ABSTRACT
Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, -3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.
Subject(s)
B-Lymphocytes/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Viral Matrix Proteins/immunology , fas Receptor/metabolism , src-Family Kinases/metabolism , Animals , Apoptosis , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Cell Line , Enzyme Activation , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/immunology , Herpesvirus 4, Human/pathogenicity , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mutation , Protein-Tyrosine Kinases/genetics , Receptors, Antigen, B-Cell/metabolism , Syk Kinase , Viral Matrix Proteins/genetics , src-Family Kinases/geneticsABSTRACT
The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.
Subject(s)
Acyl-CoA Dehydrogenase/metabolism , Tumor Suppressor Protein p53/metabolism , Acyl-CoA Dehydrogenase/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line, Tumor , Cell Survival/drug effects , Gene Regulatory Networks , Glucose/pharmacology , Humans , Mice , Mice, Nude , Molecular Sequence Data , Neoplasms/metabolism , Neoplasms/pathology , Oxidative Phosphorylation/drug effects , Protein Structure, Tertiary , RNA Interference , Sequence Alignment , Stress, Physiological , Transcriptional Activation , Transplantation, Heterologous , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/geneticsABSTRACT
p53 is a crucial tumour suppressor that responds to diverse stress signals by orchestrating specific cellular responses, including transient cell cycle arrest, cellular senescence and apoptosis, which are all processes associated with tumour suppression. However, recent studies have challenged the relative importance of these canonical cellular responses for p53-mediated tumour suppression and have highlighted roles for p53 in modulating other cellular processes, including metabolism, stem cell maintenance, invasion and metastasis, as well as communication within the tumour microenvironment. In this Opinion article, we discuss the roles of classical p53 functions, as well as emerging p53-regulated processes, in tumour suppression.
Subject(s)
Genes, Tumor Suppressor , Neoplasms/genetics , Neoplasms/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis , Cell Cycle Checkpoints , Cellular Senescence , DNA Damage , Humans , Mice , Neoplasm Invasiveness , Neoplasm Metastasis , Signal Transduction , Stem Cells/metabolism , Transcription, Genetic , Transcriptional Activation , Tumor Suppressor Protein p53/geneticsABSTRACT
The mechanisms by which the p53 tumor suppressor acts remain incompletely understood. To gain new insights into p53 biology, we used high-throughput sequencing to analyze global p53 transcriptional networks in primary mouse embryo fibroblasts in response to DNA damage. Chromatin immunoprecipitation sequencing reveals 4785 p53-bound sites in the genome located near 3193 genes involved in diverse biological processes. RNA sequencing analysis shows that only a subset of p53-bound genes is transcriptionally regulated, yielding a list of 432 p53-bound and regulated genes. Interestingly, we identify a host of autophagy genes as direct p53 target genes. While the autophagy program is regulated predominantly by p53, the p53 family members p63 and p73 contribute to activation of this autophagy gene network. Induction of autophagy genes in response to p53 activation is associated with enhanced autophagy in diverse settings and depends on p53 transcriptional activity. While p53-induced autophagy does not affect cell cycle arrest in response to DNA damage, it is important for both robust p53-dependent apoptosis triggered by DNA damage and transformation suppression by p53. Together, our data highlight an intimate connection between p53 and autophagy through a vast transcriptional network and indicate that autophagy contributes to p53-dependent apoptosis and cancer suppression.
Subject(s)
Autophagy/genetics , DNA Damage/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Up-Regulation , Animals , Cell Cycle Checkpoints/genetics , Cell Survival/genetics , Cells, Cultured , Embryo, Mammalian , Gene Expression Regulation, Developmental/genetics , Genome-Wide Association Study , Mice , Protein Binding , Sequence Analysis, RNAABSTRACT
We measured anti-Haemophilus influenzae type a capsular polysaccharide serum immunoglobulin G antibodies in cord blood sera from Mexican (n = 68) and Chilean mothers (n = 72) by enzyme-linked immunosorbent assay. Measurable antibodies were found in 79.3% of samples. Immunoglobulin G antibodies correlated with serum bactericidal activity (r = 0.66). This enzyme-linked immunosorbent assay can be used for the evaluation of adaptive immune responses to Haemophilus influenzae type a and serosurveillance studies in populations at risk.
Subject(s)
Antibodies, Bacterial/blood , Bacterial Capsules/immunology , Fetal Blood/chemistry , Haemophilus Infections/immunology , Haemophilus influenzae/immunology , Immunoglobulin G/blood , Enzyme-Linked Immunosorbent Assay/methods , Female , Fetal Blood/immunology , Humans , Membrane Transport Proteins , Pregnancy , Sensitivity and Specificity , Serum Bactericidal Test , Statistics, NonparametricABSTRACT
p53 is a pivotal tumor suppressor that induces apoptosis, cell-cycle arrest and senescence in response to stress signals. Although p53 transcriptional activation is important for these responses, the mechanisms underlying tumor suppression have been elusive. To date, no single or compound mouse knockout of specific p53 target genes has recapitulated the dramatic tumor predisposition that characterizes p53-null mice. Recently, however, analysis of knock-in mice expressing p53 transactivation domain mutants has revealed a group of primarily novel direct p53 target genes that may mediate tumor suppression in vivo. We present here an overview of well-known p53 target genes and the tumor phenotypes of the cognate knockout mice, and address the recent identification of new p53 transcriptional targets and how they enhance our understanding of p53 transcriptional networks central for tumor suppression.
