scholarly journals Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 600-605 ◽  
Author(s):  
DS Snyder ◽  
Y Wu ◽  
JL Wang ◽  
JJ Rossi ◽  
P Swiderski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Biology ◽  
Fusion Gene ◽  
Therapeutic Potential ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
A Cell

Abstract The bcr-abl fusion gene is the molecular counterpart of the Philadelphia chromosome (Ph1) and is directly involved in the pathogenesis of Ph1+ leukemia. Inhibition of bcr-abl gene expression may have profound effects on the cell biology of Ph1+ cells, as recent experiments with antisense oligonucleotides have shown. In this study we have designed and synthesized a unique ribozyme that is directed against bcr-abl mRNA. The ribozyme cleaved bcr-abl mRNA in a cell-free in vitro system. A DNA-RNA hybrid ribozyme was then incorporated into a liposome vector and transfected into EM-2 cells, a cell line derived from a patient with blast crisis of chronic myelogenous leukemia. The ribozyme decreased levels of detectable bcr-abl mRNA in these cells, inhibited expression of the bcr-abl gene product, p210bcr-abl, and inhibited cell growth. This anti-bcr-abl ribozyme may be a useful tool to study the cell biology of Ph1+ leukemia and may ultimately have therapeutic potential in treating patients with Ph1 leukemias.

scholarly journals Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 600-605 ◽  
Author(s):  
DS Snyder ◽  
Y Wu ◽  
JL Wang ◽  
JJ Rossi ◽  
P Swiderski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Biology ◽  
Fusion Gene ◽  
Therapeutic Potential ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
A Cell

The bcr-abl fusion gene is the molecular counterpart of the Philadelphia chromosome (Ph1) and is directly involved in the pathogenesis of Ph1+ leukemia. Inhibition of bcr-abl gene expression may have profound effects on the cell biology of Ph1+ cells, as recent experiments with antisense oligonucleotides have shown. In this study we have designed and synthesized a unique ribozyme that is directed against bcr-abl mRNA. The ribozyme cleaved bcr-abl mRNA in a cell-free in vitro system. A DNA-RNA hybrid ribozyme was then incorporated into a liposome vector and transfected into EM-2 cells, a cell line derived from a patient with blast crisis of chronic myelogenous leukemia. The ribozyme decreased levels of detectable bcr-abl mRNA in these cells, inhibited expression of the bcr-abl gene product, p210bcr-abl, and inhibited cell growth. This anti-bcr-abl ribozyme may be a useful tool to study the cell biology of Ph1+ leukemia and may ultimately have therapeutic potential in treating patients with Ph1 leukemias.

scholarly journals Multi-unit ribozyme-mediated cleavage of bcr-abl mRNA in myeloid leukemias

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2162-2170 ◽  
Author(s):  
LH Leopold ◽  
SK Shore ◽  
TA Newkirk ◽  
RM Reddy ◽  
EP Reddy
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Fusion Gene ◽  
Folate Receptor ◽  
Philadelphia Chromosome ◽  
Chromosome 9 ◽  
Myelogenous Leukemia ◽  
Myeloid Leukemias ◽  
Transforming Activity ◽  
Cleavage Reactions

Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.

scholarly journals Transfected leukocyte integrin CD11b/CD18 (Mac-1) mediates phorbol ester-activated, homotypic cell:cell adherence in the K562 cell line

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2537-2545 ◽  
Author(s):  
DD Hickstein ◽  
E Grunvald ◽  
G Shumaker ◽  
DM Baker ◽  
AL Back ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Activation ◽  
Blast Crisis ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Cell Clones ◽  
A Cell ◽  
Neutrophil Adherence ◽  
The Individual

