RESUMO
Several cellular proteins form stable complexes with the proteins encoded by the adenovirus early region 1A (E1A) gene in extracts derived from adenovirus infected or transformed cells. Two of the cellular proteins that bind to E1A have been identified; one, a 105-kilodalton protein (pRb), is the product of the retinoblastoma gene, and the other, a 60-kilodalton protein, is a human cyclin A. Two other proteins that bind E1A have now been shown to be related to p34cdc2. This E1A complex displayed histone H1-specific kinase activity; the kinase activity was modulated during the cell division cycle, and association of pRb with E1A apparently was not required for this activity.
Assuntos
Adenovírus Humanos/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Proteínas Oncogênicas Virais/metabolismo , Protamina Quinase/metabolismo , Proteínas Precoces de Adenovirus , Linhagem Celular , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Células HeLa/citologia , Células HeLa/fisiologia , Humanos , Proteínas Oncogênicas Virais/genética , Ligação Proteica , Recombinação GenéticaRESUMO
In this report we introduce a simple, fast, and reliable method to prepare whole cell or nuclear extracts from small numbers of cells. These extracts were used to study transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in vitro. Our results revealed that the time courses of activation of extracts derived from cells stimulated with the mitogenic lectin phytohemagglutinin (PHA) or with the tumor promoter phorbol 12-myristate 13-acetate (PMA) are different. PMA induces a rapid onset of increased in vitro transcription from the HIV-1 LTR, while PHA causes a slow and sustained response. The biochemical relevance of protein synthesis inhibition by cycloheximide treatment of cells was investigated. In these studies, PMA induction of a change in in vitro transcriptional activity is not dependent on protein synthesis. Cycloheximide alone is insufficient to induce activation. Oligonucleotide-mediated site-directed mutagenesis demonstrated that mutation of the TATA box in the LTR ablated initiation of both basal-level transcription and activation by extracts from cells stimulated with PMA. Surprisingly, mutation of both kappa B sites in the LTR reduced but did not eliminate the in vitro response to extracts prepared at early time points after PHA or PMA stimulation of Jurkat cells. The reduction was greater in extracts derived from cells treated with PMA. Deletion analysis of the HIV-1 LTR revealed at least one region (-464 to -252) capable of suppressing in vitro transcription in extracts from Jurkat cells stimulated by PMA. This result is consistent with early studies of the HIV-1 LTR in transient transfection assays. We therefore have been able to observe distinct regulatory events at early time points after cells are exposed to agents known to induce transcription of both the HIV-1 LTR reporter gene constructs and the HIV-1 provirus itself.
Assuntos
Repetição Terminal Longa de HIV/genética , HIV-1/genética , Transcrição Gênica , Linhagem Celular , Cicloeximida/farmacologia , Ampliador HIV , Humanos , Cinética , Mutagênese Sítio-Dirigida , Fito-Hemaglutininas/farmacologia , TATA Box , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Immune complexes of the product of the c-rel protooncogene and of p105, the p50 NF-kappa B precursor, isolated from human T-lymphoblastoid cell lines are comprised of multiple proteins. Only p105 and human c-Rel (hc-Rel) are common to complexes precipitated with antiserum directed against either p105 or hc-Rel. Both proteins are inducible by phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) and their subcellular distribution is affected by this induction. We demonstrate that the Rel immune complex contains a protein with a molecular weight in the 40 kDa range (p40) which apparently is exclusively cytoplasmic. We were not able to detect p40 in the p105 immune complex, though hc-Rel is present. This indicates that hc-Rel exists in different multi-protein complexes and fits a model of functional regulation mediated by differential protein-protein interaction. We also demonstrate considerable isoform diversity of both hc-Rel and p105. We show that this heterogeneity is, in part, the result of phosphorylation. Furthermore, we demonstrate that p105 and hc-Rel are tyrosine kinase substrates. This finding indicates a role for both proteins in intracellular signal transduction pathways which are modulated by modification of their phosphorylation status.
