Biomedical subjects

Andreas E Kulozik

Publications and source records attributed to Andreas E Kulozik.

At least 19 recordsLinked to original sources

PubMed2008

Long-term remission of children with relapsed and secondary anaplastic large cell non-Hodgkin lymphoma (ALCL) following treatment with pulsed dexamethasone and low dose etoposide.

Anaplastic large cell lymphoma (ALCL) accounts for approximately 15% of childhood NHL. Relapsed ALCL represents a formidable challenge because outcome is poor despite the use of high-dose chemotherapy regimens. We report two patients with relapsed T-type and 0-type ALCL who achieved long-term 3rd and 4th remissions with 4-weekly oral dexamethasone (DEX) and etoposide pulses for 2 years. This regimen also induced and maintained remission in a third patient with Nijmegen breakage syndrome (NBS) with secondary T-type ALCL. These patients demonstrate that low-intensity oral chemotherapy can induce long-term remissions and offer a curative perspective in refractory, relapsed and secondary ALCL.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2008

Treatment of neuroblastoma-related opsoclonus-myoclonus-ataxia syndrome with high-dose dexamethasone pulses.

Opsoclonus-myoclonus-ataxia-syndrome (OMS) represents a rare neuroblastoma-associated paraneoplastic syndrome that commonly results in neurologic deficits despite tumor resection and immunosuppressive therapy. We describe the response of five such children to high-dose dexamethasone pulses including two patients in whom previous glucocorticoids, rituximab, and cytostatic drugs were not successful. All patients had MYCN non-amplified tumors that were detected 1 to 7 months after the onset of the OMS or ataxia. This treatment resulted in a good partial response in three and in complete remission in two patients. Our results show that dexamethasone pulses are likely to be useful for both, first-line- and salvage-therapy for OMS-patients.

Adrenal Gland Neoplasms↗

PubMed2006

A chemiluminescence-based reporter system to monitor nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that mediates rapid degradation of transcripts bearing premature translation termination codons (PTCs) and thereby limits the expression of unproductively processed mRNAs and the synthesis of C-terminally truncated peptides. Both its importance as a means to control gene expression and in the context of genetic and acquired human diseases call for an exploration of the mammalian NMD pathway using chemical biology approaches. Here, we describe a novel cell-based chemiluminescence reporter system that recapitulates the hallmark features of mammalian NMD. The assay is characterized by its high sensitivity, robustness, and its potential for automated handling. Limiting NMD efficiency by RNAi-mediated depletion of the essential NMD factor UPF1 markedly and specifically increased the NMD reporter mRNA level and resulted in a proportional increase in protein expression reflected by Renilla luminescence. The PI 3-kinase inhibitor wortmannin has previously been found to up-modulate PTC-containing transcripts by inhibiting the UPF1 kinase SMG1. Wortmannin treatment enhanced NMD reporter expression in our system in a dose-dependent way, illustrating its utility for small molecule screening.

Androstadienes↗

PubMed2006

Expression of late cell cycle genes and an increased proliferative capacity characterize very early relapse of childhood acute lymphoblastic leukemia.

PURPOSE: In childhood acute lymphoblastic leukemia (ALL), approximately 25% of patients suffer from relapse. In recurrent disease, despite intensified therapy, overall cure rates of 40% remain unsatisfactory and survival rates are particularly poor in certain subgroups. The probability of long-term survival after relapse is predicted from well-established prognostic factors (i.e., time and site of relapse, immunophenotype, and minimal residual disease). However, the underlying biological determinants of these prognostic factors remain poorly understood. EXPERIMENTAL DESIGN: Aiming at identifying molecular pathways associated with these clinically well-defined prognostic factors, we did gene expression profiling on 60 prospectively collected samples of first relapse patients enrolled on the relapse trial ALL-REZ BFM 2002 of the Berlin-Frankfurt-Münster study group. RESULTS: We show here that patients with very early relapse of ALL are characterized by a distinctive gene expression pattern. We identified a set of 83 genes differentially expressed in very early relapsed ALL compared with late relapsed disease. The vast majority of genes were up-regulated and many were late cell cycle genes with a function in mitosis. In addition, samples from patients with very early relapse showed a significant increase in the percentage of S and G(2)-M phase cells and this correlated well with the expression level of cell cycle genes. CONCLUSIONS: Very early relapse of ALL is characterized by an increased proliferative capacity of leukemic blasts and up-regulated mitotic genes. The latter suggests that novel drugs, targeting late cell cycle proteins, might be beneficial for these patients that typically face a dismal prognosis.

