Biomedical subjects

Elisabeth Kremmer

Publications and source records attributed to Elisabeth Kremmer.

At least 37 records · Page 2Linked to original sources

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

Biochemical characterisation of the proteins encoded by the DiGeorge critical region 6 (DGCR6) genes.

The DiGeorge critical region 6 (DGCR6) gene exists in two highly homologous copies (DGCR6 and DGCR6L) on chromosome 22q11 and is deleted in patients with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). The DGCR6 mRNA levels are increased in metastatic mammary tumour cells and regulate the expression of neighbouring genes at the 22q11 region. Newly developed monoclonal antibodies detected predominantly nuclear phosphoproteins of approximately 25 kDa, with low expression levels in the cytoplasm. Both proteins have half-lives of about 2.5 h. Exogenously expressed DGCR6 and DGCR6L migrated with slightly different mobility in SDS-gels in accordance with two immunoreactive bands observed for the endogenous proteins. DGCR6 is found at low levels in primary human fibroblasts or peripheral blood mononuclear cells, while tumour cells, B-cells transformed by EBV as well as activated normal human T cells, contain elevated levels of the proteins. The proteins are differentially expressed in mammalian tissues, with high protein levels in heart, liver and skeletal muscle. These observations are important as some patients with DGCR6 syndrome exhibit a T-cell deficiency and/or cardiac malformations. As the DGCR6 protein(s) influence gene expression in trans, we analysed the influence of DGCR6/DGCR6L on the Epstein-Barr virus-encoded oncoproteins EBNA2 and EBNA3c in the activation of the viral LMP1 promoter, as well as LMP1-mediated activation of NFkB, but found no effect in either setting.

Animals↗

PubMed2005

PARP-10, a novel Myc-interacting protein with poly(ADP-ribose) polymerase activity, inhibits transformation.

The proto-oncoprotein c-Myc functions as a transcriptional regulator that controls different aspects of cell behavior, including proliferation, differentiation, and apoptosis. In addition, Myc proteins have the potential to transform cells and are deregulated in the majority of human cancers. Several Myc-interacting factors have been described that mediate part of Myc's functions in the control of cell behavior. Here, we describe the isolation of a novel 150 kDa protein, designated PARP-10, that interacts with Myc. PARP-10 possesses domains with homology to RNA recognition motifs and to poly(ADP-ribose) polymerases (PARP). Molecular modeling and biochemical analysis define a PARP domain that is capable of ADP-ribosylating PARP-10 itself and core histones, but neither Myc nor Max. PARP-10 is localized to the nuclear and cytoplasmic compartments that is controlled at least in part by a Leu-rich nuclear export sequence (NES). Functionally, PARP-10 inhibits c-Myc- and E1A-mediated cotransformation of rat embryo fibroblasts, a function that is independent of PARP activity but that depends on a functional NES. Together, our findings define a novel PARP enzyme involved in the control of cell proliferation.

Amino Acid Sequence↗

PubMed2005

Mad1 function in cell proliferation and transcriptional repression is antagonized by cyclin E/CDK2.

The transcription factors of the Myc/Max/Mad network play essential roles in the regulation of cellular behavior. Mad1 inhibits cell proliferation by recruiting an mSin3-corepressor complex that contains histone deacetylase activity. Here we demonstrate that Mad1 is a potent inhibitor of the G(1) to S phase transition, a function that requires Mad1 to heterodimerize with Max and to bind to the corepressor complex. Cyclin E/CDK2, but not cyclin D and cyclin A complexes, fully restored S phase progression. In addition inhibition of colony formation and gene repression by Mad1 were also efficiently antagonized by cyclin E/CDK2. This was the result of cyclin E/CDK2 interfering with the interaction of Mad1 with HDAC1 and reducing HDAC activity. Our findings define a novel interplay between the cell cycle regulator cyclin E/CDK2 and Mad1 and its associated repressor complex and suggests an additional mechanism how cyclin E/CDK2 affects the G(1) to S phase transition.

