Biomedical subjects

Michael Hölzel

Publications and source records attributed to Michael Hölzel.

9 recordsLinked to original sources

PubMed2007

C-myc activation impairs the NF-kappaB and the interferon response: implications for the pathogenesis of Burkitt's lymphoma.

Deregulation of the proto-oncogene c-myc is a key event in the pathogenesis of many tumors. A paradigm is the activation of the c-myc gene by chromosomal translocations in Burkitt lymphoma (BL). Despite expression of a restricted set of Epstein-Barr viral (EBV) antigens, BL cells are not recognized by antigen-specific cytotoxic T cells (CTLs) because of their inability to process and present HLA class I-restricted antigens. In contrast, cells of EBV-driven posttransplant lymphoproliferative disease (PTLD) are recognized and rejected by EBV-specific CTLs. It is not known whether the poor immunogenicity of BL cells is due to nonexpression of viral antigens, overexpression of c-myc, or both. To understand the basis for immune recognition and escape, we have compared the mRNA expression profiles of BL and EBV-immortalized cells (as PTLD model). Among the genes expressed at low level in BL cells, we have identified many genes involved in the NF-kappaB and interferon response that play a pivotal role in antigen presentation and immune recognition. Using a cell line in which EBNA2 and c-myc can be regulated at will, we show that c-MYC negatively regulates STAT1, the central player linking the Type-I and Type-II interferon response. Switching off c-myc expression leads to STAT1 induction through a direct and indirect mechanism involving induction of Type-I interferons. c-MYC thus masks an interferon-inducing activity in these cells. Our findings imply that immune escape of tumor cells is not only a matter of in vivo selection but may be additionally promoted by activation of a cellular oncogene.

Blotting, Western↗

PubMed2006

The BRCT domain of mammalian Pes1 is crucial for nucleolar localization and rRNA processing.

The nucleolar protein Pes1 interacts with Bop1 and WDR12 in a stable complex (PeBoW-complex) and its expression is tightly associated with cell proliferation. The yeast homologue Nop7p (Yph1p) functions in both, rRNA processing and cell cycle progression. The presence of a BRCT-domain (BRCA1 C-terminal) within Pes1 is quite unique for an rRNA processing factor, as this domain is normally found in factors involved in DNA-damage or repair pathways. Thus, the function of the BRCT-domain in Pes1 remains elusive. We established a conditional siRNA-based knock-down-knock-in system and analysed a panel of Pes1 truncation mutants for their functionality in ribosome synthesis in the absence of endogenous Pes1. Deletion of the BRCT-domain or single point mutations of highly conserved residues caused diffuse nucleoplasmic distribution and failure to replace endogenous Pes1 in rRNA processing. Further, the BRCT-mutants of Pes1 were less stable and not incorporated into the PeBoW-complex. Hence, the integrity of the BRCT-domain of Pes1 is crucial for nucleolar localization and its function in rRNA processing.

Cell Line, Tumor↗

PubMed2006

Rapid conditional knock-down-knock-in system for mammalian cells.

RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down-knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein.

Cell Cycle Proteins↗

PubMed2006

Dominant-negative Pes1 mutants inhibit ribosomal RNA processing and cell proliferation via incorporation into the PeBoW-complex.

The nucleolar PeBoW-complex, consisting of Pes1, Bop1 and WDR12, is essential for cell proliferation and processing of ribosomal RNA in mammalian cells. Here we have analysed the physical and functional interactions of Pes1 deletion mutants with the PeBoW-complex. Pes1 mutants M1 and M5, with N- and C-terminal truncations, respectively, displayed a dominant-negative phenotype. Both mutants showed nucleolar localization, blocked processing of the 36S/32S precursors to mature 28S rRNA, inhibited cell proliferation, and induced high p53 levels in proliferating, but not in resting cells. Mutant M1 and M5 proteins associated with large pre-ribosomal complexes and co-immunoprecipitated Bop1 and WDR12 proteins indicating their proper incorporation into the PeBoW-complex. We conclude that the dominant-negative effect of the M1 and M5 mutants is mediated by the impaired function of the PeBoW-complex.

