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ABSTRACT: A block of single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity associated) gene is associated with variation in body weight. Previous works suggest that increased expression of FTO, which encodes a 2-oxoglutarate-dependent nucleic acid demethylase, leads to increased body weight, although the underlying mechanism has remained unclear. To elucidate the function of FTO, we examined the consequences of altered FTO levels in cultured cells and murine brain. Here we show that a knockdown of FTO in HEK293 cells affects the transcripts levels of genes involved in the response to starvation, whereas overexpression of FTO affects the transcript levels of genes related to RNA processing and metabolism. Subcellular localization of FTO further strengthens the latter notion. Using immunocytochemistry and confocal laser scanning microscopy, we detected FTO in nuclear speckles and - to a lesser and varying extent - in the nucleoplasm and nucleoli of HEK293, HeLa and MCF-7 cells. Moreover, RNA modification analyses revealed that loss of Fto affects the 3-methyluridine/uridine and pseudouridine/uridine ratios in total brain RNA. We conclude that altered levels of FTO have multiple and diverse consequences on RNA modifications and the transcriptome.European Journal of Human Genetics advance online publication, 8 August 2012; doi:10.1038/ejhg.2012.168.
European journal of human genetics: EJHG 08/2012; · 3.56 Impact Factor
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ABSTRACT: Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38-/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL).
PLoS ONE 01/2012; 7(3):e34347. · 4.09 Impact Factor
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ABSTRACT: Miz-1 is a Broad-complex, Tramtrack and Bric-à-brac/pox virus zinc finger domain (BTB/POZ)-containing protein expressed in lymphoid precursors that can activate or repress transcription. We report in this article that mice expressing a nonfunctional Miz-1 protein lacking the BTB/POZ domain (Miz-1(ΔPOZ)) have a severe differentiation block at the pre-T cell "β-selection" checkpoint, evident by a drastic reduction of CD4(-)CD8(-) double-negative-3 (DN3) and DN4 cell numbers. T cell-specific genes including Rag-1, Rag-2, CD3ε, pTα, and TCRβ are expressed in Miz-1-deficient cells and V(D)J recombination is intact, but few DN3/DN4 cells express a surface pre-TCR. Miz-1-deficient DN3 cells are highly apoptotic and do not divide, which is consistent with enhanced expression of p53 target genes such as Cdkn1a, PUMA, and Noxa. However, neither coexpression of the antiapoptotic protein Bcl2 nor the deletion of p21(CIP1) nor the combination of both relieved Miz-1-deficient DN3/DN4 cells from their differentiation block. Only the coexpression of rearranged TCRαβ and Bcl2 fully rescued Miz-1-deficient DN3/DN4 cell numbers and enabled them to differentiate into DN4TCRβ(+) and double-positive cells. We propose that Miz-1 is a critical factor for the β-selection checkpoint and is required for both the regulation of p53 target genes and proper expression of the pre-TCR to support the proliferative burst of DN3 cells during T cell development.
The Journal of Immunology 08/2011; 187(6):2982-92. · 5.79 Impact Factor
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Christian Schwerk,
Rüdiger Adam,
Julia Borkowski,
Henriette Schneider,
Michael Klenk,
Sascha Zink,
Natascha Quednau,
Nicole Schmidt,
Carolin Stump,
Anubha Sagar,
Barbara Spellerberg,
Tobias Tenenbaum,
Dirk Koczan, Ludger Klein-Hitpass,
Horst Schroten
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ABSTRACT: The Gram-positive zoonotic bacterium Streptococcus suis (S. suis) is responsible for a wide range of diseases including meningitis in pigs and humans. The blood-cerebrospinal fluid (CSF) barrier is constituted by the epithelial cells of the choroid plexus, which execute barrier function also after bacteria have entered the central nervous system (CNS). We show that the bacterial capsule, a major virulence factor, strongly attenuates adhesion of S. suis to the apical side of porcine choroid plexus epithelial cells (PCPEC). Oligonucleotide microarray analysis and quantitative PCR surprisingly demonstrated that adherent wild-type and capsule-deficient S. suis influenced expression of a pronounced similar pattern of genes in PCPEC. Investigation of purified capsular material provided no evidence for a significant role of the capsule. Enriched among the regulated genes were those involved in "inflammatory response", "defense response" and "cytokine activity". These comprised several cytokines and chemokines including the interleukins 6 and 8, which could be detected on protein level. We show that after infection with S. suis the choroid plexus contributes to the immune response by actively producing cytokines and chemokines. Other virulence factors than the bacterial capsule may be relevant in inducing a strong inflammatory response in the CNS during S. suis meningitis.
