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1.
Oncogene ; 35(1): 69-82, 2016 Jan 07.
Article in English | MEDLINE | ID: mdl-25772236

ABSTRACT

Although modulation of the cellular tumor-suppressor p53 is considered to have the major role in E1A/E1B-55K-mediated tumorigenesis, other promyelocytic leukemia nuclear body (PML-NB)/PML oncogenic domain (POD)-associated factors including SUMO, Mre11, Daxx, as well as the integrity of these nuclear bodies contribute to the transformation process. However, the biochemical consequences and oncogenic alterations of PML-associated E1B-55K by SUMO-dependent PML-IV and PML-V interaction have so far remained elusive. We performed mutational analysis to define a PML interaction motif within the E1B-55K polypeptide. Our results showed that E1B-55K/PML binding is not required for p53, Mre11 and Daxx interaction. We also observed that E1B-55K lacking subnuclear PML localization because of either PML-IV or PML-V-binding deficiency was no longer capable of mediating E1B-55K-dependent SUMOylation of p53, inhibition of p53-mediated transactivation or efficiently transforming primary rodent cells. These results together with the observation that E1B-55K-dependent SUMOylation of p53 is required for efficient cell transformation, provides evidence for the idea that the SUMO ligase activity of the E1B-55K viral oncoprotein is intimately linked to its growth-promoting oncogenic activities.


Subject(s)
Adenoviridae/genetics , Cell Transformation, Viral/genetics , Leukemia, Promyelocytic, Acute/genetics , Leukemia, Promyelocytic, Acute/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Proteins/metabolism , Adenovirus E1B Proteins/genetics , Adenovirus E1B Proteins/metabolism , Animals , HEK293 Cells , Humans , Mutation , Nuclear Proteins/genetics , Promyelocytic Leukemia Protein , Protein Isoforms , Rats , Transcription Factors/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics
2.
Oncogene ; 32(13): 1626-37, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-22614022

ABSTRACT

Since the discovery of post-translational modification (PTM) by the small ubiquitin-related modifiers (SUMOs), a multitude of proteins have been described to be reversibly modified, resulting in the alteration of several cellular pathways. Interestingly, various pathogens gain access to this modification system, although the molecular mechanisms and functional consequences are barely understood. We show here that the adenoviral oncoprotein E1B-55K is a substrate of the SUMO conjugation system, which is directly linked to its C-terminal phosphorylation. This regulative connection is indispensable for modulation of the tumor suppressor p53/chromatin-remodeling factor Daxx by E1B-55K and, consequently, its oncogenic potential in primary mammalian cells. In virus infection, E1B-55K PTMs are necessary for localization to viral transcription/replication sites. Furthermore, we identify the E2 enzyme Ubc9 as an interaction partner of E1B-55K, providing a possible molecular explanation for SUMO-dependent modulation of cellular target proteins. In conclusion, these results for the first time provide evidence how E1B-55K PTMs are regulated and subsequently facilitate exploitation of the host cell SUMOylation machinery.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Protein Kinases/physiology , Sumoylation/physiology , Viral Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/physiology , Adenoviridae/genetics , Adenoviridae/metabolism , Amino Acid Sequence , Animals , Animals, Newborn , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Co-Repressor Proteins , HEK293 Cells , Humans , Models, Biological , Molecular Chaperones , Molecular Sequence Data , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Phosphorylation/genetics , Phosphorylation/physiology , Phylogeny , Protein Kinases/metabolism , Protein Processing, Post-Translational/genetics , Protein Processing, Post-Translational/physiology , Rats , Receptor Cross-Talk/physiology , Sequence Homology, Amino Acid , Sumoylation/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/physiology , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/physiology
3.
Cancer Gene Ther ; 20(1): 25-32, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23196273

