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Michaela Scherr

Publications and source records attributed to Michaela Scherr.

17 recordsLinked to original sources
PubMed2007

Comprehensive analysis of homeobox genes in Hodgkin lymphoma cell lines identifies dysregulated expression of HOXB9 mediated via ERK5 signaling and BMI1.

Many members of the nearly 200-strong homeobox gene family have been implicated in cancer, mostly following ectopic expression. In this study we analyzed homeobox gene expression in Hodgkin lymphoma (HL) cell lines. Both reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers and microarray profiling identified consistently up-regulated HOXB9 expression. Analysis of HOXB9 regulation in HL cells revealed E2F3A and BMI1 as activator and repressor, respectively. Furthermore, a constitutively active ERK5 pathway was identified in all HL cell lines analyzed as well as primary HL cells. Our data show that ERK5 probably mediates HOXB9 expression by repressing BMI1. In addition, expression analysis of the neighboring microRNA gene mir-196a1 revealed coregulation with HOXB9. Functional analysis of HOXB9 by knockdown and overexpression assays indicated their influence on both proliferation and apoptosis in HL cells. In summary, we identified up-regulation of HOXB9 in HL mediated by constitutively active ERK5 signaling which may represent novel therapeutic targets in HL.

Base Sequence↗
PubMed2006

LEF-1 is crucial for neutrophil granulocytopoiesis and its expression is severely reduced in congenital neutropenia.

We demonstrate here that lymphoid enhancer-binding factor 1 (LEF-1) mediates the proliferation, survival and differentiation of granulocyte progenitor cells. We initially documented the importance of this transcription factor in the bone marrow of individuals with severe congenital neutropenia (CN) with a 'differentiation block' at the promyelocytic stage of myelopoiesis. LEF-1 expression was greatly reduced or even absent in CN arrested promyelocytes, resulting in defective expression of the LEF-1 target genes CCND1, MYC and BIRC5, encoding cyclin D1 (ref. 2), c-Myc and survivin, respectively. In contrast, healthy individuals showed highest LEF-1 expression in promyelocytes. Reconstitution of LEF-1 in early hematopoietic progenitors of two individuals with CN corrected the defective myelopoiesis and resulted in the differentiation of these progenitors into mature granulocytes. Repression of endogenous LEF-1 by specific short hairpin RNA inhibited proliferation and induced apoptosis of CD34(+) progenitors from healthy individuals and of cells from two myeloid lines (HL-60 and K562). C/EBPalpha, a key transcription factor in granulopoiesis, was directly regulated by LEF-1. These observations indicate that LEF-1 is an instructive factor regulating neutrophilic granulopoiesis whose absence plays a critical role in the defective maturation program of myeloid progenitors in individuals with CN.

Antigens, CD↗
PubMed2006

RNA-mediated gene silencing in hematopoietic cells.

In the past few years, the discovery of RNA-mediated gene silencing mechanisms, like RNA interference (RNAi), has revolutionized our understanding of eukaryotic gene expression. These mechanisms are activated by double-stranded RNA (dsRNA) and mediate gene silencing either by inducing the sequence-specific degradation of complementary mRNA or by inhibiting mRNA translation. RNAi now provides a powerful experimental tool to elucidate gene function in vitro and in vivo, thereby opening new exciting perspectives in the fields of molecular analysis and eventually therapy of several diseases such as infections and cancer. In hematology, numerous studies have described the successful application of RNAi to better define the role of oncogenic fusion proteins in leukemogenesis and to explore therapeutic approaches in hematological malignancies. In this review, we highlight recent advances and caveats relating to the application of this powerful new methodology to hematopoiesis.

Journal Article↗
PubMed2005

Enhanced sensitivity to inhibition of SHP2, STAT5, and Gab2 expression in chronic myeloid leukemia (CML).