Cell Cycle↗

PubMed2006

Internal ribosome entry sequence-mediated translation initiation triggers nonsense-mediated decay.

In eukaryotes, a surveillance pathway known as nonsense-mediated decay (NMD) regulates the abundance of messenger RNAs containing premature termination codons (PTCs). In mammalian cells, it has been asserted that the NMD-relevant first round of translation is special and involves initiation by a specific protein heterodimer, the nuclear cap-binding complex (CBC). Arguing against a requirement for CBC-mediated translation initiation, we show that ribosomal recruitment by the internal ribosomal entry sequence of the encephalomyocarditis virus triggers NMD of a PTC-containing transcript under conditions in which ribosome entry from the cap is prohibited. These data generalize the previous model and suggest that translation per se, irrespective of how it is initiated, can mediate NMD.

Codon, Nonsense↗

PubMed2006

The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD).

Platelet production is induced by the cytokine thrombopoietin (TPO). It is physiologically critical that TPO expression is tightly regulated, because lack of TPO causes life-threatening thrombocytopenia while an excess of TPO results in thrombocytosis. The plasma concentration of TPO is controlled by a negative feedback loop involving receptor-mediated uptake of TPO by platelets. Furthermore, TPO biosynthesis is limited by upstream open reading frames (uORFs) that curtail the translation of the TPO mRNA. uORFs are suggested to activate RNA degradation by nonsense-mediated decay (NMD) in a number of physiological transcripts. Here, we determine whether NMD affects TPO expression. We show that reporter mRNAs bearing the seventh TPO uORF escape NMD. Importantly, endogenously expressed TPO mRNA from HuH7 cells is unaffected by abrogation of NMD by RNAi. Thus, regulation of TPO expression is independent of NMD, implying that mRNAs bearing uORFs cannot generally be considered to represent NMD targets.

Cell Line, Tumor↗

PubMed2006

Activating NOTCH1 mutations predict favorable early treatment response and long-term outcome in childhood precursor T-cell lymphoblastic leukemia.

Activating mutations of the transmembrane receptor NOTCH1 are common in precursor T-cell lymphoblastic leukemia (T-ALL). We systematically analyzed the impact of activating NOTCH1 mutations on early treatment response and long-term outcome in 157 patients with T-ALL of the pediatric ALL-Berlin-Frankfurt-Munster (BFM) 2000 study. We confirm previous results that NOTCH1 mutations occur in more than 50% of T-ALL in children. In 82 patients (82/157; 52.2%), activating NOTCH1 mutations were identified either in the heterodimerization (55/82; 67.1%), in the PEST (13/82; 15.9%), or in both domains (14/82; 17.0%). The presence of NOTCH1 mutations was significantly correlated with a good prednisone response and favorable minimal residual disease (MRD) kinetics, which was independent from sex, age, white blood cell count, and T-cell immunophenotype at the time of diagnosis. Furthermore, activating NOTCH1 mutations specified a large subgroup of patients with an excellent prognosis. These findings indicate that in the context of the ALL-BFM 2000 treatment strategy, NOTCH1 mutations predict a more rapid early treatment response and a favorable long-term outcome in children with T-ALL.

Adolescent↗

PubMed2006

Functions of hUpf3a and hUpf3b in nonsense-mediated mRNA decay and translation.

The exon-junction complex (EJC) components hUpf3a and hUpf3b serve a dual function: They promote nonsense-mediated mRNA decay (NMD), and they also regulate translation efficiency. Whether these two functions are interdependent or independent of each other is unknown. We characterized the function of the hUpf3 proteins in a lambdaN/boxB-based tethering system. Despite the high degree of sequence similarity between hUpf3b and hUpf3a, hUpf3a is much less active than hUpf3b to induce NMD and to stimulate translation. We show that induction of NMD by hUpf3 proteins requires interaction with Y14, Magoh, BTZ, and eIF4AIII. The protein region that mediates this interaction and discriminates between hUpf3a and hUpf3b in NMD function is located in the C-terminal domain and fully contained within a small sequence that is highly conserved in Upf3b but not Upf3a proteins. Stimulation of translation is independent of this interaction and is determined by other regions of the hUpf3 protein, indicating the presence of different downstream pathways of hUpf3 proteins either in NMD or in translation.

Amino Acid Sequence↗

PubMed2006

A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA).