Microbes and Infection 06/2011; 13(11):953-62. · 3.10 Impact Factor
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ABSTRACT: In addition to mutations in both alleles of the retinoblastoma gene (RB1) alleles, retinoblastomas frequently show additional alterations including loss of chromosome arm 16q. In a previous study, the presence of 16q alterations was found to be associated with diffuse vitreous seeding of this tumor. This growth pattern is clinically important as it determines therapeutic decisions. The present study was designed to test this association and to narrow down the list of candidate genes in the minimal region of genomic loss on chromosome arm 16q. Our data confirm the association of 16q loss and diffuse vitreous seeding and define a minimal region of genomic loss of 6.6 Mb on 16q containing 86 known genes. As retinoblastoma is an embryonic tumor, we assumed that any gene relevant for its progression is likely to show regulated expression during retinogenesis. Microarray expression analysis of RNA from a continuous developmental series of murine retinas identified murine orthologs with regulated expression and these data helped to narrow the number of candidate genes in minimal region to 35. Analysis of gene expression in retinoblastomas with and without the loss of heterozygosity (LOH) on chromosome 16q further reduced this number to 26 candidate genes. One of these genes is cadherin 13 (CDH13) and notably, downregulation of CHD13 has previously been associated with poorer prognosis in various other cancers.
Genes Chromosomes and Cancer 02/2011; 50(5):327-37. · 3.31 Impact Factor
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ABSTRACT: Dysfunction of hepatocyte nuclear factor 4α (HNF4α) has been linked to maturity onset diabetes of the young (MODY1), diabetes type II and possibly to renal cell carcinoma (RCC). Whereas diabetes causing mutations are well known, there are no HNF4A mutations found in RCC. Since so far analyses have been constricted to the promoter and open reading frame of HNF4A, we performed a systematic analysis of the human HNF4A 3'UTR. We identified a short (1724 nt) and long (3180 nt) 3'UTR that are much longer than the open reading frame and conferred a repressive effect in luciferase reporter assays in HEK293 and INS-1 cells. By dissecting the 3'UTR into several pieces, we located two distinct elements of about 400 nt conferring a highly repressive effect. These negative elements A and B are counteracted by a balancer element of 39 nt located within the 5' end of the HNF4A 3'UTR. Dicer knock-down experiments implied that the HNF4A 3'UTR is regulated by miRNAs. More detailed analysis showed that miR-34a and miR-21 both overexpressed in RCC cooperate in downregulation of the HNF4A mRNA. One of the identified miR-34a binding sites is destroyed by SNP rs11574744. The identification of several regulatory elements within the HNF4A 3'UTR justifies the analysis of the 3'UTR sequence to explore the dysfunction of HNF4α in diabetes and RCC.