Although targeting the BCR-ABL tyrosine kinase activity by imatinib mesylate has rapidly become first-line therapy in chronic myeloid leukemia (CML), drug resistance suggests that combination therapy directed to a complementing target may significantly improve treatment results. To identify such potential targets, we used lentivirus-mediated RNA interference (RNAi) as a tool for functional genomics in cell lines as well as primary normal and CML CD34+ cells. In a conditional cell culture model, we demonstrate that RNAi-mediated reduction of SHP2, STAT5, and Gab2 protein expression inhibits BCR-ABL-dependent but not cytokine-dependent proliferation in a dose-dependent manner. Similarly, colony formation of purified primary CML but not of normal CD34+ colony-forming cells is specifically reduced by inhibition of SHP2, STAT5, and Gab2 expression, respectively. In addition, coexpression of both anti-BCR-ABL and anti-SHP2 shRNAs from a single lentiviral vector induces stronger inhibition of colony formation as compared to either shRNA alone. The data indicate that BCR-ABL expression may affect the function of normal signaling molecules. Targeting these molecules may harbor significant therapeutic potential for the treatment of patients with CML.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Activation of HLXB9 by juxtaposition with MYB via formation of t(6;7)(q23;q36) in an AML-M4 cell line (GDM-1).

Mutation or dysregulation of related homeobox genes occurs in leukemia. Using RT-PCR, we screened members of the EHG family of homeobox genes, comprising EN1 (at 2q14), GBX2 (at 2q36), and EN2, GBX1, and HLXB9 (at 7q36), for dysregulation in acute myeloid leukemia (AML) cell lines indicated by chromosomal breakpoints at these sites. Only one EHG-family gene was expressed, HLXB9, in cell line GDM-1 (AML-M4). Karyotypic analysis of GDM-1 revealed a unique t(6;7)(q23;q35), also present in the patient. Fluorescence in situ hybridization analysis showed chromosomal breakpoints close to the region upstream of HLXB9, at 7q36, a region rearranged in certain AML patients, and at 6q23 upstream of MYB, a gene activated in leukemia. Detailed expression analysis suggested ectopic activation of HLXB9 occurred via juxtaposition with regions upstream of MYB, which was highly expressed in GDM-1. Our data identified a cell line model for a novel leukemic translocation involving MYB with HLXB9, further implicating HLXB9 in leukemogenesis.

Cell Line, Tumor↗
PubMed2005

Modulation of gene expression by siRNA in hematopoietic cells.

RNA interference (RNAi) has been established as a powerful tool for identifying gene function in many biological processes and can be used for genome-wide functional genetic screens in mammalian cells. For such purposes, expression cassettes encoding RNAi triggers can be efficiently introduced into the host cell genome utilizing viral vector systems, resulting in long-term silencing of target gene expression. Transient gene silencing can also be induced by exogenous delivery of suitable RNAi triggers to target cells. However, similarly to other reverse genetic tools, there are technical challenges and limitations associated with RNAi, some of which are specific to hematopoietic cells. In this review we discuss the rational design of effective RNAi triggers, different approaches for their efficient delivery, and the value of RNAi both as a potential therapeutic strategy and as a tool for functional genomics and target validation in hematopoietic cells.

Animals↗
PubMed2004

RNAi in functional genomics.

There has been a lack of powerful tools for systematic analysis of mammalian gene function, but RNA interference (RNAi) may now provide such a strategy. Stable transcription of RNAi triggers from suitable expression cassettes integrated into the host cell genome by viral gene transfer can induce long-term and heritable gene silencing in mammalian cells. However, the use of RNAi as a genetic tool is limited by difficulties in identifying efficient RNAi triggers, the problem of effective delivery and off-target effects, as well as potential genotoxic side effects of viral gene transfer strategies. Recent insights into the molecular mechanisms of silencing processes mediated by either siRNA or miRNA will allow further optimization of RNAi triggers as genetic tools.

Animals↗
PubMed2003

Inhibition of GM-CSF receptor function by stable RNA interference in a NOD/SCID mouse hematopoietic stem cell transplantation model.

