Down-Modulation of P210bcr/abl Induces Apoptosis/Differentiation in K562 Leukemic Blast Cells

1997 ◽  
Vol 83 (4) ◽  
pp. 756-761 ◽  
Author(s):  
Arunkumar B. Deora ◽  
Michelle B. Miranda ◽  
S.G. Anand Rao
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Leukemic Blast ◽  
Tyrosine Kinase Activity ◽  
Abl Tyrosine Kinase ◽  
Blast Cells ◽  
Induced Apoptosis

Aims and background K562 cells are growth factor independent and neither function as stem cells nor differentiate into functional end cells. They are blast cells. There is evidence that the constitutively expressed bcr-abl tyrosine kinase might be responsible for the maintenance of the blast state of CML cells. We have studied the effect of two tyrosine kinase inhibitors, quercetin and genistein, on K562 cells. Methods K562 cells were treated with quercetin/genistein for a period of 72 hrs and then subjected to staining for apoptosis and erythroid differentiation and Western blotting with c-abl and phosphotyrosine monoclonal antibodies. Results The IC50 value was found to be 9.2 μg/ml for quercetin and 11.8 μg/ml for genistein. Quercetin-treated cells did not show any differentiation but showed 68% apoptosis as compared to 7% in control. Genistein-treated cells showed 16% apoptosis and 15% erythroid differentiation. Quercetin reduced the level of p210 by 74% and its phosphotyrosine content by 67.6%. Genistein reduced p210 by 77.8% and its phosphotyrosine content by 16%. Conclusion Both quercetin and genistein are able to down-modulate the tyrosine kinase activity of p210 as well as bring about a decrease in the content of the protein with different effects: quercetin induced apoptosis while genistein brought about both differentiation and apoptosis.

scholarly journals Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic-nuclear transport

1996 ◽  
Vol 93 (26) ◽  
pp. 15174-15179 ◽  
Author(s):  
J. M. Lewis ◽  
R. Baskaran ◽  
S. Taagepera ◽  
M. A. Schwartz ◽  
J. Y. J. Wang
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Nuclear Transport ◽  
Tyrosine Kinase Activity ◽  
Abl Tyrosine Kinase ◽  
Integrin Regulation

scholarly journals Transmission of growth cone traction force through apCAM–cytoskeletal linkages is regulated by Src family tyrosine kinase activity

2001 ◽  
Vol 155 (3) ◽  
pp. 427-438 ◽  
Author(s):  
Daniel M. Suter ◽  
Paul Forscher
Keyword(s):  
Tyrosine Kinase ◽  
Growth Cone ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Traction Force ◽  
Tyrosine Kinase Activity ◽  
Cellular Mechanisms ◽  
Traction Forces ◽  
Kinase Activation ◽  
Neuronal Growth Cone

Tyrosine kinase activity is known to be important in neuronal growth cone guidance. However, underlying cellular mechanisms are largely unclear. Here, we report how Src family tyrosine kinase activity controls apCAM-mediated growth cone steering by regulating the transmission of traction forces through receptor–cytoskeletal linkages. Increased levels of tyrosine phosphorylation were detected at sites where beads coated with apCAM ligands were physically restrained to induce growth cone steering, but not at unrestrained bead binding sites. Interestingly, the rate and level of phosphotyrosine buildup near restrained beads were decreased by the myosin inhibitor 2,3-butanedione-2-monoxime, suggesting that tension promotes tyrosine kinase activation. While not affecting retrograde F-actin flow rates, genistein and the Src family selective tyrosine kinase inhibitors PP1 and PP2 strongly reduced the growth cone's ability to apply traction forces through apCAM–cytoskeletal linkages, assessed using the restrained bead interaction assay. Furthermore, increased levels of an activated Src family kinase were detected at restrained bead sites during growth cone steering events. Our results suggest a mechanism by which growth cones select pathways by sampling both the molecular nature of the substrate and its ability to withstand the application of traction forces.

