Generation and Application of a CML-Specific Recombinant Adeno-Associated Virus (rAAV) Vector.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4417-4417
Author(s):  
Marius Stiefelhagen ◽  
Marlon R. Veldwijk ◽  
Anna Jauch ◽  
Volker Eckstein ◽  
Stephanie Laufs ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Myelogenous Leukemia ◽  
Raav Vector ◽  
Adeno Associated Virus ◽  
Transfer Rates ◽  
Primary Material ◽  
Leukemic Cell Lines

Abstract Introduction: Chronic myelogenous leukemia can be controlled but in most patients not be cured by tyrosine kinase inhibition. Direct targeting using gene therapy vectors combined with vaccination strategies may allow to eradicate residual leukemic progenitors. Adeno-associated virus (AAV) vectors are stable DNA vectors which were proven to be effective in the clinical gene therapy for e.g. coagulation disorders. The various AAV serotypes lack specificity for BCR-ABL+ leukemia cells. Recently developed AAV-library techniques allow a retargeting of vectors. We generated a set of rAAV vectors specific for BCR-ABL+ cells. Material and Methods: After four selection rounds on BCR-ABL+ cells, the peptide sequences of the persisting clones were cloned into an AAV helper plasmid. rAAV-GFP stocks (2 K562-specific, 1 random) of each of the mutants were produced. Titers were determined using real time PCR-based titration assay. Both, a panel of leukemic cell lines and CML primary material were transduced with these vectors and gene transfer was determined by FACS analysis. Specificity was tested in a competitive transduction assay using BCR-ABL+ and BCR-ABL− leukemic cell lines. Transduction of primary CML cells was confirmed using FACS-sorted GFP+ cells and subsequent BCR-ABL-FISH. Results: Using the CML-specific rAAV clone on a panel of BCR-ABL+ cell lines, gene transfer rates of >60% could be obtained (random clone: <1%; rAAV-2: <5%), whereas the BCR-ABL− cell lines were not susceptible to these vectors (gene transfer < 1 %). Admixing BCR-ABL− to BCR-ABL+ cells did not result in a significant drop of the gene transfer rates in the BCR-ABL− cell lines, suggesting that vector particles were not blocked by unspecific binding. Using primary material, significant gene transfer was observed (>5%; 6x more efficient than rAAV-2). In those cells, the CML-genotype was confirmed by BCR-ABL-FISH. Conclusion: In this study, we were able to generate and apply a CML-specific rAAV vector on CML cell lines and primary material. Efficient and selective gene transfer in these cells could be obtained compared to standard rAAV-2 vectors and randomly generated clones. These data hold promise for future developments.

Complex Regulation of the Homeobox Transcription Factor TGIF: Differential Expression of Multiple 5′ Isoforms.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4254-4254
Author(s):  
Rizwan Hamid ◽  
Johnequia Patterson ◽  
Danko Martincic ◽  
Stephen J. Brandt
Keyword(s):  
Cell Lines ◽  
Expressed Sequence Tag ◽  
Genomic Structure ◽  
Complex Structure ◽  
Leukemic Cell ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
Splice Isoforms ◽  
Multiple Promoters ◽  
Leukemic Cell Lines

Abstract TG-interacting factor (TGIF) is a transcriptional repressor belonging to the TALE (three amino acid loop extension) class of homeobox proteins. In addition to its involvement by mutation or deletion in the inherited craniofacial disorder holoprosencephaly, we have shown that expression of TGIF is highly predictive of relapse and survival in acute myelogenous leukemia (AML). To better understand how TGIF expression is regulated, we characterized its genomic structure using expressed sequence tag analysis, reverse transcriptase-coupled PCR, and rapid amplification of cDNA ends. These studies revealed a complex pattern of gene expression, with 15 splice isoforms and 11 alternative 5′ exons spread over 40 kb of DNA, suggesting the presence of multiple promoters. Real-time and semi-quantitative PCR analysis showed these isoforms were differentially expressed in various adult tissues, leukemic cell lines, and AML blasts. Further, isoform C was found to be the major RNA product in hematopoietic cells, contributing significantly to total TGIF expression in leukemic cell lines TF1a, U937, AML-193, KG-1a, Kasumi-1, K562, GDM-1, HL-60 and AML blasts. This analysis suggests that altered splicing and/or expression of specific isoforms could be responsible for the reduced levels of TGIF message observed in AML blasts and cell lines. The unusually complex structure of the TGIF gene may enable its precise regulation during normal hematopoiesis and may be relevant to its reduced expression in myeloid leukemias.