RNA interference (RNAi) describes a highly conserved mechanism of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA (dsRNA). Whereas RNAi is applied to study gene function in different organisms and in variant cell types, little is known about RNAi in human hematopoietic stem and progenitor cells and their myeloid progeny. To address this issue, short hairpin RNAs (shRNA) were designed to target the common beta-chain of the human receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (betaGMR). These receptors regulate proliferation, survival, differentiation, and functional activity of hematopoietic cells. In addition to markedly inhibiting mRNA and protein expression, anti-beta-GMR shRNAs were also found to inhibit receptor function in a cell culture model. Furthermore, lentiviral gene transfer of shRNA expression cassettes into primary normal CD34+ cells selectively inhibited colony formation of transduced progenitors when stimulated with GM-CSF/IL-3 but not when stimulated with cytokines that do not signal via beta-GMR. Finally, anti-beta-GMR shRNAs had no detectable effect on engraftment or lineage composition of lentivirally transduced human CD34+ cells transplanted into NOD/SCID mice. However, the growth defect of transduced colony-forming cells under stimulation with GM-CSF/IL-3 remains unchanged in bone marrow cells harvested from individual NOD/SCID mice 6 weeks after transplantation. These data indicate that lentiviral gene transfer of shRNA expression cassettes may be used to induce long-term RNAi in human hematopoietic stem and progenitor cells for functional genetics and potential therapeutic intervention.

Animals↗
PubMed2003

Gene silencing mediated by small interfering RNAs in mammalian cells.

RNA interference (RNAi) as a protecting mechanism against invasion by foreign genes was first described in C. elegans and has subsequently been demonstrated in diverse eukaryotes such as insects, plants, fungi and vertebrates. RNAi is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNAs (dsRNA) homologous to the gene being suppressed. dsRNAs are processed by Dicer, a cellular ribonuclease III, to generate duplexes of about 21 nt with 3'-overhangs (small interfering RNA, siRNA) which mediate sequence-specific mRNA degradation. In mammalian cells siRNA molecules are capable of specifically silencing gene expression without induction of the unspecific interferon response pathway. Thus, siRNAs have become a new and powerful alternative to other genetic tools such as antisense oligonucleotides and ribozymes to analyze loss-of-function phenotypes. Application of siRNA duplexes to interfere with the expression of a specific gene requires knowledge of target accessibility, highly effective delivery of siRNAs into target cells and for some applications long-term siRNA expression. Effective strategies to deliver siRNAs to target cells in cell culture include transduction by physical or chemical transfection. An alternative strategy uses the endogenous expression of siRNAs by various Pol III promoter expression cassettes that allow transcription of functional siRNAs or their precursors. This review summarizes some genetic and biochemical aspects of RNAi, the delivery and application of siRNAs to target cells, the kinetics of RNAi and the utility of siRNAs as analytical and potential therapeutic tools.

Animals↗
PubMed2002

Specific inhibition of bcr-abl gene expression by small interfering RNA.

Small interfering RNAs (siRNAs) were designed to target the bcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl-positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti-bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl-positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl-positive and normal CD34(+) cells, respectively. Finally, anti-bcr-abl siRNA inhibited BCR-ABL-dependent, but not cytokine-dependent, proliferation in a bcr-abl-positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells.

Animals↗
PubMed2002

High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter.

Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.

Animals↗
PubMed2002

Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells.

This study reports a lentiviral gene transfer protocol for efficient transduction of adult human peripheral blood (PB)-derived CD34+ NOD/SCID-repopulating cells (SRCs) using vesicular stomatitis virus-G protein (VSV-G)-pseudotyped lentiviruses encoding for enhanced green fluorescence protein (eGFP). Lentiviral stocks were concentrated by anion exchange chromatography, and transduction was performed under serum-free conditions at a multiplicity of infection (MOI) between 3 and 50. Similar transduction efficiencies were achieved in the presence and absence of cytokines. Transduction of PB-derived CD34+ cells at a MOI of 3 resulted in gene transfer efficiencies into SRCs of 9.2% and 12.0% in the absence and presence of cytokines, respectively. Using improved lentiviral vectors, transduction frequency varied between 42.0% (MOI 10) and 36.0% (MOI 50) with multilineage transgene expression within SRC-derived myeloid and lymphoid cells. The protocol described can be adapted for clinical application of lentiviral gene transfer into PB-derived CD34+ cells from adult patients.

Adult↗
PubMed2002

Invasive drug delivery.