Subject(s)
Gene Regulatory Networks , Neoplasms/metabolism , Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis , Humans , Neoplasms/genetics , Transcriptional Activation , Tumor Suppressor Protein p53/geneticsABSTRACT
The link between EBV infection and Burkitt lymphoma (BL) is strong, but the mechanism underlying that link has been elusive. We have developed a mouse model for EBV-associated BL in which LMP2A, an EBV latency protein, and MYC are expressed in B cells. Our model has demonstrated the ability of LMP2A to accelerate tumor onset, increase spleen size, and bypass p53 inactivation. Here we describe the results of total gene expression analysis of tumor and pretumor B cells from our transgenic mouse model. Although we see many phenotypic differences and changes in gene expression in pretumor B cells, the transcriptional profiles of tumor cells from LMP2A/λ-MYC and λ-MYC mice are strikingly similar, with fewer than 20 genes differentially expressed. We evaluated the functional significance of one of the most interesting differentially expressed genes, Egr1, and found that it was not required for acceleration of tumor onset by LMP2A. Our studies demonstrate the remarkable ability of LMP2A to affect the pretumor B-cell phenotype and tumorigenesis without substantially altering gene expression in tumor cells.
Subject(s)
Burkitt Lymphoma/genetics , Burkitt Lymphoma/virology , Gene Expression Profiling , Herpesvirus 4, Human/physiology , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , B-Lymphocytes/virology , Burkitt Lymphoma/metabolism , Disease Models, Animal , Flow Cytometry , Genes, myc/genetics , Herpesvirus 4, Human/genetics , Host-Pathogen Interactions , Humans , Leukocyte Common Antigens/genetics , Leukocyte Common Antigens/metabolism , Leukosialin/genetics , Leukosialin/metabolism , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Spleen/metabolism , Spleen/pathology , Spleen/virology , Viral Matrix Proteins/geneticsABSTRACT
Burkitt's lymphoma (BL) is characterized by translocation of the MYC gene to an immunoglobulin locus. Transgenic mouse models have been used to study the molecular changes that are necessary to bypass tumor suppression in the presence of translocated MYC. Inactivation of the p53 pathway is a major step to tumor formation in mouse models that is also seen in human disease. Human BL is often highly associated with Epstein-Barr virus (EBV). The EBV latency protein latent membrane protein 2A (LMP2A) is known to promote B cell survival by affecting levels of pro-survival factors. Using LMP2A transgenic mouse models, we have identified a novel mechanism that permits lymphomagenesis in the presence of an intact p53 pathway. This work uncovers a contribution of EBV to molecular events that have documented importance in BL pathogenesis, and may underlie the poorly understood link between EBV and BL.
Subject(s)
Burkitt Lymphoma/virology , Herpesvirus 4, Human/metabolism , Viral Matrix Proteins/physiology , Animals , Apoptosis , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/isolation & purification , Humans , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Protein p53/metabolism , Viral Matrix Proteins/metabolismABSTRACT
Although Epstein-Barr virus (EBV) is linked to Burkitt's lymphoma (BL), the role of the virus in lymphomagenesis is unclear. LMP2A, encoded by EBV, can be detected in BL biopsies and has prosurvival functions. We generated mice expressing MYC and LMP2A in B cells. LMP2A/lambda-MYC mice show greatly accelerated tumor onset. Similar to previous work, we found p53 mutations in lambda-MYC tumors; however, we detected no mutations in the rapidly arising LMP2A/lambda-MYC tumors. We further demonstrate that the p53 pathway is functionally intact in LMP2A/lambda-MYC tumors, which have increased levels of PUMA and sensitivity to p53 activation by Nutlin. This work shows that LMP2A can permit tumorigenesis in the presence of an intact p53 pathway, identifying an important contribution of EBV to BL.
Subject(s)
Burkitt Lymphoma/etiology , Genes, myc , Genes, p53 , Herpesvirus 4, Human/pathogenicity , Viral Matrix Proteins/genetics , Viral Matrix Proteins/physiology , Animals , Apoptosis , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , B-Lymphocytes/virology , Burkitt Lymphoma/genetics , Burkitt Lymphoma/virology , Disease Models, Animal , Genes, Viral , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/physiology , Humans , Mice , Mice, Transgenic , Models, Biological , MutationABSTRACT
ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.
Subject(s)
Bacterial Proteins/metabolism , Cell Membrane/metabolism , Peptide Fragments/metabolism , 3T3 Cells , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/toxicity , HeLa Cells , Humans , Mice , Phospholipases A/metabolism , Protein Transport , TransfectionABSTRACT
We developed fluorescent mono- and multivalent opsonophagocytic assays (fOPA and fmOPA, respectively) specific for seven Streptococcus pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). Bacterial survival was quantitated with alamar blue, a fluorescent metabolic indicator. Both fOPA and fmOPA allow for determination of viability endpoints for up to seven serotypes with high levels of agreement to the reference method. The fmOPA eliminates colony counting, reduces serum volume, and produces results in 1 day.