PLoS ONE 01/2011; 6(11):e27438. · 4.09 Impact Factor
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Ludger Sellmann,
Rene Scholtysik,
Markus Kreuz,
Sandra Cyrull,
Enrico Tiacci,
Jens Stanelle,
Alexander Carpinteiro,
Holger Nückel,
Tanja Boes,
Stefan Gesk,
Reiner Siebert, Ludger Klein-Hitpass,
Ulrich Dührsen,
Jan Dürig,
Ralf Küppers
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ABSTRACT: To understand the influence of chromosomal alterations on gene expression in a genome-wide view, chromosomal imbalances detected by single nucleotide polymorphism (SNP) chips were compared with global gene expression in 16 cases of chronic lymphocytic leukemia (CLL). A strong concordance between chromosomal gain or loss and increased or reduced expression of genes in the affected regions was found, respectively. Regions of uniparental disomy (UPD) were rare and had usually no consistent influence on gene expression, but in one instance, a large UPD was associated with a downregulation of most genes in the affected chromosome. The frequently deleted miRNAs, MIRN15A and MIRN16-1, did not show a reduced expression in cases with monoallelic deletions. The BCL2 protein, considered to be downregulated by these miRNAs, was upregulated not only in CLL with biallelic deletion of MIRN15A and MIRN16-1, but also in cases with monoallelic deletion. This suggests a complex regulation of BCL2 levels in CLL cells. Taken together, in CLL, a global gene dosage effect exists for chromosomal gains and deletions and in some instances for UPDs. We did not confirm a consistent correlation between MIRN15A and MIRN16-1 expression levels and BCL2 protein levels, indicating a complex regulation of BCL2 expression.
Cancer genetics and cytogenetics 12/2010; 203(2):149-60. · 1.54 Impact Factor
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Adrien Daigeler,
Ansgar M Chromik,
Kathrin Haendschke,
Sabine Emmelmann,
Monica Siepmann,
Karin Hensel,
Georg Schmitz, Ludger Klein-Hitpass,
Hans U Steinau,
Marcus Lehnhardt,
Joerg Hauser
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ABSTRACT: Sonodynamic therapy, in combination with ultrasound contrast agents, proved to enhance the uptake of chemotherapeutics in malignant cells. HT1080 fibrosarcoma cells were treated in vitro with a combination of ultrasound SonoVue™-microbubbles and taurolidine (TRD) plus tumor necrosis factor related apoptosis inducing ligand (TRAIL). Apoptosis was measured by TdT-mediated dUTP-biotin nick end labelling (TUNEL) assay and fluorescence activated cell sorting (FACS) analysis. Gene expression was analysed by RNA-microarray. The apoptotic effects of TRD and TRAIL on human fibrosarcoma are enhanced by sonodynamic therapy and additional application of contrast agents, such as SonoVue™ by 25%. A broad change in the expression of genes related to apoptotic pathways is observed when ultrasound and microbubbles act synchronously in combination with the chemotherapeutics (e.g. BIRC3, NFKBIA and TNFAIP3). Some of these genes have already been proven to play a role in programmed cell death in human fibrosarcoma (HSPA1A/HSPA1B, APAF1, PAWR, SOCS2) or were associated with sonication induced apoptosis (CD44). Further studies are needed to explore the options of sonodynamic therapy on soft tissue sarcoma and its molecular mechanisms.
Ultrasound in medicine & biology 11/2010; 36(11):1893-906. · 2.02 Impact Factor
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ABSTRACT: As first shown more than 100 years ago, fertilization of an aged (overripe) egg increases the rate of malformations and embryonic loss in several vertebrates, including possibly humans as well. Since the molecular events in aging eggs may be similar in these species, we established in the frog Xenopus tropicalis a defined protocol for delayed fertilization of eggs. A three-hour delayed fertilization led to a dramatic increase in malformation and mortality. Gene expression profiling revealed that 14% of the polyadenylated maternal transcripts were downregulated upon aging. These transcripts were not degraded, but rather deadenylated as shown for specific maternal mRNAs. The affected transcripts are characterized by a relatively short 3'UTR and a paucity of cytoplasmic polyadenylation elements (CPE) and polyadenylation signals (PAS). Furthermore, maternal mRNAs known to be deadenylated during egg maturation as well as after fertilization were preferentially deadenylated in aged eggs. Taken together our analysis of aging eggs reveals that unfertilized eggs are in a dynamic state that was previously not realized. On the one hand deadenylation of transcripts that are typically deadenylated during egg maturation continues and this implies overripeness of the aged egg in the truest sense of the word. On the other hand transcripts that normally are deadenylated after fertilization loose their poly(A) in the aged egg and this implies that the egg awaiting fertilization starts processes that are normally only observed after fertilization. Based on our novel finding we postulate that the imbalance of the polyadenylated maternal transcripts upon egg aging contributes to the loss of developmental potential. Based on this hypothesis the developmental consequences of downregulation of specific transcripts can be analyzed in future.