ABSTRACT

Coxsackie adenovirus receptor (CAR) is the primary receptor to which oncolytic adenoviruses have to bind for internalization and viral replication. A total of 171 neuroendocrine lung tumors in form of multitissue arrays have been analyzed resulting in a positivity of 112 cases (65.5%). Immunostaining correlated statistically significant with histopathology and development of recurrence. The subtype small cell lung cancer (SCLC) showed the highest CAR expression (77.6%), moreover the CAR level was correlated to the disease-free survival. Further, high CAR expression level in SCLC cell lines was found in vitro and in vivo when cell lines had been transplanted into immunodeficient mice. A correlation between CAR expression in the primary tumors and metastases development in the tumor model underlined the clinical relevance. Cell lines with high CAR level showed a high infectivity when infected with a replication-deficient adenovirus. Low levels of CAR expression in SCLC could be upregulated with Trichostatin A, a histone deacetylase inhibitor. As a result of the unaltered poor prognosis of SCLC and its high CAR expression it seems to be the perfect candidate for oncolytic therapy. With our clinically relevant tumor model, we show that xenograft experiments are warrant to test the efficiency of oncolytic adenoviral therapy.


Subject(s)
Coxsackie and Adenovirus Receptor-Like Membrane Protein/biosynthesis , Lung Neoplasms/metabolism , Neuroendocrine Tumors/metabolism , Oncolytic Virotherapy , Small Cell Lung Carcinoma/metabolism , Adenoviridae/genetics , Animals , Cell Line, Tumor , Female , Gene Expression/drug effects , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Hydroxamic Acids/pharmacology , Kaplan-Meier Estimate , Lung Neoplasms/mortality , Lung Neoplasms/therapy , Male , Mice , Neuroendocrine Tumors/mortality , Neuroendocrine Tumors/therapy , Oncolytic Viruses/genetics , Proportional Hazards Models , Small Cell Lung Carcinoma/mortality , Small Cell Lung Carcinoma/therapy , Xenograft Model Antitumor Assays
4.
Oncogene ; 29(40): 5511-22, 2010 Oct 07.
Article in English | MEDLINE | ID: mdl-20639899

ABSTRACT

The E1B-55K product from human adenovirus is a substrate of the small ubiquitin-related modifier (SUMO)-conjugation system. SUMOylation of E1B-55K is required to transform primary mammalian cells in cooperation with adenovirus E1A and to repress p53 tumour suppressor functions. The biochemical consequences of SUMO1 conjugation of 55K have so far remained elusive. Here, we report that E1B-55K physically interacts with different isoforms of the tumour suppressor protein promyelocytic leukaemia (PML). We show that E1B-55K binds to PML isoforms IV and V in a SUMO1-dependent and -independent manner. Interaction with PML-IV promotes the localization of 55K to PML-containing subnuclear structures (PML-NBs). In virus-infected cells, this process is negatively regulated by other viral proteins, indicating that binding to PML is controlled through reversible SUMOylation in a timely coordinated manner. These results together with earlier work are consistent with the idea that SUMOylation regulates targeting of E1B-55K to PML-NBs, known to control transcriptional regulation, tumour suppression, DNA repair and apoptosis. Furthermore, they suggest that SUMO1-dependent modulation of p53-dependent growth suppression through E1B-55K PML-IV interaction has a key role in adenovirus-mediated cell transformation.


Subject(s)
Adenovirus E1B Proteins/metabolism , Cell Transformation, Viral/physiology , Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , SUMO-1 Protein/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cell Line , Fluorescent Antibody Technique, Indirect , Gene Expression , Gene Expression Regulation, Neoplastic/physiology , Humans , Immunoblotting , Immunoprecipitation , Promyelocytic Leukemia Protein , Protein Binding , Protein Isoforms/metabolism , Rats , Transfection
5.
J Virol ; 72(10): 7960-71, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9733834

ABSTRACT

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.


