dots and cytoplasmic filaments. Their expression in human B cells was upregulated by Epstein-Barr virus (EBV) infection or by exposure to anti-CD40 antibodies and IL4. Phenotypically representative (type I) Burkitt's lymphoma (BL) cell lines do not express pNDCFs or, if they do, the proteins accumulate preferentially in nuclear dots. Type III BL cell lines that have drifted to a more immunoblastic phenotype during in vitro passage and EBV-transformed lymphoblastoid cell lines (LCLs) of non-neoplastic origin express these proteins regularly at high levels. They are preferentially but not exclusively associated with vimentin filaments in the cytoplasm. Here we show that all five pNDCFs can be upregulated by expressing the EBV-encoded membrane protein LMP1 in type I BLs. Three of them could also be upregulated in the human keratinocyte cell line RHEK-1 by LMP1 transfection. This upregulation was paralleled by the LMP1-induced increase in vimentin expression in both cell types. One of the pNDCFs, detected by the MAb DM_4A6, accumulated in cap-like structures under the cell membrane that colocalized with membrane patches of LMP1, in addition to its localization in nuclear dots and in association with cytoplasmic vimentin filaments.
Assuntos
Transformação Celular Viral , Proteínas do Citoesqueleto/biossíntese , Herpesvirus Humano 4/fisiologia , Proteínas Nucleares/biossíntese , Vimentina/biossíntese , Proteínas da Matriz Viral/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos B , Linfoma de Burkitt , Antígenos CD40/imunologia , Antígenos CD40/fisiologia , Linhagem Celular Transformada , Feminino , Humanos , Interleucina-4/farmacologia , Queratinócitos , Neoplasias Mamárias Experimentais , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteínas da Matriz Viral/biossínteseRESUMO
Mouse monoclonal antibodies raised against nuclear bodies isolated from an EBV-immortalized lymphoblastoid cell line (LCL) known to contain several viral and cellular proteins (Jiang et al., Exp. Cell Res., 197: 314-318, 1991; Szekely et al., J. Gen. Virol., 76: 2423-2432, 1995; Szekely et al., J. Virol., 70: 2562-2568, 1996). Seventy six clones gave detectable immunofluorescence staining on LCLs. Five independent monoclonal antibodies detected a group of apparently novel, high M(r) (> 200,000) proteins that shared common features of subcellular distribution. In LCLs, these proteins were preferentially associated with vimentin filaments in the cytoplasm and with distinct nuclear foci. The appearance of the latter differed from the premyelocytic leukemia-associated protein, EBV nuclear antigen #5, and retinoblastoma-protein-positive bodies that were used for immunization. They seemed to be connected to the cytoplasmic filaments through thin fibrillar nuclear structures. In mitotic cells, these complex structures rearranged into a perichromosomal basket that was associated with vimentin filaments. The target proteins, operationally designated as proteins associated with nuclear dots and cytoplasmic filaments (pNDCFs), were not present in resting human B cells or were expressed at a low level. The level increased considerably after EBV infection or mitogenic stimulation by interleukin 4 and anti-CD40 antibodies. In Burkitt lymphoma (BL) type I lines phenotypically representative of the in vivo tumors, the pNDCFs were either absent or exclusively localized to the nucleus, usually to well-defined nuclear foci. EBV-positive type I BLs often shift to a more LCL-like (type III) phenotype during prolonged in vitro propagation. Type I cells express only EBV nuclear antigen 1 and the surface markers CD10 and CD77, whereas type III express all nine growth-associated EBV-encoded proteins and a gamut of B-cell activation markers. Most of the type III BL cell lines contained increased amounts of pNDCFs bound to cytoplasmic filaments, as seen in the LCLs. We propose that the expression of vimentin-associated pNDCFs should be included in the definition of type III BL phenotype.
Assuntos
Linfoma de Burkitt/metabolismo , Núcleo Celular/metabolismo , Transformação Celular Viral , Citoesqueleto/metabolismo , Herpesvirus Humano 4 , Biossíntese de Proteínas , Linfoma de Burkitt/ultraestrutura , Linhagem Celular Transformada , Humanos , Proteínas Nucleares/metabolismo , Células Tumorais CultivadasRESUMO
Small DNA tumour viruses, such as simian virus 40 (SV40), papilloma viruses and adenoviruses, encode proteins that form complexes with and inactivate the p53 and retinoblastoma (RB) proteins. This convergent evolution reflects the common need of these viruses to inactivate these two important regulators of cell cycle progression and cell survival. Polyomavirus, a close relative of SV40, is different. Its large T protein complexes only with RB, not with p53. We have examined whether this is compensated by the frequent appearance of p53 mutations in polyomavirus-induced tumours. We tested the p53 status of 15 polyomavirus-induced sarcomas. Two sarcomas were p53-negative while six carried mutant p53. Another six sarcomas expressed low levels of wild-type p53. One tumour expressed high levels of wild-type p53 protein as shown by DNA sequencing and immunofluorescence staining. MDM2 amplification was not detected in any of the tumours, but Northern blotting showed that MDM2 was overexpressed in at least two tumours that expressed wild-type p53 and in one tumour that expressed both wild-type and mutant p53. Treatment with the DNA-damaging agent mitomycin C caused p53 protein accumulation followed by induction of MDM2 and WAF1/p21 mRNA in four of the tumours expressing wild-type p53, indicating that p53-mediated transcriptional activation was unaltered in these tumours. However, p53-mediated transactivation of WAF1/p21 was impaired in the wild-type p53-expressing tumours that expressed elevated levels of MDM2. These results demonstrate that p53 mutation and inactivation are frequently but not invariably involved in polyomavirus-induced tumorigenesis.