Gene Therapy of Chronic Myelogenous Leukemia Using Pseudotyped Recombinant Adeno-Associated Viral Vectors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4506-4506
Author(s):  
Leopold Sellner ◽  
Marlon R Veldwijk ◽  
Jürgen A. Kleinschmidt ◽  
Stephanie Laufs ◽  
Julian Topaly ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Cell Lines ◽  
Chronic Myelogenous Leukemia ◽  
Safety Profile ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Transduction Efficiency ◽  
Vector Systems ◽  
Imatinib Resistant

Abstract Abstract 4506 Introduction Gene transfer into malignant leukemia cells can be of relevance to overcome conventional therapy-resistance. Either suicide or immune stimulating gene therapy vectors may be a tool for second line treatment of imatinib-resistant chronic myelogenous leukemia (CML). Unfortunately, for gene transfer into CML cells, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Conventional adeno-associated virus (AAV) based vectors have an advantageous safety profile, yet lack the required efficiency. Methods Pseudotyped recombinant adeno-associated viruses of the serotypes 2/1 to 2/6 (rAAV2/1 to rAAV2/6) were screened on a panel of human CML cell lines and primary CML cells to determine their gene transfer efficacy. Additionally, double-stranded self complementary rAAV (scAAV) were used to determine possible second strand synthesis limitations. Results On CML cell lines, generally rAAV2/2 and rAAV2/6 were most efficient (Fig. 1). For both, an interesting difference in transduction efficiency between the imatinib-resistant LAMA84-R and imatinib-sensitive LAMA84-S cells were observed. On primary human CML cells, rAAV2/6 proved to be significantly more efficient than the other tested vectors (4.6% ± 5.3% GFP-positive cells, p=0.011). Additionally, the transduction efficiency could be significantly increased by using scAAV vectors (scAAV2/6: 43.1% ± 25.9% GFP-positive cells, p<0.001 vs rAAV2/6; Fig. 2). Our data suggest that both the conversion of the single stranded AAV genome to double stranded DNA, as well as cell binding/entry are rate limiting steps in efficient transduction of primary human CML cells with AAV vectors. Furthermore, donor-dependent differences in gene transfer efficiency were observed. Of note, data from experiments on CML cell lines seems to provide limited information about the transduction efficiency of rAAV vectors on primary CML cells. Conclusions Overall, pseudotyped rAAV and scAAV vectors offer efficient clinically relevant gene transfer into human CML cells with a potential for future clinical application. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lineage infidelity of a human myelogenous leukemia cell line

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1059-1063 ◽  
Author(s):  
A Palumbo ◽  
J Minowada ◽  
J Erikson ◽  
CM Croce ◽  
G Rovera
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Chain Gene ◽  
B Cell Differentiation ◽  
Light Chain Gene ◽  
Leukemic Cell Lines ◽  
Atypical Cells

Abstract We have analyzed the organization and expression of the immunoglobulin heavy and light chain gene in the human myeloblastic leukemic sublines, ML1, ML2, and ML3, and in the human myeloid leukemic cell lines, HL-60, U937, THP1, and K562. ML1, ML2, and ML3 cells, despite a predominant granulocytic phenotype, express a rearrangement of the immunoglobulin heavy chain gene that typically occurs during the early stages of the B cell differentiation pathway. No rearrangement was found in any of the other cell lines tested. These findings strongly support the notion that, at least in some cases, acute myeloid leukemia (AML) cells represent highly atypical cells with profoundly altered gene expression, rather than cells arrested at a well-defined stage of the myeloid lineage.