Animals↗

PubMed2005

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system.

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1.

Animals↗

PubMed2005

Mammalian WDR12 is a novel member of the Pes1-Bop1 complex and is required for ribosome biogenesis and cell proliferation.

Target genes of the protooncogene c-myc are implicated in cell cycle and growth control, yet the linkage of both is still unexplored. Here, we show that the products of the nucleolar target genes Pes1 and Bop1 form a stable complex with a novel member, WDR12 (PeBoW complex). Endogenous WDR12, a WD40 repeat protein, is crucial for processing of the 32S precursor ribosomal RNA (rRNA) and cell proliferation. Further, a conditionally expressed dominant-negative mutant of WDR12 also blocks rRNA processing and induces a reversible cell cycle arrest. Mutant WDR12 triggers accumulation of p53 in a p19ARF-independent manner in proliferating cells but not in quiescent cells. Interestingly, a potential homologous complex of Pes1-Bop1-WDR12 in yeast (Nop7p-Erb1p-Ytm1p) is involved in the control of ribosome biogenesis and S phase entry. In conclusion, the integrity of the PeBoW complex is required for ribosome biogenesis and cell proliferation in mammalian cells.

Animals↗

PubMed2005

Dissection of transcriptional programmes in response to serum and c-Myc in a human B-cell line.

Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix-loop-helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.

B-Lymphocytes↗

PubMed2003

A role for c-Myc in the regulation of ribosomal RNA processing.

The proto-oncogene c-myc encodes a basic helix-loop-helix leucine zipper transcription factor (c-Myc) that has a profound role in growth control and cell cycle progression. Previous microarray studies identified various classes of c-Myc target genes, including genes involved in ribosome biogenesis. By screening the human B-cell line P493-6 and rat fibroblasts conditionally expressing c-Myc, we could substantially extend the list of c-Myc target genes, particularly those required for ribosome biogenesis. The identification of 38 new c-Myc target genes with nucleolar function, prompted us to investigate processing of ribosomal RNA (rRNA). Using pulse-chase labelling experiments we show that c-Myc regulates the efficiency of rRNA maturation. In serum-stimulated P493-6 cells, only the processing of the 47S rRNA precursor to mature 18S and 28S rRNA, but not the synthesis of the 47S transcript, was dependent on the presence of c-Myc. As processing of rRNA is sensitive to inhibition of cyclin-dependent kinase (cdk) activity by roscovitine, we conclude that c-Myc regulates cell growth and proliferation by the coordinated induction of cdk activity and rRNA processing.

Animals↗

PubMed2003

FANCD2 protein is expressed in proliferating cells of human tissues that are cancer-prone in Fanconi anaemia.

Fanconi anaemia (FA) is an inherited form of progressive pancytopenia associated with developmental defects, chromosomal instability, and cancer predisposition. At least seven distinct FA proteins function in concert to protect the genome, a key step being the activation of FANCD2 by mono-ubiquitination. This paper reports an immunohistochemical analysis of FANCD2 expression in normal human tissue. The highest expression was observed in maturing spermatocytes and fetal oocytes (consistent with a role for FANCD2 in meiosis) and in germinal centre cells of the spleen, tonsil, and lymph nodes (consistent with a role in proliferation). FANCD2 expression was also seen in tissues predisposed to cancer development in FA patients: haematopoietic cells, especially in the fetus, and squamous cell epithelia, particularly in the head and neck region and uterine cervix. FANCD2 expression was also occasionally seen in the breast and Fallopian tube epithelium, the respiratory epithelium of the trachea, and the exocrine cells of the pancreas, indicating that these tissues may also be cancer-prone in FA. FANCD2 expression is frequently expressed in proliferating cells as demonstrated by Ki-67 immunofluorescence double staining, consistent with a function of FANCD2 in DNA replication.

Adult↗