Abstract The CD11b/CD18 leukocyte integrin molecule mediates diverse neutrophil adherence-related functions, including cell:cell and cell:extracellular matrix attachments. To study the individual role of this leukocyte integrin in cell adherence in hematopoietic cells, we expressed the CD11b/CD18 complex on the surface of K562 cells, a cell line derived from an individual with chronic myelogenous leukemia in blast crisis. We used an amphotrophic retroviral vector designated LCD18SN, harboring the complete coding sequence for the CD18 subunit, to transfer the CD18 cDNA into K562 cells and select stable cell lines. The CD11b subunit in the expression plasmid pREP4 was transfected into these K562/CD18 cells by electroporation and stable cell clones were selected. These K562 cells possessed RNA and intracellular protein for each subunit, and they expressed the CD11b/CD18 heterodimer on the cell surface. When CD11b/CD18 expressing K562 cells were stimulated with phorbol myristate acetate (50 ng/mL) for 24 to 48 hours, these K562 cells formed dense cell:cell aggregates. This homotypic aggregation required both activation of the CD11b/CD18 complex and the induction of the counter- receptor for CD11b/CD18 on the conjugate cell. This cell line will (1) enable the structure-function relationships between cell activation and homotypic adherence to be assessed, (2) provide the opportunity to identify accessory molecules required for activation of the CD11b/CD18 complex, and (3) facilitate the identification of novel ligands for the CD11b/CD18 complex.

Application of FASTTM Fragment-Based Lead Discovery and Structure-Guided Design to Discovery of Small Molecule Inhibitors of BCR-ABL Tyrosine Kinase Active Against the T315I Imatinib-Resistant Mutant.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 698-698 ◽  
Author(s):  
Stephen K. Burley
Keyword(s):  
Tyrosine Kinase ◽  
Fusion Gene ◽  
Clinical Success ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Lead Discovery ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant

Abstract The Philadelphia chromosome translocation creates a BCR-ABL fusion gene that encodes a constitutively active BCR-ABL tyrosine kinase, which gives rise to chronic myelogenous leukemia (CML). The clinical success of imatinib (Gleevec) demonstrated that BCR-ABL tyrosine kinase inhibitors can provide effective treatment for CML. However, some CML patients treated with imatinib develop resistance leading to disease progression. The majority of resistance is due to point mutations in BCR-ABL, which give rise to active mutant enzymes that are insensitive to imatinib. In all, ~30 imatinib-resistant BCR-ABL mutants have been identified in clinical isolates. The T315I mutant represents ~20% of clinically observed mutations, making it one of the most common causes of resistance. Second-generation BCR-ABL inhibitors, including AMN-107 and BMS-354825, inhibit many of the clinically relevant mutants but not T315I. Mutant T315I BCR-ABL is, therefore, an important and challenging target for discovery of CML therapeutics. We have applied a proprietary X-ray crystallographic fragment-based lead discovery platform (FASTTM) and structure-guided lead optimization to identify potent inhibitors of wild-type BCR-ABL and the four most common mutants, including T315I. Our lead discovery efforts yielded five chemical series that inhibit both wild-type (WT) and T315I BCR-ABL. Compounds in our most advanced lead series potently inhibit proliferation of K562 cells and Ba/F3 cells with WT BCR-ABL and the four major clinically relevant BCR-ABL mutations (T315I, E255K, M351T, Y253F; see below). Further details describing in vitro and in vivo profiling of these novel BCR-ABL T315I inhibitors will be presented. Ba/F3 cell proliferation for BCR-ABL Inhibitors (EC50, nM) BCR-ABL Form Imatinib AMN-107 BMS-354825 SGX-70430 WT 790 33 12 11 T315I > 10000 > 10000 > 10000 21 Y253F 5700 370 8 334 E255K 8300 350 7 77 M351T 2000 38 28 15 Control Assay Ba/F3 (T315I) + IL3 > 10000 > 10000 > 10000 > 10000

The Philadelphia Chromosome Is Not a Stable Structure and Is Vulnerable to Further Rearrangements during Disease Progression in CML

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4141-4141
Author(s):  
Anna Virgili ◽  
Diana Brazma ◽  
Anastasios Chanalaris ◽  
Colin Grace ◽  
Elisabeth Nacheva
Keyword(s):  
Disease Progression ◽  
Cell Line ◽  
Cell Lines ◽  
Copy Number ◽  
Fusion Gene ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
High Copy Number ◽  
Haematopoietic Stem Cell ◽  
Ph Chromosome