PLoS ONE 01/2010; 5(10):e13532. · 4.09 Impact Factor
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Stefanie Bug,
Jan Dürig,
Florian Oyen, Ludger Klein-Hitpass,
Jose I Martin-Subero,
Lana Harder,
Michael Baudis,
Norbert Arnold,
Uwe Kordes,
Ulrich Dührsen,
Reinhard Schneppenheim,
Reiner Siebert
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ABSTRACT: In T-cell prolymphocytic leukemia (T-PLL), chromosomal imbalances affecting the long arm of chromosome 22 are regarded as typical chromosomal aberrations secondary to a TCRAD-TCL1A fusion due to inv(14) or t(14;14). We analyzed recently obtained data from conventional karyotyping, SNP-chip array copy number mapping, genome-wide expression profiling, and interphase fluorescence in situ hybridization (FISH) of inv(14)-positive T-PLL with respect to structural aberrations on chromosome 22. Combined gene chip and interphase FISH analyses revealed interstitial deletions on 22q in 4 of 12 cases, with one case additionally showing a terminal copy number gain. A minimally deleted region of approximately 9.1 Mb was delineated, from 16.2 Mb (22cen) to 25.3 Mb (22q12.1). The distal borders of copy number alterations spread over a region of approximately 8.8 Mb, from 25.2 Mb (22q12.1) to 34 Mb (22q12.3). Mutation screening of candidate tumor suppressor genes SMARCB1 and CHEK2 mapping to the minimally deleted and the breakpoint regions, respectively, in cases with hemizygous deletion, revealed no inactivating mutations. With gene expression profiling, no significantly downregulated genes were identified in the minimally deleted region. We therefore assume that haploinsufficiency or alternative pathomechanisms underlie chromosome 22 aberrations in T-PLL.
Cancer genetics and cytogenetics 08/2009; 192(1):44-7. · 1.54 Impact Factor
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ABSTRACT: The objective of this study was to confirm previous results regarding the differential expression and prognostic significance of the circadian gene CRY1 in chronic lymphocytic leukemia (CLL) patients and its relationship with the expression of other circadian genes and well-established prognostic markers. We also aimed to investigate whether the peripheral circadian machinery may be deregulated in CLL cells. The expression of CRY1, PER1, and PER2 was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in 116 CLL patients. The expression at sequential time points over a 24-h period was measured in six CLL patients and six normal donors. We confirmed the differential expression of CRY1 in ZAP-70(+)/CD38(+) and ZAP-70(-)/CD38(-) CLL samples. Subgroups formed according to CRY1 expression levels differed significantly in time to treatment. This difference was even more pronounced for subgroups stratified by a CRY1 : PER2 expression ratio and the ratio was an independent prognostic marker in a multivariate model. Furthermore, our data indicate disturbances in the periodic expression of circadian genes in CLL cells. Because of their role in the expression of cell cycle-related and DNA-damage response genes, we suggest that the deregulated expression of circadian genes may be linked to the molecular pathogenesis of CLL.