Assuntos
NF-kappa B/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Humanos , Fosforilação , Fito-Hemaglutininas , Proteínas Proto-Oncogênicas c-rel , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The T cell line, CEM-E5, acutely and chronically infected with HIV-1, was used as a target cell in a standard 51Cr release HIV-1-specific antibody-dependent cellular cytotoxicity (ADCC) assay. CEM-E5, acutely infected with HIV-1, showed peak sensitivity to lysis in an HIV-1 specific ADCC assay on day 9 after infection. CEM-E5 clones, chronically infected with HIV-1, that productively express the virus were better ADCC targets than infected clones that do not express HIV-1. One clone, C5D7, was identified which was particularly sensitive to lysis in the ADCC assay. This cell line grows continually and is stable in culture and appears to be the best target for HIV-1-specific ADCC in our system.
Assuntos
Especificidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Testes Imunológicos de Citotoxicidade , Anticorpos Anti-HIV/análise , Linhagem Celular , Células Clonais/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Soropositividade para HIV/imunologia , Humanos , Soros Imunes , Imunidade InataRESUMO
Rabbits were injected with cell walls obtained from crown-gall tumor tissue or the corresponding cell walls from normal potato tissue. The serum obtained from rabbits 53 days after they were injected with tumor cell walls contained immunoglobins that reacted with both tumor and normal cell walls as well as with the cells from the inciting strain of Agrobacterium tumefaciens. When this serum was repeatedly absorbed against normal cell walls and the cells of the inciting strain of Agrobacterium tumefaciens, only tumor-specific immunoglobins remained. These immunoglobins did not react with cell walls obtained from meristematic (nontumorous) potato tissue. Yet this same serum reacted with crown-gall tumor cell walls obtained from turnip and carrot discs.
RESUMO
We evaluated a cytofluorometric method for determining the number of antigens expressed on the cell surface of human lymphocytes. Using beads that have a known number of binding sites for mouse immunoglobulin and monoclonal antibodies specific for various antigens on human lymphocytes, we found that this system is quite reproducible, reliable and technically easy to perform. The greatest source of variation in expression of cell surface antigens is interdonor variability.
Assuntos
Antígenos de Superfície/biossíntese , Citometria de Fluxo , Linfócitos/imunologia , Anticorpos Monoclonais , Reações Antígeno-Anticorpo/efeitos dos fármacos , Antígenos CD/análise , Imunofluorescência , Formaldeído/farmacologia , Humanos , Polímeros/farmacologiaRESUMO
HIV-1 is capable of infecting many different cell types that express the CD4 molecule. In vivo and in vitro this infection is associated with profound immunologic defects. We have examined the effect of HIV-1 infection on the expression of MHC class I (MHC-I) molecules to explore the possibility that this important immune system molecule is perturbed after HIV-1 infection. Our data show that in vitro, HIV-1 infection of CD4+ PBL, and the CD4+ cell lines, CEM-E5, HT, and U937, results in decreased expression of MHC-I molecules on the cell surface. This down-modulation is transient, occurring 18 h after HIV-1 infection of CD4+ PBL and returning to normal expression by 24 h. In CEM-E5, MHC-I down-modulation occurs over the course of days, reaching its greatest decrease (40%) about the time the cells are producing the most virus. Reversal of MHC-I expression to normal levels occurs as viral production decreases. Down-regulation during the time periods examined appear to be specific for MHC-I and does not occur with other cell-surface Ag nor is it caused by selection of a preexisting cell population with low MHC-I expression. Radioimmunoprecipitation of MHC-I protein from CEM-E5 indicated that the decrease of surface MHC-I is caused by decreased total protein secondary to a decrease in the level of mRNA for MHC-I. These decreased levels of MHC-I are biologically relevant because HIV-1 infected CEM-E5 cells are less susceptible to CTL lysis determined by the use of MHC-I cytolytic T cell clones and with the use of cold target-inhibition assay.