Abstract Chronic myeloid leukaemia (CML) is a pluripotent haematopoietic stem cell disorder characterized by the expression of the BCR/ABL1 fusion gene, which commonly results from formation of the Philadelphia chromosome (Ph) after a t(9;22)(q34;q11) or related variant rearrangement. BCR/ABL1 is a constitutively activated tyrosine kinase and its amplification has been described in association with resistance to imatinib in CML patients. BAC array CGH analysis on CML patients and CML cell lines (Brazma et al., 2007) revealed unexpected genomic imbalances in form of duplications and high copy number gains affecting the region immediately downstream of the ABL1 gene at the Philadelphia (Ph) chromosome in patients at the blast crisis stage. We aimed to confirm and map these amplifications by fluorescence in situ hybridization (FISH) on 19 CML patients in accelerated phase/blast crisis and 10 CML cell lines (KU812, K562, MEG-01, MC3, BV173, EM-2, LAMA-84, KCL-22, JK-1 and CML-T1) with more than 1 copy of the BCR/ABL1 fusion gene. We used a range of BAC probes and 9q and 22q sub-telomeric probes in order to do the FISH mapping. While the majority of the analysed patients and cell lines (12/19 patients and 6/10 CML cell lines) had 2 identical Ph chromosomes, 2 main groups of abnormalities were identified. Firstly, gains of the Ph chromosome taking the form of ider(22)t(9;22) chromosome were detected in 1 or more copies in a subset of bone marrow cells of 5/19 patients and, secondly, high copy number gains were seen in 2/19 patients and 2/10 cell lines (K562 and KU812). The amplified region was variable in size spanning from 400 Kb up to 1.5 Mb downstream of the ABL1 gene. In 1 patient, 7 different cell sub-clones harbouring increasing levels of amplification were found. The gains resulted in formation of different chromosome structures-from an ider(22)t(9;22) to markers with tandem amplifications, which included the BCR/ABL1 fusion with variable in length sequences downstream of the ABL1. Duplication of some 571 Kb downstream of ABL1 was also detected in 1 of the 2 apparently normal Ph chromosomes in the MC3 cell line, while a larger duplication (5.16 Mb) was found in another cell line (MEG-01). These findings confirm the presence of duplications and high level amplifications at the der(22) t(9;22) in CML patients and that the sequences involved are variable in length, indicating that the Ph chromosome is an unstable structure and vulnerable to further rearrangements during disease progression.

TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3624-3624
Author(s):  
Chiara Palmi ◽  
Grazia Fazio ◽  
Ilaria Brunati ◽  
Valeria Cazzaniga ◽  
Valentina André ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Expression System ◽  
Leukemic Cells ◽  
Calcium Flux ◽  
Actin Dynamics ◽  
Childhood Leukaemia

Abstract Abstract 3624 Introduction: The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aim of our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods: the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results: The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions: in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rare occurrence of N-ras point mutations in Philadelphia chromosome positive chronic myeloid leukemia

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 1028-1032 ◽  
Author(s):  
SJ Collins ◽  
M Howard ◽  
DF Andrews ◽  
E Agura ◽  
J Radich
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Point Mutations ◽  
Myelogenous Leukemia ◽  
Nucleotide Sequencing ◽  
Ras Oncogene ◽  
Acute Myelogenous

Point mutations of the N-ras oncogene are relatively common in acute myelogenous leukemia (AML) cells, occurring in some 25% to 50% of patient samples. We used a technique involving the direct nucleotide sequencing of in vitro amplified N-ras genomic fragments to determine the frequency of N-ras point mutations in chronic myeloid leukemia (CML) cells at various stages of the disease. This approach will detect N-ras point mutations in a mixed population of cells if the mutation is present in 25% or more of the cells. We could not demonstrate any point mutation at N-ras codons 12,13 or 59–63 in any of the 44 CML cases analyzed, which included 21 blast crisis samples. In contrast with AML N-ras point mutations are exceedingly rare in CML.

scholarly journals Transfected leukocyte integrin CD11b/CD18 (Mac-1) mediates phorbol ester-activated, homotypic cell:cell adherence in the K562 cell line

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2537-2545
Author(s):  
DD Hickstein ◽  
E Grunvald ◽  
G Shumaker ◽  
DM Baker ◽  
AL Back ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Activation ◽  
Blast Crisis ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Cell Clones ◽  
A Cell ◽  
Neutrophil Adherence ◽  
The Individual