3T3-L1 Cells↗

PubMed2005

Dendritic cells govern induction and reprogramming of polarized tissue-selective homing receptor patterns of T cells: important roles for soluble factors and tissue microenvironments.

Tissue-selective homing is established during naive T cell activation by the tissue microenvironment and tissue-specific dendritic cells (DC). The factors driving induction and maintenance of T cell homing patterns are still largely unknown. Here we show that soluble factors produced during the interaction of T cells with CD11c(+) DC isolated from skin- or small intestine-associated tissues differentially modulate expression of the corresponding tissue-selective homing receptors (E-selectin ligands and alpha4beta7 integrin/CCR9, respectively) on murine CD8(+) T cells. Injection of tissue-specific DC via different routes induces T cells with homing receptors characteristic of the corresponding local tissue microenvironment, independent of the origin of the DC. These data indicate an important role for signals delivered in trans. Moreover, DC can reprogram the homing receptor expression on T cells previously polarized in vitro for homing to skin or small intestine. Importantly, skin-homing memory T cells stimulated directly ex vivo can also be reprogrammed by intestinal DC to a gut-homing phenotype. Our results show that tissue-selective homing receptor expression on effector and memory T cells is governed by inductive as well as suppressive signals from both DC and tissue microenvironments.

Animals↗

PubMed2005

Differential expression of activation-induced cytidine deaminase (AID) in nodular lymphocyte-predominant and classical Hodgkin lymphoma.

Activation-induced cytidine deaminase (AID) is indispensable for class switch recombination and somatic hypermutation of immunoglobulin genes. Expression of AID has been detected in germinal centre centroblasts and in lymphomas derived from germinal centre cells. However, in situ studies of AID expression have until now been hampered by a lack of antibodies suitable for immunohistochemistry. To overcome this problem, an AID-specific monoclonal antibody suitable for immunohistochemical staining of formalin-fixed, paraffin wax-embedded tissue sections has been generated. This antibody was shown to detect AID expression in normal germinal centre B-cells as well as in non-Hodgkin lymphomas with a putative germinal centre origin. Using this antibody, a virtually exclusive cytoplasmic localization of AID in normal and neoplastic B-cells is shown. Employing a combination of immunohistochemistry and AID-specific in situ hybridization, it is demonstrated that AID is consistently expressed in the neoplastic cells of nodular lymphocyte-predominant Hodgkin lymphoma (HLnlp) but only infrequently in classical HL (cHL). This is in keeping with the notion that tumour cells of HLnlp represent transformed germinal centre B-cells showing evidence of somatic hypermutation. AID represents an additional marker useful in the differential diagnosis of HLnlp and cHL.

Antibodies, Monoclonal↗

PubMed2005

Prominent transcription of zebrafish N-myc (nmyc1) in tectal and retinal growth zones during embryonic and early larval development.

Because of its oncogenic capacity and expression restricted to embryonic and newborn tissues, the N-myc proto-oncogene is suggested to play a key role in vertebrate organogenesis as well as in the control of cell proliferation and differentiation. To further approach the developmental function of N-myc, we cloned full-length zebrafish N-myc (nmyc1) and analyzed its expression in the embryo and early larva. nmyc1 transcription is initiated at the mid-blastula stage. At somitogenesis stages, its expression was detected in the retina, midbrain, posterior hindbrain and presumptive spinal cord. nmyc1 was also transcribed in the endoderm and its derivatives as well as in branchial arches. At later developmental stages, posterior neural expression of nmyc1 was switched off, but expression remained intense in the brain, mainly in the optic tectum, cerebellar plate and dorsal rhombomere 2. Comparison of nmyc1 transcription with proliferation zones using a M phase mitotic marker revealed that nmyc1 expression is specifically associated with mitosis in the optic tectum and the retina. This result contrasts with previous studies in other vertebrates where N-myc expression can persist in differentiating cells.

Amino Acid Sequence↗

PubMed2005

Sex-dependent susceptibility to Listeria monocytogenes infection is mediated by differential interleukin-10 production.