Lineage infidelity of a human myelogenous leukemia cell line

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1059-1063 ◽  
Author(s):  
A Palumbo ◽  
J Minowada ◽  
J Erikson ◽  
CM Croce ◽  
G Rovera
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Chain Gene ◽  
B Cell Differentiation ◽  
Light Chain Gene ◽  
Leukemic Cell Lines ◽  
Atypical Cells

We have analyzed the organization and expression of the immunoglobulin heavy and light chain gene in the human myeloblastic leukemic sublines, ML1, ML2, and ML3, and in the human myeloid leukemic cell lines, HL-60, U937, THP1, and K562. ML1, ML2, and ML3 cells, despite a predominant granulocytic phenotype, express a rearrangement of the immunoglobulin heavy chain gene that typically occurs during the early stages of the B cell differentiation pathway. No rearrangement was found in any of the other cell lines tested. These findings strongly support the notion that, at least in some cases, acute myeloid leukemia (AML) cells represent highly atypical cells with profoundly altered gene expression, rather than cells arrested at a well-defined stage of the myeloid lineage.

Novel triterpenoid CDDO-Me is a potent inducer of apoptosis and differentiation in acute myelogenous leukemia

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 326-335 ◽  
Author(s):  
Marina Konopleva ◽  
Twee Tsao ◽  
Peter Ruvolo ◽  
Irina Stiouf ◽  
Zeev Estrov ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Acute Myelogenous Leukemia ◽  
Leukemic Cell ◽  
Myelogenous Leukemia ◽  
Monocytic Differentiation ◽  
Leukemic Cell Lines ◽  
Acute Myelogenous ◽  
Myeloid Leukemic Cell ◽  
Mdr 1

It has been shown that the novel synthetic triterpenoid CDDO inhibits proliferation and induces differentiation and apoptosis in myeloid leukemia cells. In the current study the effects of the C-28 methyl ester of CDDO, CDDO-Me, were analyzed on cell growth and apoptosis of leukemic cell lines and primary acute myelogenous leukemia (AML). CDDO-Me decreased the viability of leukemic cell lines, including multidrug resistant (MDR)-1–overexpressing, p53null HL-60-Dox and of primary AML cells, and it was 3- to 5-fold more active than CDDO. CDDO-Me induced a loss of mitochondrial membrane potential, induction of caspase-3 cleavage, increase in annexin V binding and DNA fragmentation, suggesting the induction of apoptosis. CDDO-Me induced pro-apoptotic Bax protein that preceded caspase activation. Furthermore, CDDO-Me inhibited the activation of ERK1/2, as determined by the inhibition of mitochondrial ERK1/2 phosphorylation, and it blocked Bcl-2 phosphorylation, rendering Bcl-2 less anti-apoptotic. CDDO-Me induced granulo-monocytic differentiation in HL-60 cells and monocytic differentiation in primary cells. Of significance, colony formation of AML progenitors was significantly inhibited in a dose-dependent fashion, whereas normal CD34+ progenitor cells were less affected. Combinations with ATRA or the RXR-specific ligand LG100268 enhanced the effects of CDDO-Me on cell viability and terminal differentiation of myeloid leukemic cell lines. In conclusion, CDDO-Me is an MDR-1– and a p53-independent compound that exerts strong antiproliferative, apoptotic, and differentiating effects in myeloid leukemic cell lines and in primary AML samples when given in submicromolar concentrations. Differential effects of CDDO-Me on leukemic and normal progenitor cells suggest that CDDO-Me has potential as a novel compound in the treatment of hematologic malignancies.