The CD11b/CD18 leukocyte integrin molecule mediates diverse neutrophil adherence-related functions, including cell:cell and cell:extracellular matrix attachments. To study the individual role of this leukocyte integrin in cell adherence in hematopoietic cells, we expressed the CD11b/CD18 complex on the surface of K562 cells, a cell line derived from an individual with chronic myelogenous leukemia in blast crisis. We used an amphotrophic retroviral vector designated LCD18SN, harboring the complete coding sequence for the CD18 subunit, to transfer the CD18 cDNA into K562 cells and select stable cell lines. The CD11b subunit in the expression plasmid pREP4 was transfected into these K562/CD18 cells by electroporation and stable cell clones were selected. These K562 cells possessed RNA and intracellular protein for each subunit, and they expressed the CD11b/CD18 heterodimer on the cell surface. When CD11b/CD18 expressing K562 cells were stimulated with phorbol myristate acetate (50 ng/mL) for 24 to 48 hours, these K562 cells formed dense cell:cell aggregates. This homotypic aggregation required both activation of the CD11b/CD18 complex and the induction of the counter- receptor for CD11b/CD18 on the conjugate cell. This cell line will (1) enable the structure-function relationships between cell activation and homotypic adherence to be assessed, (2) provide the opportunity to identify accessory molecules required for activation of the CD11b/CD18 complex, and (3) facilitate the identification of novel ligands for the CD11b/CD18 complex.

Study on the Resistant Mechanisms of K562 Cells Induced by STI571.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4366-4366
Author(s):  
Xiaoli Liu ◽  
Song Zhang ◽  
Qingfeng Du ◽  
Wei Fan ◽  
Rong Li
Keyword(s):  
Cell Line ◽  
Fusion Gene ◽  
Homo Sapiens ◽  
Blast Crisis ◽  
Cdna Array ◽  
K562 Cells ◽  
Blue Staining ◽  
Fish Analysis ◽  
Mdr 1

Abstract STI571 is a highly effective drug for the therapy of CML, but there is still drug resistance, especially in blast crisis. To study the possible mechanisms of resistance to STI571, we established the BCR/ABL+ cell line with resistance to STI571 (K562-R) in vitro by culturing a wild-type K562 cells (K562-W) in gradually increased concentrations of STI571 over a period of months. Trypan blue staining, MTT assay and Hoechst 33342 staining confirmed that K562-R can live steadily at 0.5umol/L STI571. Furthermore MDR-1 expression assay, sequence analysis, fluorescence in situ hybridization(FISH) and cDNA array were used to study the potential mechanisms of acquired resistance. The MDR-1 expression percentages of K562-W and K562-R with FASC analysis were 2.68% and 1.39% respectively. No point mutant in the BCR/ABL ATP-binding site was detected and the copies of BCR/ABL fusion gene were found increased in K562-R by FISH analysis. By a expression profile of cDNA microarray, 327 genes’ expression were found down-regulating including one of homo sapiens protein tyrosine phosphatase genes(PTPRF) and 335 genes up-regulating including homo sapiens hematopoietic cell-specific Lyn substrate 1 gene(HCLS1). Our studies proved the possible mechanism of K562-R resistance involved amplification of BCR/ABL fusion gene and increase of phosphorylation activity in this cell line.

scholarly journals A Comparison of Gene Expression Signatures from Breast Tumors and Breast Tissue Derived Cell Lines

2001 ◽  
Vol 17 (2) ◽  
pp. 99-109 ◽  
Author(s):  
Douglas T. Ross ◽  
Charles M. Perou
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Biology ◽  
Expression Patterns ◽  
Tumor Biology ◽  
Clinical Samples ◽  
Analysis Tool

Cell lines derived from human tumors have historically served as the primary experimental model system for exploration of tumor cell biology and pharmacology. Cell line studies, however, must be interpreted in the context of artifacts introduced by selection and establishment of cell linesin vitro. This complication has led to difficulty in the extrapolation of biology observed in cell lines to tumor biologyin vivo. Modern genomic analysis tool like DNA microarrays and gene expression profiling now provide a platform for the systematic characterization and classification of both cell lines and tumor samples. Studies using clinical samples have begun to identify classes of tumors that appear both biologically and clinically unique as inferred from their distinctive patterns of expressed genes. In this review, we explore the relationships between patterns of gene expression in breast tumor derived cell lines to those from clinical tumor specimens. This analysis demonstrates that cell lines and tumor samples have distinctive gene expression patterns in common and underscores the need for careful assessment of the appropriateness of any given cell line as a model for a given tumor subtype.

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