European Journal Of Haematology 07/2009; 83(4):320-7. · 2.61 Impact Factor
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Langenbeck s Archives of Surgery 06/2009; 394(3):589. · 1.81 Impact Factor
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Johannes H Schulte,
Soyoung Lim,
Alexander Schramm,
Nicolaus Friedrichs,
Jan Koster,
Rogier Versteeg,
Ingrid Ora,
Kristian Pajtler, Ludger Klein-Hitpass,
Steffi Kuhfittig-Kulle,
Eric Metzger,
Roland Schüle,
Angelika Eggert,
Reinhard Buettner,
Jutta Kirfel
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ABSTRACT: Aberrant epigenetic changes in DNA methylation and histone acetylation are hallmarks of most cancers, whereas histone methylation was previously considered to be irreversible and less versatile. Recently, several histone demethylases were identified catalyzing the removal of methyl groups from histone H3 lysine residues and thereby influencing gene expression. Neuroblastomas continue to remain a clinical challenge despite advances in multimodal therapy. Here, we address the functional significance of the chromatin-modifying enzyme lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA-mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene-specific H3K4 methylation. Moreover, LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation and growth inhibition of neuroblastoma cells in vitro. Finally, targeting LSD1 reduced neuroblastoma xenograft growth in vivo. Here, we provide the first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. We show that inhibition of LSD1 reprograms the transcriptome of neuroblastoma cells and inhibits neuroblastoma xenograft growth. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy.
Cancer Research 03/2009; 69(5):2065-71. · 7.86 Impact Factor
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Adrien Daigeler,
Daigeler Adrien, Ludger Klein-Hitpass,
Klein-Hitpass Ludger,
Ingo Stricker,
Stricker Ingo,
Oliver Müller,
Müller Oliver,
Cornelius Kuhnen,
Kuhnen Cornelius,
Ansgar Michael Chromik,
Chromik Ansgar Michael,
Lars Steinstraesser,
Steinstraesser Lars,
Ole Goertz,
Goertz Ole,
Hans-Ulrich Steinau,
Steinau Hans-Ulrich,
Marcus Lehnhardt,
Lehnhardt Marcus
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ABSTRACT: The new classification of malignant fibrous histiocytoma leaves only a small group of tumors without further line of differentiation, so-called pleomorphic sarcomas, not otherwise specified (NOS) as a pseudo-entity. This study focused on these tumors and analyzed the association of gene expression profiles to clinical outcome.
Ten fresh samples of pleomorphic NOS sarcomas were evaluated histopathologically and by means of microarray analysis. Analysis of expression profiles was performed by clustering methods as well as by statistical analysis of primary vs recurrent tumors, irradiated vs nonirradiated tumors, tumors of patients above and below 60 years of age, male and female, and of tumors that developed metastatic or recurrent disease during the clinical course and those that did not.
Tumor clustering did not correlate to any histopathological or clinical finding. Detailed gene expression analysis showed a variety of genes whose upregulation (platelet-derived growth factor receptor alpha polypeptide, solute carrier family 39 member 14, solute carrier family 2 member 3, pleiotrophin, trophinin, pleckstrin and Sec7 domain containing 3, enolase 2, biglycan, SH3 and cysteine-rich domain, matrix metalloproteinases 16) and whose downregulation (tissue inhibitor of metalloproteinase 4, hairy/enhancer of split related with YRPW motif 2, protein tyrosine phosphatase receptor-type Z polypeptide 1, SH3 domain GRB2-like 2, microtubule-associated protein 7, potassium voltage-gated channel shaker-related subfamily member 1, RUN and FYVE domain containing 3, Sin3A-associated protein 18 kDa, proline-rich 4, calcium/calmodulin-dependent protein kinase ID, myeloid/lymphoid or mixed-lineage leukemia translocated to 3, insulin-like growth factor binding protein 5, nucleoside diphosphate-linked moiety X-type motif 9, NudC domain containing 3, imprinted in Prader-Willi syndrome, TAF6-like RNA polymerase II p300/CBP-associated factor 65 kDa, WD repeat and SOCS box-containing 2, adenosine diphosphate ribosylation factor 3, KRR1, proliferation-associated 2G4; CD36, complement component (3b/4b) receptor 1, solute carrier family 4 sodium bicarbonate cotransporter member 4, lipoprotein lipase (LPL), GATA binding protein 3, LPL, glutathione peroxidase 3, D: -aspartate oxidase, apolipoprotein E, sphingomyelin phosphodiesterase acid-like 3A) were associated with poor clinical outcome in terms of development of metastatic or recurrent disease.