Assuntos
Infecções por Polyomavirus/genética , Polyomavirus/isolamento & purificação , Sarcoma Experimental/genética , Proteína Supressora de Tumor p53/genética , Infecções Tumorais por Vírus/genética , Animais , Divisão Celular , Camundongos , Mutação Puntual , Sarcoma Experimental/patologia , Sarcoma Experimental/virologia , Transcrição GênicaRESUMO
Exposure of normal resting B lymphocytes to EBV in vitro leads to activation, subsequent immortalization, and the establishment of lymphoblastoid cell lines (LCLs). The endemic form of Burkitt's lymphoma (BL) is associated with EBV. EBV-positive BL lines maintain the original tumor phenotype (group I BL) initially and express only one EBV-encoded protein, EBV nuclear antigen (EBNA)-1. Most of them drift toward a LCL-like phenotype during in vitro culturing and express all nine EBV-encoded growth transformation-associated proteins (group III BL). Cyclin D2 and D3 have been found previously to differ in their mRNA expression in BL and LCL. Cyclin D2 expression has been attributed to EBV gene expression and to EBNA-2 and EBNA-5 in particular. We have studied cyclin D2/D3 expression in larger series of LCLs, BL lines, and freshly EBV-infected peripheral blood B lymphocytes, both at the mRNA and protein levels. The predominant cyclin D2 expression in the LCLs and group III BLs correlated with an activated B-cell phenotype. In contrast, only cyclin D3 was expressed in the group I BL lines. EBV-carrying group II BL lines that retained the BL-associated CD10 did not express cyclin D2. A collection of in vitro EBV-converted sublines of originally EBV-negative BLs that expressed a full set of the growth transformation-associated EBV proteins maintained their CD10 and cyclin D3 expression. Blood B lymphocytes expressed no D2 or D3 as judged by immunostaining. Cyclin D2 could be detected by immunostaining in a proportion of the activated blasts 40 h postinfection. Cyclin D3 that is expressed at a low level in the established LCLs was stained easily in B blasts at this time. The level of cyclin D3 at 72 h postinfection was two to three times higher than in the exponentially growing LCLs, as detected by immunoblotting. It was still 5-10 times lower than in group I BLs. Mitogen stimulation of primary B cells induced cyclin D3 at a similar level as in the LCLs. Our data are consistent with the notion of cell lineage-specific D-type cyclin expression. They also indicate that B cells may switch their D-type cyclin expression during differentiation.
Assuntos
Linfócitos B/citologia , Ciclinas/genética , Linfócitos B/fisiologia , Linfócitos B/virologia , Northern Blotting , Western Blotting , Linfoma de Burkitt , Linhagem Celular/citologia , Linhagem Celular/fisiologia , Linhagem Celular/virologia , Ciclina D2 , Ciclina D3 , Ciclinas/análise , Citometria de Fluxo , Expressão Gênica/fisiologia , Herpesvirus Humano 4 , Humanos , Imuno-Histoquímica , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Fenótipo , RNA Mensageiro/metabolismo , Infecções Tumorais por VírusRESUMO
Wild type (wt) p53 expressed from a temperature-sensitive construct (ts p53) can induce both G1 cell cycle arrest and apoptosis in the p53-negative J3D mouse T lymphoma line (Wang et al, 1995). The human papillomavirus (HPV) 16 E7 protein has been shown to prevent p53-induced G1 cell cycle arrest following DNA damage. We asked whether inhibition of p53-induced G1 arrest by overexpression of the HPV16 E7 protein in the ts p53-transfected J3D cells would interfere with p53-induced apoptosis. Whereas a majority of the ts p53-expressing J3D cells were arrested in the G1 phase 22 h after induction of wt p53 by temperature shift to 32 degrees C, the E7/ts p53-expressing cells showed only a minor increase in the number of cells in G1 at this time point. In addition, the E7/ts p53-expressing cells showed a much less dramatic reduction in number of cells in S phase than the ts p53-expressing cells. This demonstrates that E7 at least partially rescues the cells from p53-induced G1 arrest. In contrast, overexpression of HPV16 E7 did not have any effect on the kinetics nor the frequency of p53-triggered apoptotic death, as shown by FACS analysis, trypan blue exclusion, and DNA fragmentation analysis. These findings support the notion that p53-induced G1 arrest and p53-induced apoptosis are two separate independent pathways.