The central nervous system is a very attractive target for new therapeutic strategies since many genes involved in neurological diseases are known and often only local low level gene expression is required. However, as the blood brain barrier on one hand prevents some therapeutic agents given systematically from exerting their activity in the CNS, it also provides an immune privileged environment. Neurosurgical technology meanwhile allows the access of nearly every single centre of the CNS and provides the surgical tool for direct gene delivery via minimal invasive surgical approaches to the brain. Successful therapy of the central nervous system requires new tools for delivery of therapeutics in vitro and in vivo (Fig. 1). The application of therapeutic proteins via pumps into the CSF was shown to be only of limited value since the protein mostly is not sufficiently transported within the tissue and the half life of proteins limits the therapeutic success. Direct gene delivery into the host cell has been a main strategy for years, and in the beginning the direct DNA delivery or encapsulation in liposomes or other artificial encapsulation have been applied with different success. For several years the most promising tools have been vectors based on viruses. Viruses are able to use the host cell machinery for protein synthesis, and some of them are able to stably insert into the host cell genome and provide long term transgene expression as long as the cell is alive. The increasing knowledge of viruses and their live cycle promoted the development of viral vectors that function like a shuttle to the cell, with a single round of infection either integrating or transiently expressing the transgene. Viral vectors have proven to be one of the most efficient and stable transgene shuttle into the cell and have gained increasing importance. The limitations of some viral vectors like the adenoviral vector and adeno-associated viral vector have been improved by new constructs like HIV-1 based lentiviral vectors. The immune response caused by expression of viral proteins, or the inability of some viral vectors like the retroviral vector to infect only dividing cells have been overcome by these new constructs. Lentiviral vectors allow an efficient and stable transgene expression over years in vivo without effecting transgene expression or immune response. In this Chapter we will describe synthetic vectors, give an overview of the most common viral vectors and focus our attention on lentiviral vectors, since we consider them to be the most efficient tool for gene delivery in the CNS.

Central Nervous System↗
PubMed2002

Gene transfer into hematopoietic stem cells using lentiviral vectors.

Gene transfer into hematopoietic cells is currently being used to modulate immune responses, to protect hematopoietic cells against cytotoxic drugs or viral genes, and to restore gene deficiencies due to either inborn genetic defects or acquired loss of regular gene function. In particular, gene addition strategies for inherited severe combined immunodeficiencies (SCID) due to adenosine deaminase (ADA) deficiency or defects of the interleukin-2 receptor gamma-chain represent potentially curative strategies based on gene transfer into hematopoietic cells using recombinant retroviral vectors. Since long-term correction of genetic defects in hematopoietic cells often requires transduction of hematopoietic stem cells, an effective gene transfer into stem cells with efficient long-term and multi-lineage transgene expression is the desired goal for these therapeutic strategies. However, gene transfer strategies with retroviral vectors unable to integrate into non-cycling cells are limited by the quiescent state of the stem cells that have to be stimulated by cytokines to induce cell cycle progression. To circumvent these barriers, lentiviral vector systems based on HIV-1 have recently been developed which are able to deliver and express genes in non-dividing cells both in vitro and in vivo. This review outlines the development and improvement of lentivirus-based gene transfer protocols and discusses the use of lentiviral vectors in preclinical gene therapy studies.

Adenosine Deaminase↗
PubMedDate not supplied

Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA.

RNA interference (RNAi) mediates sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA. RNAi is an effective tool for functional genomics in C. elegans, whereas biochemical characteristics of RNAi hamper its use in mammalian cells. We here analyze kinetic and quantitative aspects of mammalian RNAi in different cell lines using novel lentiviral constructs with double H1-shRNA expression cassettes located in the U3 region of the LTR. Using enhanced green fluorescence protein (EGFP) as a target gene for RNAi and red fluorescence protein (RFP) as surrogate marker for intracellular siRNA expression, we show that long-term siRNA expression mediates stable RNAi. Furthermore, RNAi-induced gene silencing varies from minimal to complete loss-of-function phenotypes within homogeneous and between different cell populations. Interestingly, the extent of gene silencing correlates to lentiviral integrations as well as siRNA expression levels in target cells. Finally, we demonstrate functional gene silencing of a cytokine receptor gene in normal CD34+ hematopoietic progenitor cells. These data suggest that the use of suitable lentiviral constructs to quantify siRNA expression in living cells may allow analysis of loss-of-function phenotypes in a dose-dependent manner in a wide variety of target cells.

Animals↗