The classification of these tumors may undergo further changes in the future. Gene expression profiling can provide additional information to categorize pleomorphic sarcoma (NOS) and reveal potential prognostic factors in this "entity."
Langenbeck s Archives of Surgery 02/2009; 395(3):261-75. · 1.81 Impact Factor
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Adrien Daigeler,
Christina Brenzel,
Daniel Bulut,
Anne Geisler,
Christoph Hilgert,
Marcus Lehnhardt,
Hans U Steinau,
Annegret Flier,
Lars Steinstraesser, Ludger Klein-Hitpass,
Ulrich Mittelkötter,
Waldemar Uhl,
Ansgar M Chromik
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ABSTRACT: Disseminated soft tissue sarcoma still represents a therapeutic dilemma because effective cytostatics are missing. Therefore we tested TRAIL and Tarolidine (TRD), two substances with apoptogenic properties on human fibrosarcoma (HT1080).
Viability, apoptosis and necrosis were visualized by TUNEL-Assay and quantitated by FACS analysis (Propidiumiodide/AnnexinV staining). Gene expression was analysed by RNA-Microarray and the results validated for selected genes by rtPCR. Protein level changes were documented by Western Blot analysis. NFKB activity was analysed by ELISA and proliferation assays (BrdU) were performed.
The single substances TRAIL and TRD induced apoptotic cell death and decreased proliferation in HT1080 cells significantly. Gene expression of several genes related to apoptotic pathways (TRAIL: ARHGDIA, NFKBIA, TNFAIP3; TRD: HSPA1A/B, NFKBIA, GADD45A, SGK, JUN, MAP3K14) was changed. The combination of TRD and TRAIL significantly increased apoptotic cell death compared to the single substances and lead to expression changes in a variety of genes (HSPA1A/B, NFKBIA, PPP1R15A, GADD45A, AXL, SGK, DUSP1, JUN, IRF1, MYC, BAG5, BIRC3). NFKB activity assay revealed an antipodal regulation of the several subunits of NFKB by TRD and TRD+TRAIL compared to TRAIL alone.
TRD and TRAIL are effective to induce apoptosis and decrease proliferation in human fibrosarcoma. A variety of genes seems to be involved, pointing to the NFKB pathway as key regulator in TRD/TRAIL-mediated apoptosis.
Journal of Experimental & Clinical Cancer Research 01/2009; 27:82. · 2.15 Impact Factor
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Katja Specht,
Nadia Harbeck,
Jan Smida,
Katja Annecke,
Ulrike Reich,
Joerg Naehrig,
Rupert Langer,
Joerg Mages,
Raymonde Busch,
Elisabeth Kruse, Ludger Klein-Hitpass,
Manfred Schmitt,
Marion Kiechle,
Heinz Hoefler
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ABSTRACT: Cyclophosphamide, methotrexate and 5-fluorouracile (CMF)-based chemotherapy for adjuvant treatment of breast cancer reduces the risk of relapse. In this exploratory study, we tested the feasibility of identifying molecular markers of recurrence in CMF-treated patients. Using Affymetrix U133A GeneChips, RNA samples from 19 patients with primary breast cancer who had been uniformly treated with adjuvant CMF chemotherapy were analyzed. Two supervised class prediction approaches were used to identify gene markers that can best discriminate between patients who would experience relapse and patients who would remain disease-free. An additional independent validation set of 51 patients and 21 genes were analyzed by quantitative RT-PCR. Applying different algorithms to evaluate our microarray data, we identified two gene expression signatures of 21 and 12 genes containing eight overlapping genes, that predict recurrence in 19 cases with high accuracy (94%). Quantitative RT-PCR demonstrated that six genes from the combined signatures (CXCL9, ITSN2, GNAI2, H2AFX, INDO, and MGC10986) were significantly differentially expressed in the recurrence versus the non-recurrence group of the 19 cases and the independent breast cancer patient cohort (n = 51) treated with CMF. High expression levels of CXCL9, ITSN2, and GNAI2 were associated with prolonged disease-free survival (DFS) (P = 0.029, 0.018 and 0.032, respectively). When patients were stratified by combined CXCL9/ITSN2 or CXCL9/FLJ22028 tumor levels, they exhibited significantly different disease-free survival curves (P = 0.0073 and P = 0.005, respectively). Finally, the CXCL9/ITSN2 and CXCL9/FLJ22028 ratio was an independent prognostic factor (P = 0.034 and P = 0.003, respectively) for DFS by multivariate Cox analysis in the 70-patient cohort. Our data highlight the feasibility of a prognostic assay that is applicable to therapeutic decision-making for breast cancer. Whether the biomarker profile is chemotherapy-specific or whether it is a more general indicator of bad prognosis of breast cancer patients remains to be explored.