Distinct DNA Methylation Patterns of HOX Genes in Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2238-2238
Author(s):  
Amanda J. Saraf ◽  
Samantha F. Lau ◽  
Rong He ◽  
Jean-Pierre J. Issa ◽  
Jaroslav Jelinek
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Blood Cells ◽  
Hox Genes ◽  
Cpg Islands ◽  
Leukemic Cell ◽  
Epigenetic Silencing ◽  
Myelogenous Leukemia ◽  
Leukemic Cell Lines ◽  
Normal Controls

Abstract The HOX subset of homeobox gene family comprises 39 genes organized in 4 clusters A-D on 4 different chromosomes and in 13 paralog groups defined across the clusters. HOX genes are important regulators of development and tissue differentiation. Dysregulated expression disturbs hematopoiesis and HOX genes are frequent partners in chromosomal translocations involved in leukemia. DNA methylation of promoter-associated CpG islands is an epigenetic modification resulting in transcriptional silencing of affected genes. Methylation pattern is faithfully copied during cell replication to subsequent cell generations and hypermethylation thus represents a permanent mark functionally equivalent to a loss-of-function mutation. Epigenetic silencing of HOX genes may disrupt normal development of blood cells and thus be involved in the development of leukemia, as was recently reported for HOXA5. We performed a comprehensive DNA methylation analysis of 22 HOX genes with CpG islands in promoter to exon 1 regions: HOXA1, A4, A5, A7, A9, A10, A11; B1, B4, B5, B7, B8, B9, B13; C4, C5, C8, C9, C10, C13; D1 and D10 in 16 leukemic cell lines, 24 samples from patients with acute myelogenous leukemia (AML), 20 samples from patients with acute lymphoblastic leukemia (ALL), and 15 control samples of normal blood cells. We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Overall, lymphoblastic leukemic cell lines (TALL, Raji, ALL1, JTAg, CEM, BJAB, Jurkat) showed the highest degree of HOX genes methylation (average 60%), followed by myeloid leukemic cell lines (HL60, KG1a, ML1, HEL, TF-1, OCI-AML3, K562, TF1i, KG1, average methylation 40%), p=0.000. Analysis of ALL and AML patient samples revealed significant differences in methylation levels of several HOX genes. Consistently with their reported overexpression in leukemia, HOX A9, B4, and also HOXB5 and HOXD10 were seldom methylated in AML patients (2/24, 0/24, 1/12, 7/24, respectively), while hypermethylation over a 10% cutoff value was observed frequently in ALL patients (10/20, 6/19, 6/17, 9/19, respectively). The differences in methylation were statistically significant (p<0.05). On the other hand, HOXA4 showed significantly higher methylation densities in AML (median 74%, range 35–95%) and ALL (median 49%, range 12–91%) than in normal controls (median 34%, range 17–59%), p<0.01. HOXC4 was more frequently methylated in normal controls (11/13) and AML (21/24) than in ALL (12/20 patients). ALL patients also showed significantly lower methylation densities of HOXC4 (median 12%, range 5–39%) than AML patients (median 32%, range 4–54%, p=0.001), underscoring the importance of HOXC4 for lymphoid cells. Unsupervised hierarchical clustering of methylation densities of all analyzed HOX genes clearly separated AML from ALL patients. We propose that epigenetic silencing of HOX genes by DNA methylation plays a role in the process of leukemic transformation of hematopoietic cells. Further analyses will help to gain a better insight into the role of HOX gene dysregulation in leukemia.

Repositioning of Difluorinated Propanediones as Inhibitors of Histone Methyltransferases and their Biological Evaluation in Human Leukemic Cell Lines

2019 ◽  
Vol 18 (13) ◽  
pp. 1892-1899 ◽  
Author(s):  
Tanushree Pal ◽  
Asmita Sharda ◽  
Bharat Khade ◽  
C. Sinha Ramaa ◽  
Sanjay Gupta
Keyword(s):  
Cell Lines ◽  
Physiological Role ◽  
Leukemic Cell ◽  
Docking Studies ◽  
Biological Evaluation ◽  
Leukemic Cell Lines ◽  
Histone Lysine ◽  
Histone Lysine Methyltransferase ◽  
Subsequent Decrease