Subject(s)
Adenoviridae/genetics , Adenovirus E1B Proteins/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Adenovirus E1B Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Biological Transport , Cell Line , DNA, Complementary , HeLa Cells , Humans , Molecular Sequence Data , Protein Binding , RNA, Messenger/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , Rats , Sequence Homology, Amino Acid
6.
Genes Chromosomes Cancer ; 4(1): 69-74, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1377011

ABSTRACT

Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line J1 was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity.


Subject(s)
Burkitt Lymphoma/genetics , Chromosomes, Human, Pair 8/ultrastructure , Nucleic Acid Hybridization , Translocation, Genetic , Burkitt Lymphoma/pathology , Chromosome Banding , Chromosomes, Human, Pair 2/ultrastructure , DNA Probes , Fluorescent Dyes , Humans , Interphase , Male , Metaphase , Microscopy, Fluorescence , Oncogenes , Plasmids , Tumor Cells, Cultured/ultrastructure
7.
Proc Natl Acad Sci U S A ; 86(9): 3257-60, 1989 May.
Article in English | MEDLINE | ID: mdl-2470097

ABSTRACT

Chromosomal translocations in Burkitt lymphoma and mouse plasmacytomas typically lie within or near the protooncogene MYC. In some instances, however, these tumors contain variant translocations with breakpoints located more distant from and downstream of MYC, in a domain commonly known as pvt-1. Until now, there has been no evidence that pvt-1 marks the location of a functional gene. Here we report the identification of a large transcriptional unit in human DNA that includes pvt-1. We have designated this unit as PVT. PVT begins 57 kilobase pairs downstream of MYC and occupies a minimum of 200 kilobase pairs of DNA. Some of the translocations that occur downstream of MYC in Burkitt lymphoma transect PVT; others lie between the two genes. None of the translocations we have studied appear to enhance transcription from an intact allele of PVT (indeed, they may inactivate that transcription), but some are associated with the production of abundant and anomalous 0.8- to 1.0-kilobase RNAs that contain the 5' exon of PVT and sequences transcribed from the constant region of an immunoglobulin gene (the reciprocal participant in the translocation). Identification of PVT should facilitate the exploration of how translocations downstream of MYC and insertions of retroviral DNA in the vicinity of pvt-1 might contribute to tumorigenesis.


Subject(s)
Burkitt Lymphoma/genetics , Genetic Variation , Transcription, Genetic , Translocation, Genetic , Base Composition , Base Sequence , Chimera , Cloning, Molecular , Cosmids , DNA/genetics , DNA/isolation & purification , Exons , Humans , Introns , Molecular Sequence Data , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-myc , Proto-Oncogenes , RNA/genetics , Sequence Homology, Nucleic Acid
8.
Mol Cell Biol ; 9(5): 2105-13, 1989 May.
Article in English | MEDLINE | ID: mdl-2747644

ABSTRACT

The variant translocations t(2;8) in Burkitt's lymphoma cells join band q24 of chromosome 8, distal from c-myc, to the Igkappa locus, with considerable variation in the location of the breakpoints on chromosome 8. We report the cloning and molecular characterization of a chromosome 8 region, distal from the c-myc locus, which encompasses the breakpoints of the Burkitt's lymphoma cell lines BL64, BL21, and LY91 within 11 kilobase pairs, termed provisionally bvr-1 (Burkitt's variants' rearranging region 1). Using probes from the c-myc, the bvr-1, and the human pvt-1 loci obtained by chromosome walking coupled with pulsed-field gel electrophoresis, we have constructed a physical map of the region 3' of c-myc. We map bvr-1 and pvt-1 about 140 and 260 kilobase pairs, respectively, distal from c-myc.


Subject(s)
Burkitt Lymphoma/genetics , Chromosomes, Human, Pair 2 , Chromosomes, Human, Pair 8 , Translocation, Genetic , Base Sequence , Cell Line , DNA, Neoplasm/genetics , Genetic Variation , Humans , Molecular Sequence Data , Proto-Oncogenes , Restriction Mapping
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