Breast Cancer Research and Treatment 11/2008; 118(1):45-56. · 4.43 Impact Factor
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ABSTRACT: In the childhood tumor neuroblastoma, high expression of the TrkA neurotrophin receptor is associated with a favorable prognosis and a lack of structural chromosomal changes, whereas TrkB is expressed in aggressive neuroblastomas demonstrating high genomic instability. The ability to repair DNA double-strand breaks (DSBs) is considered a central determinant of chromosomal stability with nonhomologous end joining (NHEJ) being the major pathway of DSB repair in vertebrates. Here, we used the SH-SY5Y human neuroblastoma cell line ectopically expressing either TrkA or TrkB as a model system to analyze the impact of Trk receptor expression on NHEJ-mediated DSB repair. In a cell-free NHEJ assay, SY5Y-TrkA cells displayed a significantly higher efficiency for NHEJ compared to SY5Y-TrkB cells. To detect possible underlying mechanisms, gene expression data (Affymetrix U95A microarray chips) obtained from the same SY5Y-TrkA/TrkB model system were reanalyzed focussing on genes involved in DNA repair. Expression of XRCC4, a central component of NHEJ, was significantly upregulated in SY5Y-TrkA compared to SY5Y-TrkB cells. Expression data were confirmed using real-time PCR and western blotting. Additionally, XRCC4 expression was enhanced in most primary neuroblastomas with high TrkA expression. The TrkA-induced increase in NHEJ activity could be reverted by XRCC4 knock-down, confirming the induction of XRCC4 by TrkA to be essential for the observed phenotype. Our data provide the first evidence for a functional relationship between tyrosine kinase receptor signaling and NHEJ-mediated DSB repair in cancer cells, potentially contributing to their genomic stability.
DNA Repair 09/2008; 7(10):1757-64. · 4.14 Impact Factor
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ABSTRACT: PRAME is a tumor-associated antigen, which belongs to the family of cancer-testis antigens (CTA). The expression of CTA is mainly restricted to the testis and various tumors. In contrast to other CTA, PRAME expression is also frequently detected in acute and chronic leukemias. Due to this expression pattern, PRAME has attracted great interest as a prognostic tumor marker that can be used for the detection of minimal residual disease and as a potential target for immunotherapy. In acute myeloid leukemia (AML), PRAME expression has been observed in 30-64% of cases. To evaluate whether epigenetic mechanisms contribute to PRAME activation in AML, we studied DNA methylation of 15 CpG dinucleotides within a CpG-rich region located in the intron 1 of the PRAME gene. DNA methylation was determined by sequence analysis of cloned PCR products generated from bisulfite-treated genomic DNA. Methylation patterns were correlated with PRAME mRNA levels as determined by microarray analysis and real-time PCR. We found almost complete methylation in mononuclear blood cells from two healthy donors and in bone marrow cells of four PRAME-negative AML patients. In contrast, the degree of PRAME methylation was clearly reduced in four PRAME-positive AML bone marrow samples. In particular, these samples were characterized by the presence of clones, which were completely devoid of methylation. The significant inverse correlation between the degree of methylation and PRAME expression suggests a causal role of DNA methylation in PRAME regulation. Such a role is further supported by the observation that treatment of PRAME-negative cell lines U-937 and THP-1 with the demethylating agent 5'-Aza-2'dC resulted in a dose-related upregulation of PRAME expression.