It is well documented that sex-dependent factors affect susceptibility to infection, with most mouse models demonstrating higher resistance in females. We made the unexpected observation that infection with the intracellular bacterium Listeria monocytogenes showed an opposite pattern in several commonly used inbred mouse strains: female C57BL/6J, BALB/c, C3H/HeN, and CBA/J mice were significantly more susceptible to Listeria infection. The pronounced sensitivity of females to Listeria, which was revealed by significantly higher lethality rates, correlated also with increased bacterial numbers in organ tissues (spleen and liver) and several immunological changes in peripheral blood samples. Surprisingly, increased severity of infection in females was associated with elevated interleukin-10 (IL-10) levels in plasma. Experiments using Il10 knockout mice, for which no differences between the susceptibilities of males and females to Listeria infection could be detected, confirmed the important role of this immunosuppressive cytokine for the outcome of disease. Our findings are likely to have clinical relevance, since similar sex differences with regard to infection with Listeria monocytogenes and other intracellular pathogens have been reported for humans.

Animals↗

PubMed2005

A somatic knockout of CBF1 in a human B-cell line reveals that induction of CD21 and CCR7 by EBNA-2 is strictly CBF1 dependent and that downregulation of immunoglobulin M is partially CBF1 independent.

CBF1 is a cellular highly conserved DNA binding factor that is ubiquitously expressed in all tissues and acts as a repressor of cellular genes. In Epstein-Barr virus growth-transformed B-cell lines, CBF1 serves as a central DNA adaptor molecule for several viral proteins, including the viral transactivator Epstein-Barr virus nuclear antigen 2 (EBNA-2). EBNA-2 binds to CBF1 and thereby gains access to regulatory regions of target genes and activates transcription. We have inactivated the CBF1 gene by homologous recombination in the human B-cell line DG75 and characterized changes in cellular gene expression patterns upon loss of CBF1 and activation of EBNA-2. CBF1-negative DG75 cells were viable and proliferated at wild-type rates. Loss of CBF1 was not sufficient to release repression of the previously described EBNA-2 target genes CD21 or CCR7, whereas induction of both target genes by EBNA-2 required CBF1. In contrast, repression of immunoglobulin M by EBNA-2 was mainly CBF1 independent. CBF1-negative DG75 B cells thus provide an excellent tool to dissect CBF1-dependent and -independent functions exerted by the EBNA-2 protein in future studies.

Alleles↗

PubMed2005

The Epstein-Barr virus EBNA-LP protein preferentially coactivates EBNA2-mediated stimulation of latent membrane proteins expressed from the viral divergent promoter.

The mechanistic contribution of the Epstein-Barr virus (EBV) EBNA-LP protein to B-cell immortalization remains an enigma. However, previous studies have indicated that EBNA-LP may contribute to immortalization by enhancing EBNA2-mediated transcriptional activation of the LMP-1 gene. To gain further insight into the potential role EBNA-LP has in EBV-mediated B-cell immortalization, we asked whether it is a global or gene-specific coactivator of EBNA2 and whether coactivation requires interaction between these proteins. In type I Burkitt's lymphoma cells, we found that EBNA-LP strongly coactivated EBNA2 stimulation of LMP-1 and LMP2B RNAs, which are expressed from the viral divergent promoter. Surprisingly, the viral LMP2A gene and cellular CD21 and Hes-1 genes were induced by EBNA2 but showed no further induction after EBNA-LP coexpression. We also found that EBNA-LP did not stably interact with EBNA2 in coimmunoprecipitation assays, even though the conditions were adequate to observe specific interactions between EBNA2 and its cellular cofactor, CBF1. Colocalization between EBNA2 and EBNA-LP was not detectable in EBV-transformed cell lines or transfected type I Burkitt's cells. Finally, no significant interactions between EBNA2 and EBNA-LP were found with mammalian two-hybrid assays. From this data, we conclude that EBNA-LP is not a global coactivator of EBNA2 targets, but it preferentially coactivates EBNA2 stimulation of the viral divergent promoter. While this may require specific transient interactions between these proteins that only occur in the context of the divergent promoter, our data strongly suggest that EBNA-LP also cooperates with EBNA2 through mechanisms that do not require direct or indirect complex formation between these proteins.