MicroRNAs represent a class of short (approximately 22 nt), noncoding regulatory RNAs involved in development, differentiation, and metabolism. We describe a novel microarray platform for genome-wide profiling of mature miRNAs (miChip) using locked nucleic acid (LNA)-modified capture probes. The biophysical properties of LNA were exploited to design probe sets for uniform, high-affinity hybridizations yielding highly accurate signals able to discriminate between single nucleotide differences and, hence, between closely related miRNA family members. The superior detection sensitivity eliminates the need for RNA size selection and/or amplification. MiChip will greatly simplify miRNA expression profiling of biological and clinical samples.

Animals↗

PubMed2006

3' end processing of the prothrombin mRNA in thrombophilia.

Clinical and genetic studies have led to the discovery of specific genotypes that predispose to thromboembolism in adults and children. The exploration of the underlying pathologies has revealed a broad variety of affected molecular mechanisms. Most recently, the functional analysis of the prothrombin (F2) 20210*A variant revealed increased efficiency of 3' end processing as a novel genetic mechanism predisposing to human disease. Here, we review the 3' end processing of the human F2 mRNA and demonstrate how clinically relevant mutations in the F2 gene contribute to thrombophilia by interfering with a tightly balanced architecture of noncanonical 3' end formation signals.

3' Flanking Region↗

PubMed2005

Exon-junction complex components specify distinct routes of nonsense-mediated mRNA decay with differential cofactor requirements.

Messenger RNAs (mRNAs) bearing premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). For mammalian NMD, current models propose a linear pathway that involves the splicing-dependent deposition of exon-junction complexes (EJCs) and the sequential action of the NMD factors UPF3, UPF2, and UPF1. We show here that different EJC proteins serve as entry points for the formation of distinguishable NMD-activating mRNPs. Specifically, Y14, MAGOH, and eIF4A3 can activate NMD in an UPF2-independent manner, whereas RNPS1-induced NMD requires UPF2. We identify the relevant regions of RNPS1, eIF4A3, Y14, and MAGOH, which are essential for NMD and provide insights into the formation of complexes, that classify alternative NMD pathways. These results are integrated into a nonlinear model for mammalian NMD involving alternative routes of entry that converge at a common requirement of UPF1.

Animals↗

PubMed2005

Moyamoya syndrome associated with hemolytic anemia due to Hb Alesha.

Moyamoya angiopathy is a well-known complication of sickle cell disease but has rarely been observed in other hemoglobinopathies. The authors describe a previously unreported association of hemolytic anemia due to a rare unstable hemoglobinopathy with abnormal oxygen affinity (Hb Alesha) and moyamoya syndrome in a 10-year-old girl. At age 4 she had recurrent migraine-with-aura-like symptoms. Cranial MRI, Doppler, and EEG examinations were not conclusive. Deterioration of her neurologic symptoms prompted a renewed EEG examination at 10 years of age, which revealed a re-buildup phenomenon. MRI and MR angiography now showed moyamoya angiopathy with stenotic and occlusive lesions of both internal carotid and middle cerebral arteries. Conventional angiography confirmed these findings. Reperfusion with three extra-intracranial bypasses terminated the transient ischemic attacks. The authors suggest that chronic hypoxemia may be the cause of occlusive moyamoya angiopathy in Hb Alesha and possibly other unstable hemoglobinopathies with altered oxygen affinity.

Anemia, Hemolytic↗

PubMed2004

Profiling and functional annotation of mRNA gene expression in pediatric rhabdomyosarcoma and Ewing's sarcoma.

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewing's sarcomas (EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t-test, SAM, k-nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS. We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t-test). Genes involved in protein biosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor-specific genes reveals detailed insights into tumor biology and differentiation-specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

Biomarkers, Tumor↗

PubMed2004

The prothrombin 3'end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations.

The functional analysis of the common prothrombin 20210 G>A(F2 20210(*)A) mutation has recently revealed gain of function of 3'end processing as a novel genetic mechanism predisposing to human disease. We now show that the physiologic G at the cleavage site at position 20210 is the functionally least efficient nucleotide to support 3'end processing but has evolved to be physiologically optimal. Furthermore, the F2 3'end processing signal is characterized by a weak downstream cleavage stimulating factor (CstF) binding site with a low uridine density, and the functional efficiency of F2 3'end processing can be enhanced by the introduction of additional uridine residues. The recently identified thrombosis-related mutation (F2 20221(*)T) within the CstF binding site up-regulates F2 3'end processing and prothrombin biosynthesis in vivo. F2 20221(*)T thus represents the first example of a likely pathologically relevant mutation of the putative CstF binding site in the 3'flanking sequence of a human gene. Finally, we show that the low-efficiency F2 cleavage and CstF binding sites are balanced by a stimulatory upstream uridine-rich element in the 3'UTR. The architecture of the F2 3'end processing signal is thus characterized by a delicate balance of positive and negative signals. This balance appears to be highly susceptible to being disturbed by clinically relevant gain-of-function mutations.