Annals of Hematology 07/2008; 87(10):809-18. · 2.62 Impact Factor
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ABSTRACT: The treatment of choice for esophageal cancer is considered surgical resection, but a median survival of around 20 months after treatment is still discouraging. The value of adjuvant or neoadjuvant radiation or chemotherapy is limited and to date, benefits have only been described for certain tumor stages. Therefore, new therapeutic options are required. As alternative chemotherapeutics, we tested the antibiotic taurolidine (TRD) on KYSE 270 human esophageal carcinoma cells alone and in combination with rhTRAIL (TNF related apoptosis-inducing ligand). Viability, apoptosis and necrosis were visualized by TUNEL assay and quantitated by FACS analysis. Gene expression was analysed by RNA microarray. The most effective concentration of TRD as single substance (250 micromol/l) induced apoptosis to a maximum of 40% after 12-h dose dependently, leaving 4% viable cells after 48 h; by comparison, rhTRAIL did not have a significant effect. The combination of both substances doubled the effect of TRD alone. Gene expression profiling revealed that TRD downregulated endogenous TRAIL, TNFRSF1A, TRADD, TNFRSF1B, TNFRSF21, FADD, as well as MAP2K4, JAK2 and Bcl2, Bcl2l1, APAF1 and caspase-3. TNFRSF25, cytochrome-c, caspase-1, -8, -9, JUN, GADD45A and NFKBIA were upregulated. TRAIL reduced endogenous TRAIL, Bcl2l1 and caspase-1 expression. BIRC2, BIRC3, TNFAIP3, and NFKBIA were upregulated. The combined substances upregulated endogenous TRAIL, NFKBIA and JUN, whereas DFFA and TRAF3 were downregulated compared to TRD as single substance. We conclude that TRD overcomes TRAIL resistance in KYSE 270 cells. Synergistic effects are dependent on the same and on distinct apoptotic pathways which, jointly triggered, result in an amplified response. Several apoptotic pathways, including the TNF-receptor associated and the mitochondrial pathway, were differentially regulated by the substances on gene expression level. Additionally transcription factors seem to be influenced, NFKB in particular. Endogenous TRAIL expression is increased by the combination of substances, whereas it is reduced by each single substance. Taking into consideration that the non-toxic TRD was able to reduce rhTRAIL toxicity and dose, combined therapy with TRD and rhTRAIL may offer new options for treatment in esophageal cancer.
International Journal of Oncology 07/2008; 32(6):1205-20. · 2.40 Impact Factor
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ABSTRACT: The canonical Wnt pathway controls cell differentiation, proliferation and apoptosis by regulating the expression of a high number of target genes. The first target gene of the Wnt pathway was discovered nearly 20 years ago, when analysing gene expression patterns in the Drosophila embryo. Since the year 2002 entire transcriptomes have been screened by microarray analysis in order to identify genes, which are differentially expressed in cells with activated Wnt pathway. Recently, novel genome-based screening methods have been developed, which are less error-prone and independent from RNA. The exemplified methods STAGE (Sequence Tag Analysis Of Genomic Enrichment), ChIP-PET (chromatin immunoprecipitation with paired-end ditag), ChIP-Seq (ChIP followed by direct sequencing) and the bioinformatics approach EEL (Enhancer Element Locator) will be introduced shortly. The high number of potential target genes and regulated functions left questions unanswered, for instance how the Wnt pathway controls such a high number of genes and how it is able to regulate so many different cellular functions. In order to answer these questions we ordered the genes of the published Wnt target screenings according to their functions, and summarized the pathways, which are regulated by the Wnt pathway. This review focuses on the totality of Wnt target genes, the Wnt targetome, which is the clue to understand the manifold roles of the Wnt pathway within the cellular context.
Cellular Signalling 06/2008; 20(5):795-802. · 4.06 Impact Factor