Assuntos
Apoptose/genética , Fase G1/genética , Genes p53 , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Regulação Neoplásica da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Proteínas E7 de Papillomavirus , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Transfecção , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
Using Western blot analysis, we examined cyclin E and cyclin A protein levels in 19 patients with acute lymphoblastic leukemia ([ALL] 15 B-ALL and four T-ALL). Whereas normal, nonproliferating peripheral blood mononuclear cells (PBMCs) expressed low levels of the 50-kD cyclin E, ALL blasts in the peripheral blood, although showing low-level or no proliferation as judged by FACS/cell-cycle analysis and cyclin A protein levels, expressed high levels of cyclin E, with a mean value similar to that of the proliferating Burkitt's lymphoma cell line, Akata. The accumulation of a protein shown to shorten the G1 phase of the cell cycle, cyclin E, in growth-delayed leukemic blasts may reflect the malignant status of these cells. Before treatment, B-ALL cells expressed predominantly the 50-kD cyclin E. T-ALL samples displayed the 50-kD cyclin E protein and a smaller, approximately 43-kD cyclin E species. In paired B-ALL samples taken before treatment and at relapse, we found a significant overexpression of the 50-kD protein in relapsed samples (P < .006), plus the presence of up to four additional smaller-molecular-weight species of cyclin E, illustrating clear diagnosis versus relapse differences. Cyclin E expression in ALL blasts may correlate to the relative malignant status of the cells, with higher protein levels reflecting a more advanced stage of the disease and a greater potential to proliferate under permissive conditions.
Assuntos
Biomarcadores Tumorais/metabolismo , Ciclinas/biossíntese , Proteínas de Neoplasias/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Adolescente , Adulto , Idoso , Linfócitos B/metabolismo , Ciclo Celular , Intervalo Livre de Doença , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia-Linfoma de Células T do Adulto/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Células-Tronco Neoplásicas/metabolismo , Linfócitos T/metabolismoRESUMO
EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.
Assuntos
Antígenos Virais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas de Neoplasias , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Linhagem Celular , Cromatina/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr , Temperatura Alta , Humanos , Mitose , Proteína da Leucemia Promielocítica , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
The Epstein-Barr virus (EBV)-encoded, nuclear matrix-associated EBNA-5 protein is preferentially localized within distinct nuclear blobs in EBV-immortalized lymphoblastoid cell lines. We have previously found that the same blobs also contain retinoblastoma (Rb) protein. We now show that they contain hsp70 protein as well. Both EBNA-5 and hsp70 translocate to the nucleolus under cell density congestion or after heat shock. Both proteins relocate to their original position upon the re-establishment of normal physiological conditions. EBNA-5 is tightly bound to the nuclear matrix. The translocated EBNA-5 is also tightly associated with matrix structures, as shown by sequential elution-based cell fractionation. The Rb protein does not translocate to the nucleolus. The virally encoded EBNA-1, -2, -3 and -6, and cellular PCNA, snRNP and cyclin E are not affected either. The translocation of EBNA-5 to the nucleolus is not species- or cell type-specific since stress conditions induced the same phenomenon in EBNA-5-transfected human, mouse and rat cells of different tissue origins.
Assuntos
Antígenos Virais/metabolismo , Nucléolo Celular/metabolismo , Nucléolo Celular/virologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Herpesvirus Humano 4/metabolismo , Animais , Linfócitos B/virologia , Contagem de Células , Linhagem Celular Transformada , Antígenos Nucleares do Vírus Epstein-Barr , Resposta ao Choque Térmico , Humanos , Camundongos , Matriz Nuclear/virologia , Ratos , Especificidade da Espécie , Células Tumorais CultivadasRESUMO
Using immunofluorescence technique we have analysed the Rb, p53, EBNA-2 and EBNA-5 expression pattern in EBV infected human B-cells and established lymphoblastoid cell lines (LCL-s). Resting B-cells showed only a faint Rb and no p53 immunostaining. The expression of both Rb and p53 increased after EBV infection. The change was first detectable 6 h after infection. The frequency of brilliantly Rb positive cells increased more rapidly than p53 positives. EBNA-2 and EBNA-5 became first detectable 12 h after infection. The frequency of EBNA positive cells in the freshly infected cultures was concordant with the proportion of CD23 and PCNA positives, but remained consistently below the frequency of Rb and p53 positive cells. Double immunofluorescence staining showed that all EBNA-5 positive cells were strongly Rb and p53 positive. LCL-s did not stain for p53, whereas the Rb staining was maintained at a high level. The EBNA-5 staining pattern changed from brilliant almost homogeneous nuclear staining in the freshly infected B-cells, to a nonhomogeneous pattern with a small number of strongly fluorescent nuclear bodies in established LCL-s. There was no change in the EBNA-2 staining pattern. Our findings indicate that the immortalization of B-cells by EBV may initially involve a high expression of EBNA-5, p53 and Rb, but only cells with low p53 and focal expression of EBNA-5 in nuclear bodies have the selective advantage required to grow into immortalized lines.