B-Lymphocytes↗

PubMed2004

PIASy-deficient mice display modest defects in IFN and Wnt signaling.

Protein inhibitors of activated STATs (PIAS) represent a small family of nuclear proteins that modulate the activity of many transcription factors and act as E3 ligases for covalent modification of proteins with the small ubiquitin-like modifier (SUMO). In particular, PIASy has been shown to inhibit the activation of gene expression by the IFN-responsive transcription factor STAT1 and the Wnt-responsive transcription factor LEF1. To assess the function of PIASy in vivo, we generated and analyzed mice carrying a targeted mutation of the Piasy gene. We find that homozygous mutant mice have no obvious morphological defects and have a normal distribution of lymphocyte populations. Molecular analysis of signaling in response to IFN-gamma and Wnt agonists revealed a modest reduction in the activation of endogenous and transfected target genes. Two-dimensional analysis of total proteins and SUMO-modified proteins in transformed pre-B cells showed no significant differences between wild-type mice and homozygous mutant mice. Taken together, our data indicate that PIASy has a modest effect on cytokine and Wnt signaling, suggesting a redundancy with other members of the family of PIAS proteins.

Animals↗

PubMed2004

The Drosophila MBD2/3 protein mediates interactions between the MI-2 chromatin complex and CpT/A-methylated DNA.

Methyl-DNA binding proteins play an important role in epigenetic gene regulation. The Drosophila genome encodes a single protein (MBD2/3) with extended homologies to the vertebrate methyl-DNA binding proteins MBD2 and MBD3. However, very little is known about its functional properties. We have now characterized an MBD2/3 null mutant allele that is viable and fertile. This mutation caused a strong dominant suppression of position-effect variegation and also resulted in a high rate of chromosome segregation defects during early embryogenesis. Confocal analysis of mutant embryos showed local displacement of MI-2 from DNA and indicated that MBD2/3 is associated with only a subset of MI-2 complexes. In addition, band shift experiments demonstrated a specific binding of MBD2/3 to CpT/A-methylated DNA, which reflects the endogenous DNA methylation pattern of Drosophila. Consistently, the localization of MBD2/3 was disrupted in embryos with reduced levels of DNA methylation. Our data provide novel insights into the function of MBD2/3 proteins and strongly suggest the existence of methylation-dependent chromatin structures in Drosophila.

Adenosine Triphosphatases↗

PubMed2004

Expression of tumor necrosis factor receptor-associated factor 1 in nasopharyngeal carcinoma: possible upregulation by Epstein-Barr virus latent membrane protein 1.

EBV infection is associated with virtually all cases of undifferentiated NPC, and the EBV-encoded LMP1 is expressed in a proportion of cases. LMP1 has transforming functions similar to members of the TNF receptor family and activates intracellular signaling cascades through interaction with TRAFs. In B cells, expression of TRAF1 is in turn upregulated by LMP1. LMP1 signaling in epithelial cells may be affected by the presence or absence of TRAF1. By immunohistochemistry, we detected TRAF1 expression in 17 of 42 (40%) EBV+ undifferentiated NPCs. All 7 LMP1+ NPC biopsies were also TRAF1+. Using an RNAse protection assay, high-level TRAF1 expression was detected in an LMP1-expressing NPC-derived cell line (C15) and expression was weaker in 2 LMP1- cell lines (C17, C19). Finally, LMP1 upregulated TRAF1 expression in an EBV- keratinocyte cell line. Our results demonstrate that TRAF1 is expressed in NPC tumor cells in vivo and suggest that TRAF1 expression may be upregulated by LMP1 in NPC. An antiapoptotic function has been proposed for TRAF1, and this may be relevant for the pathogenesis of NPC.

Antigens, Viral↗

PubMed2004

Extending the working range of immunoanalysis by exploitation of two monoclonal antibodies.

