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 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 Absorption Characteristics of Combination Medication of Realgar and Indigo Naturalis: In Vitro Transport across MDCK-MDR1 Cells and In Vivo Pharmacokinetics in Mice after Oral Administration

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Miao Zhang ◽  
Lin Guo ◽  
Long-Fei Lin ◽  
Chang-Hai Qu ◽  
Xing-Bin Yin ◽  
...  
Keyword(s):  
Cell Line ◽  
K562 Cell ◽  
Computational Study ◽  
Arsenic Concentration ◽  
K562 Cells ◽  
Cell Permeability ◽  
K562 Cell Line ◽  
Apparent Permeability ◽  
Indigo Naturalis

Realgar and indigo naturalis are clinically combined to treat varieties of leukemia. Exploring the drug-drug interactions might be beneficial to find active substances and develop new targeted drugs. This study aimed at exploring the change of arsenic concentration in mice and across MDCK-MDR1 cells and the cytotoxicity on K562 cells when realgar and indigo naturalis were combined. In the presence or absence of indigo naturalis, pharmacokinetics and cell-based permeability assays were used to evaluate the change of arsenic concentration, and K562 cell line was applied to evaluate the change of cytotoxicity. The drug concentration-time profiles exhibited that the combination medication group generated higher AUC, thalf, and longer MRT for arsenic, compared with the single administration of realgar. The apparent permeability coefficients (Papp) of bidirectional transport in MDCK-MDR1 cell permeability experiments showed that arsenic permeability obviously went up when indigo naturalis was incubated together. The combination medication significantly decreased the cell viability of K562 cells when both the concentration of realgar and the concentration of indigo naturalis were nontoxic. The pharmacokinetic research, the MDCK-MDR1 based permeability study, and the K562 cytotoxicity study were united together to verify the combination medication of realgar and indigo naturalis enhanced the absorption and the permeability across cells for arsenic and effectively inhibited the proliferation of K562 cell line. The molecular binding of As4S4 and indirubin was analyzed by computational study. It is predicted that the formation of the complex [As4S4…Indirubin] involves noncovalent interaction that changes the concentration of arsenic.

scholarly journals Establishment of a novel human CIC-DUX4 sarcoma cell line, Kitra-SRS, with autocrine IGF-1R activation and metastatic potential to the lungs

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sho Nakai ◽  
Shutaro Yamada ◽  
Hidetatsu Outani ◽  
Takaaki Nakai ◽  
Naohiro Yasuda ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Fusion Gene ◽  
Primary Tumour ◽  
Chromosomal Rearrangements ◽  
Basic Research ◽  
Metastatic Potential ◽  
Original Tumour

Abstract Approximately 60–70% of EWSR1-negative small blue round cell sarcomas harbour a rearrangement of CIC, most commonly CIC-DUX4. CIC-DUX4 sarcoma (CDS) is an aggressive and often fatal high-grade sarcoma appearing predominantly in children and young adults. Although cell lines and their xenograft models are essential tools for basic research and development of antitumour drugs, few cell lines currently exist for CDS. We successfully established a novel human CDS cell line designated Kitra-SRS and developed orthotopic tumour xenografts in nude mice. The CIC-DUX4 fusion gene in Kitra-SRS cells was generated by t(12;19) complex chromosomal rearrangements with an insertion of a chromosome segment including a DUX4 pseudogene component. Kitra-SRS xenografts were histologically similar to the original tumour and exhibited metastatic potential to the lungs. Kitra-SRS cells displayed autocrine activation of the insulin-like growth factor 1 (IGF-1)/IGF-1 receptor (IGF-1R) pathway. Accordingly, treatment with the IGF-1R inhibitor, linsitinib, attenuated Kitra-SRS cell growth and IGF-1-induced activation of IGF-1R/AKT signalling both in vitro and in vivo. Furthermore, upon screening 1134 FDA-approved drugs, the responses of Kitra-SRS cells to anticancer drugs appeared to reflect those of the primary tumour. Our model will be a useful modality for investigating the molecular pathology and therapy of CDS.

Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1758-1766 ◽  
Author(s):  
Philipp le Coutre ◽  
Elena Tassi ◽  
Marileila Varella-Garcia ◽  
Rossella Barni ◽  
Luca Mologni ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Amplification ◽  
Marker Chromosome ◽  
Kinase Domain ◽  
Leukemic Cells ◽  
Tyrosine Kinase Domain ◽  
Fish Analysis ◽  
Human Leukemic Cells

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenicbcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/ablinhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 μM) 10-fold higher than the IC50 (0.1 μM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3–like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro.

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.

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.

Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-xL

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 1014-1022 ◽  
Author(s):  
Charles Perkins ◽  
Caryn N. Kim ◽  
Guofu Fang ◽  
Kapil N. Bhalla
Keyword(s):  
Myeloid Leukemia ◽  
Blast Crisis ◽  
K562 Cells ◽  
Cell Types ◽  
Multidrug Resistant ◽  
Leukemia Cells ◽  
Protein Levels ◽  
Growth Inhibitory ◽  
Induced Apoptosis

We investigated the in vitro growth inhibitory and apoptotic effects of clinically achievable concentrations of As2O3 (0.5 to 2.0 μmol/L) against human myeloid leukemia cells known to be resistant to a number of apoptotic stimuli. These included chronic myelocytic leukemia (CML) blast crisis K562 and HL-60/Bcr-Abl cells, which contain p210 and p185 Bcr-Abl, respectively, and HL-60 cell types that overexpress Bcl-2 (HL-60/Bcl-2), Bcl-xL(HL-60/Bcl-xL), MDR (HL-60/VCR), or MRP (HL-60/AR) protein. The growth-inhibitory IC50 values for As2O3 treatment for 7 days against all these cell types ranged from 0.8 to 1.5 μmol/L. Exposure to 2 μmol/L As2O3 for 7 days induced apoptosis of all cell types, including HL-60/Bcr-Abl and K562 cells. This was associated with the cytosolic accumulation of cyt c and preapoptotic mitochondrial events, such as the loss of inner membrane potential (▵Ψm) and the increase in reactive oxygen species (ROS). Treatment with As2O3 (2 μmol/L) generated the activities of caspases, which produced the cleavage of the BH3 domain containing proapoptotic Bid protein and poly (ADP-ribose) polymerase. Significantly, As2O3-induced apoptosis of HL-60/Bcr-Abl and K562 cells was associated with a decline in Bcr-Abl protein levels, without any significant alterations in the levels of Bcl-xL, Bax, Apaf-1, Fas, and FasL. Although As2O3 treatment caused a marked increase in the expression of the myeloid differentiation marker CD11b, it did not affect Hb levels in HL-60/Bcr-Abl, K562, or HL-60/neo cells. However, in these cells, As2O3 potently induced hyper-acetylation of the histones H3 and H4. These findings characterize As2O3 as a growth inhibiting and apoptosis-inducing agent against a variety of myeloid leukemia cells resistant to multiple apoptotic stimuli.

scholarly journals Vitamin E in Chronic Myeloid Leukemia (CML) Prevention

2021 ◽  
Author(s):  
Lyudmyla Shvachko ◽  
Michael Zavelevich ◽  
Daniil Gluzman ◽  
Gennadii Telegeev
Keyword(s):  
Alkaline Phosphatase ◽  
Vitamin E ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Blast Cells ◽  
Differentiation Block

The resistance to inhibitors of tyrosine kinase necessitates novel approaches to the therapy of chronic myeloid leukemia (CML). The progression of CML to blast crisis is associated with down-regulation of C/EBP-alpha being involved in the differentiation block in leukemic blast cells. Moreover, lowered C/EBP-alpha expression correlates with resistance to imatinib in CML. We have demonstrated that vitamin E up-regulates expression of C/EBP-alpha and down-regulates expression of Snail transcription factor in K562 cells in vitro contributing to the putative recovery of myeloid differentiation potential. In parallel with increased CEBP alpha expression, Vitamin E treatment results in the decreasing expression of placental-like alkaline phosphatase and increasing expression of tissue non-specific alkaline phosphatase. We suggest that vitamin E could be used as the plausible biological modulator to prevent the progression to blast crisis and to overcome drug resistance of leukemic cells in CML.

Protein kinase A mediates novel serine-584 phosphorylation of HDAC4

2019 ◽  
Vol 97 (5) ◽  
pp. 526-535 ◽  
Author(s):  
Shanmukha K. Doddi ◽  
Githavani Kummari ◽  
Jagannadham M.V. ◽  
Arunasree M. Kalle
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Line ◽  
Target Genes ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Wild Type ◽  
Kinase A

Given the well-established diversified signaling pathways for histone deacetylase 4 (HDAC4) and the regulation of HDAC4 by several post-translational modifications (PTMs), including phosphorylation, sumoylation, and ubiquitination, an unbiased and detailed analysis of HDAC4 PTMs is needed. In this study, we used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) to describe phosphorylation at serine 584 (Ser584) along with already-known dual phosphorylation at serines 265 and 266 (Ser265/266), that together regulate HDAC4 activity. Overexpression of site-specific HDAC4 mutants (S584A, S265/266A) in HEK 293T cells, followed by HDAC activity assays, revealed the mutants to be less active than the wild-type protein. In vitro kinase assays have established that Ser584 and Ser265/266 are phosphorylated by protein kinase A (PKA). Luciferase assays driven by the myocyte enhancer factor 2 (MEF2) promoter and real-time PCR analysis of the MEF2 target genes show that the S584A and S265/266A mutants are less repressive than the wild-type. Furthermore, treatment with PKA activators such as 8-Bromo-cAMP and forskolin, and silencing either by shRNA or its inhibitor H-89 in a mouse myoblast cell line (C2C12) and in a non-muscle human cell line (K562), confirmed in vivo phosphorylation of HDAC4 in C2C12 but not in K562 cells, indicating the specific functional significance of HDAC4 phosphorylation in muscle cells. Thus, we identified PKA-induced Ser584 phosphorylation of HDAC4 as a yet unknown regulatory mechanism of the HDAC4–MEF2 axis.

Sign in / Sign up

Export Citation Format

Share Document