3' Untranslated Regions↗

PubMed2004

Nonsense-mediated mRNA decay: from vacuum cleaner to Swiss army knife.

Nonsense-mediated mRNA decay (NMD) downmodulates mRNAs that have in-frame premature termination codons and prevents translation of potentially harmful truncated proteins from aberrant mRNAs. Two new approaches have identified physiological NMD substrates, and suggest that NMD functions as a multipurpose tool in the modulation of gene expression.

Animals↗

PubMed2004

Nonsense-mediated decay approaches the clinic.

Nonsense-mediated decay (NMD) eliminates mRNAs containing premature termination codons and thus helps limit the synthesis of abnormal proteins. New results uncover a broader role of NMD as a pathway that also affects the expression of wild-type genes and alternative-splice products. Because the mechanisms by which NMD operates have received much attention, we discuss here the emerging awareness of the impact of NMD on the manifestation of human genetic diseases. We explore how an understanding of NMD accounts for phenotypic differences in diseases caused by premature termination codons. Specifically, we consider how the protective function of NMD sometimes benefits heterozygous carriers and, in contrast, sometimes contributes to a clinical picture of protein deficiency by inhibiting expression of partially functional proteins. Potential 'NMD therapeutics' will therefore need to strike a balance between the general physiological benefits of NMD and its detrimental effects in cases of specific genetic mutations.

Alternative Splicing↗

PubMed2004

Impact of pre-analytical handling on bone marrow mRNA gene expression.

Large clinical trials on leukaemia, require the transport of bone marrow (BM) from participating clinics to central diagnostic laboratories. We have investigated the impact of RNA extraction protocols and time delays between sample aspiration and RNA extraction on RNA quality and gene expression profiles. Intact RNA can be extracted from BM samples stored at room temperature for up to 48 h. Gene expression analyses using Affymetrix U95Av2 GeneChips and a custom-designed cDNA array in parallel showed that even short-term storage of BM has dramatic effects on mRNA expression of individual transcripts. Many probe sets/genes showed either reproducible deregulation (18.8%, analysis of variance <0.05), or inconsistent expression that differed from patient to patient (38.4%). Moderate alterations were observed in 42.8% genes, with a maximum fold change <2.0 in all experiments and at all time points. These profound effects complicate the use of unstabilized, shipped BM samples for gene expression analyses. The comparison of a variety of RNA stabilization reagents (e.g. PAXgene) resulted in partial conservation of the mRNA expression patterns. Immediate density centrifugation or erythrocyte lysis and freezing at -80 degrees C represent simple procedures that reliably preserved mRNA gene expression patterns in BM.

Bone Marrow↗

PubMed2004

Using the lambdaN peptide to tether proteins to RNAs.

Proteins interacting with messenger RNAs (mRNAs) affect their nuclear processing, export, translation efficiency, stability, or cytoplasmic localization. Such RNA-binding proteins are often modular, containing RNA-binding domain(s) and other functional modules. To analyze the function of such proteins independent of their normal RNA-binding domains or to introduce effector modules to defined RNA-binding regions, a number of tethering approaches have been developed, often based on the use of large proteins and their specifically interacting RNA sequences. Here we report the use of a versatile system to tether proteins to mRNAs. The 22 amino acid RNA-binding domain of the lambda bacteriophage antiterminator protein N (lambdaN-(1-22) or lambdaN peptide) is used to tag the protein of interest, and its specific 19 nt binding site (boxB) is inserted into the target RNA recruiting the properties of the fusion protein to the RNA. The major advantage of this system derives from the small size of the peptide and its target sequence, which facilitates cloning and its use for biochemical experiments and diminishes possible interferences with the fused protein. The chapter illustrates the use of this system to create dedicated mRNA-specific factors involved in processes, such as mRNA translation and nonsense-mediated mRNA decay.

3' Untranslated Regions↗