A newly established rat monoclonal antibody (mAb) for isoproturon, namely, IOC 10G7, is described. This mAb shows a standard curve for isoproturon in phosphate-buffered saline with a test midpoint of 5.5 +/- 1.8 microg/L (n = 15). In combination with the formerly developed mAb IOC 7E1, IOC 10G7 can be exploited to extend the working range for the analysis of isoproturon. Both antibodies were formatted into a competitive enzyme-linked immunosorbent assay (ELISA), using the same enzyme-tracer. MAb IOC 7E1 and mAb IOC 10G7 have different affinities for the target compound, but the signal-response curves of the single antibodies overlap. Cross-reactivity (CR) patterns of both antibodies are comparable, showing the highest CR for the metabolite 1-(4-isopropylphenyl)-3-methylurea (IOC 10G7, 9%; IOC 7E1, 19%). The system described here includes the combined, but individual, usage of both assays on one microtiter plate, as well as the strategy for mixing the two antibodies for the utilization in one assay. When standards are performed in Milli-Q water, the working range for isoproturon with the individual ELISAs using mAb IOC 7E1 is from 0.01 to 1 microg/L (test midpoint = 0.11 +/- 0.03 microg/L; n = 17) and with IOC 10G7, it is 1-100 microg/L (test midpoint = 10.3 +/- 1.6 microg/L; n = 32). The working range with mixed antibodies is usually on the order of 0.03-30 microg/L (test midpoint = 0.5 +/- 0.2 microg/L; n = 17). These strategies (mAbs individually and mixed) cover a range of 4 and 3 orders of magnitude, respectively. As a demonstration, water samples of different origins and an extract of mixed sediment were analyzed. The advantages of these strategies are discussed.

Animals↗

PubMed2004

Protein arginine methylation during lytic adenovirus infection.

Arginine methylation of proteins affects major processes in the cell, including transcriptional regulation, mRNA metabolism, signal transduction and protein sorting. Arginine methylation of Ad (adenovirus) E1B 55-kDa-associated protein E1B-AP5 was recently described by us [Kzhyshkowska, Schutt, Liss, Kremmer, Stauber, Wolf and Dobner (2001) Biochem. J. 358, 305-314]. In this first example of protein arginine methylation analysis in Ad-infected cells, we investigated methylation of the E1B-AP5 and the viral L4-100 kDa protein. We demonstrate that E1B-AP5 methylation is enhanced during the course of infection in a cell-type-specific manner. We also show that L4-100 kDa is efficiently methylated in Ad-infected cells. L4-100 kDa formed complex with methyltransferase in vivo during productive infection, and can be methylated by HRMT1L2 (human protein arginine methyltransferase 1) in vitro. Comparative analysis of E1B-AP5 and L4-100 kDa protein methylation in Ad-infected HeLa, MCF-7 and H1299 cells revealed that the profile of protein arginine methylation correlates with the efficiency of Ad proteins production. Our results suggest that protein arginine methylation is an important host-cell function required for efficient Ad replication.

Adenoviridae↗

PubMed2004

Cell death triggered by alpha-emitting 213Bi-immunoconjugates in HSC45-M2 gastric cancer cells is different from apoptotic cell death.