Background: At present, ‘pharmaco-epigenomics’ constitutes the hope in cancer treatment owing to epigenetic deregulation- a reversible process and playing a role in malignancy. Objective: Chemotherapy has many limitations like host-tissue toxicity, drug resistance. Hence, it is imperative to unearth targets to better treat cancer. Here, we intend to repurpose a set of our previously synthesized difluorinated Propanediones (PR) as Histone lysine Methyltransferase inhibitors (HMTi). Methods: The cell lines of leukemic origin viz. histiocytic lymphoma (U937) and acute T-cell leukemia (JURKAT) were treated with PR-1 to 7 after docking studies with active pocket of HMT. The cell cycle analysis, in vitro methylation and cell proliferation assays were carried out to delineate their physiological role. Results: A small molecule PR-4, at 1 and 10µM, has shown to alter the methylation of histone H3 and H4 in both cell lines. Also, treatment shows an increase in G2/M population and a subsequent decrease in the G0/G1 population in U937. In JURKAT, an increase in both G2/M and S phase population was observed. The sub-G1 population showed a steady rise with increase in dose and prolonged time intervals in U937 and JURKAT cell lines. In SRB assay, the PR showed a cell growth of 42.6 and 53.4% comparable to adriamycin; 44.5 and 53.2% in U937 and JURKAT, respectively. The study suggests that PR-4 could emerge as a potential HMT inhibitor. Conclusion: The molecule PR-4 could be a lead in developing more histone lysine methyltransferases inhibitors with potential to be pro-apoptotic agents.

A novel peptide isolated from garlic shows anticancer effect against leukemic cell lines via interaction with Bcl‐2 family proteins

2021 ◽  
Vol 97 (5) ◽  
pp. 1017-1028
Author(s):  
Karunaithas Rasaratnam ◽  
Chanin Nantasenamat ◽  
Narumon Phaonakrop ◽  
Sittiruk Roytrakul ◽  
Dalina Tanyong
Keyword(s):  
Cell Lines ◽  
Leukemic Cell ◽  
Anticancer Effect ◽  
Leukemic Cell Lines

scholarly journals 30-Normedicagenic Acid Glycosides from Chenopodium Foliosum

2012 ◽  
Vol 7 (11) ◽  
pp. 1934578X1200701
Author(s):  
Paraskev T. Nedialkov ◽  
Zlatina Kokanova-Nedialkova ◽  
Daniel Bücherl ◽  
Georgi Momekov ◽  
Jörg Heilmann ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Cell Lines ◽  
Interleukin 2 ◽  
Leukemic Cell ◽  
2D Nmr ◽  
Spectroscopic Methods ◽  
Dioic Acid ◽  
Leukemic Cell Lines ◽  
Aerial Parts

Two new glycosides of 30-normedicagenic acid, namely 3- O-[ β-D-glucuronopyranosyl methyl ester]-2 β,3 β-dihydroxy-30-noroleane-12,20(29)-diene-23,28-dioic acid 28- O-β-D-glucopyranosyl ester, and 3- O-β-D-glucopyranosyl-2 β,3 β-dihydroxy-30-noroleane-12,20(29)-diene-23,28-dioic acid, together with the known 3- O-β-glucopyranosyl-2 β,3 β-dihydroxy-30-noroleane-12,20(29)-diene-23,28-dioic acid 28- O-β-glucopyranosyl ester, and 3- O-β-glucuronopyranosyl-2 β,3 β-dihydroxy-30-noroleane-12,20(29)-diene-23,28-dioic acid 28- O-β-glucopyranosyl ester were isolated from the aerial parts of Chenopodium foliosum Asch. The structures of the compounds were determined by means of spectroscopic methods (1D and 2D NMR, UV, IR) and HRMS-ESI. The compounds were tested for cytotoxicity on three leukemic cell lines (BV-173, SKW-3, HL-60). In addition, the saponins showed moderate stimulatory effects on interleukin-2 production in PHA/PMA stimulated Jurkat E6.1 cells.

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