PURPOSE: Radioimmunotherapy with alpha-particle-emitting nuclides, such as 213Bi, is a promising concept for the elimination of small tumour nodules or single disseminated tumour cells. The aim of this study was to investigate cellular damage and the mode of cell death triggered by 213Bi-immunoconjugates. METHODS: Human gastric cancer cells (HSC45-M2) expressing d9-E-cadherin were incubated with different levels of activity of 213Bi-d9MAb targeting d9-E-cadherin and 213Bi-d8MAb, which does not bind to d9-E-cadherin. Micronucleated (M) cells, abnormal (A) cells and apoptotic (A) [(MAA)] cells were scored microscopically in the MAA assay following fluorescent staining of nuclei and cytoplasm. Chromosomal aberrations were analysed microscopically following Giemsa staining. The effect of z-VAD-fmk, known to inhibit apoptosis, on the prevention of cell death was investigated following treatment of HSC45-M2 cells with sorbitol as well as 213Bi-d9MAb. Activation of caspase 3 after incubation of HSC45-M2 cells with both sorbitol and 213Bi-d9MAb was analysed via Western blotting. RESULTS: Following incubation of HSC45-M2 human gastric cancer cells expressing d9-E-cadherin with 213Bi-d9MAb the number of cells killed increased proportional to the applied activity concentration. Microscopically visible effects of alpha-irradiation of HSC45-M2 cells were formation of micronuclei and severe chromosomal aberrations. Preferential induction of these lesions with specific 213Bi-d9MAb compared with unspecific 213Bi-d8MAb (not targeting d9-E-cadherin) was not observed if the number of floating, i.e. unbound 213Bi-immunoconjugates per cell exceeded 2 x 10(4), most likely due to intense crossfire. In contrast to sorbitol-induced cell death, cell death triggered by 213Bi-immunoconjugates was independent of caspase 3 activation and could not be inhibited by z-VAD-fmk, known to suppress the apoptotic pathway. CONCLUSION: 213Bi-immunoconjugates seem to induce a mode of cell death different from apoptosis in HSC45-M2 cells.

Alpha Particles↗

PubMed2004

Thymic T cell development and progenitor localization depend on CCR7.

T cell differentiation in the adult thymus depends on sequential interactions between lymphoid progenitors and stromal cells found in distinct regions of the cortex and medulla. Therefore, migration of T cell progenitors through distinct stromal environments seems to be a crucial process regulating differentiation and homeostasis inside the thymus. Here we show that CCR7-deficient mice are distinguished by a disturbed thymic architecture, impaired T cell development, and decreased numbers of the thymocytes. Analysis of developing double negative (CD4-CD8-) pool of wild-type thymus reveals that CCR7 expression is restricted to a CD25intCD44+ subpopulation. Correspondingly, CCR7 deficiency results in an accumulation of this population in mutant thymus. Furthermore, immunohistology shows that in CCR7-deficient mice CD25+CD44+ cells accumulate at the cortico-medullary junction, suggesting that CCR7 signaling regulates the migration of early progenitors toward the outer thymic cortex, thereby continuing differentiation. Results obtained from mixed bone marrow chimeras support this view, since the development of CCR7-deficient thymocytes is also disturbed in a morphologically intact thymus. Thus, our findings establish an essential role for CCR7 in intrathymic migration and proper T cell development.

Animals↗

PubMed2004

Erythroid differentiation regulator (EDR), a novel, highly conserved factor I. Induction of haemoglobin synthesis in erythroleukaemic cells.

In serum-free WEHI-3 supernatants an activity was detected inducing haemoglobin synthesis in human and murine erythroleukaemia cell lines. The absolute numbers of benzidine-positive cells induced with either DMSO or WEHI-3-conditioned medium were comparable. Terminal differentiation was not observed. An expression library from WEHI-3 RNA aided by PCR cloning revealed an open reading frame corresponding to a 209 amino acid protein. This was 100% identical to a sequence from human stimulated peripheral blood mononuclear cells. In contrast to human RNA, mouse RNA exhibited multiple bands of pre-mRNA in Northern blots. The gene was provisionally termed erythroid differentiation regulator (edr). In mammalian cells EDR is mostly expressed as a 56 kDa dimer showing higher activity than the recombinant monomer. The activity profile is bell-shaped. Expression was observed in many normal mouse tissues, yet in haematopoiesis it was largely confined to CD34+ cells. It was enhanced by a series of stimuli such as phorbol ester, and transformed cells generally showed a higher level of EDR expression than normal ones. The protein is localized at the inner side of the cytoplasmic membrane and is released in part via vesicles. In view of the broad range of EDR-expressing tissues the function obviously exceeds haemoglobin synthesis induction. Involvement in cell survival and growth control has been observed and will be dealt with in detail elsewhere.

Animals↗