Fluorescent monitoring of kinase activity in real time: development of a robust fluorescence-based assay for Abl tyrosine kinase activity

2001 ◽  
Vol 11 (24) ◽  
pp. 3091-3094 ◽  
Author(s):  
Roseanne M. Hofmann ◽  
Graham J. Cotton ◽  
Emmanuel J. Chang ◽  
Ephraim Vidal ◽  
Darren Veach ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Real Time ◽  
Kinase Activity ◽  
Time Development ◽  
Tyrosine Kinase Activity ◽  
Abl Tyrosine Kinase

Combined Effects of As4S4 and Imatinib on Chronic Myeloid Leukemia Cells and BCR-ABL Oncoprotein.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4651-4651
Author(s):  
Tong Yin ◽  
Ying-Li Wu ◽  
Hui-Ping Sun ◽  
Guan-Lin Sun ◽  
Ji Zhang ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Synergistic Effect ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Synergistic Effects ◽  
Chronic Phase ◽  
K562 Cells ◽  
G1 Arrest ◽  
Tyrosine Kinase Activity

Abstract Imatinib is a tailored drug for chronic myeloid leukemia (CML), which has very good effects on patients at chronic phase (CP), but not on those at accelerated phase or blast phase. In addition, even among patients at CP, Imatinib seems unable to eradicate the malignant progenitors and a significant portion of patients develops drug resistance after long time use. Arsenic compounds were known as ancient remedies for CML with certain efficacy. The aim of this study was to investigate the potential benefit of combination therapy with Imatinib and arsenic sulfide (As4S4) on BCR-ABL+ K562 cells and fresh CD34+ hematopoietic progenitor cells isolated from CML patients and non-leukemic donors. Analysis of cell proliferation and clonogenic ability showed that As4S4 and Imatinib exerted synergistic effects on both K562 cells and fresh CML cells. The effective concentrations on fresh CML cells were pharmacokinetically available in vivo but had much less inhibitory effect on CD34+ cells from the non-leukemia donors. The synergistic effect of Imatinib/As4S4 combination in terms of anti-proliferation might be connected with their distinct but complementary roles in interfering with the cell cycle progression. Our data showed that Imatinib induced G1 arrest of K562 cells, while As4S4 induced G2/M arrest. In addition, Imatinib induces significant down-regulation of phosphorylated Rb and CDK1, which is in agreement with the G1/S but not G2/M arrest under this drug. However, As4S4 shows no obvious effect on these proteins in spite of a visible effect on G2/M block. Using a number of parameters such as morphology, Annexin V/PI, mitochondrial transmembrane potential, caspase3 activity and Fas/Fas-L, the synergistic effects were revealed on induction of cell apoptosis, largely through mitochondrial pathway. What’s more, the two drugs also exhibited synergistic effect in targeting BCR-ABL protein. While As4S4 triggered its degradation and Imatinib inhibited its tyrosine kinase activity, combined use of the two led to lower protein/enzymatic activity levels of BCR-ABL. In conclusion, our study suggests As4S4 and Imatinib have synergistic effects in inhibiting proliferation, inducing apoptosis of cells and reduction the tyrosine kinase activity of BCR-ABL. Our in vitro data thus strongly suggest a potential clinical application of Imatinib/As4S4 combination on CML.

scholarly journals Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins.

1991 ◽  
Vol 11 (3) ◽  
pp. 1553-1565 ◽  
Author(s):  
J R McWhirter ◽  
J Y Wang
Keyword(s):  
Amino Acids ◽  
Tyrosine Kinase ◽  
Fusion Proteins ◽  
Kinase Activity ◽  
Lymphoblastic Leukemia ◽  
Myelogenous Leukemia ◽  
Tyrosine Kinase Activity ◽  
Abl Tyrosine Kinase ◽  
Binding Function

Chronic myelogenous leukemia and one type of acute lymphoblastic leukemia are characterized by a 9;22 chronosome translocation in which 5' sequences of the bcr gene become fused to the c-abl proto-oncogene. The resulting chimeric genes encode bcr/abl fusion proteins which have deregulated tyrosine kinase activity and appear to play an important role in induction of these leukemias. A series of bcr/abl genes were constructed in which nested deletions of the bcr gene were fused to the c-abl gene. The fusion proteins encoded by these genes were assayed for autophosphorylation in vivo and for differences in subcellular localization. Our results demonstrate that bcr sequences activate two functions of c-abl; the tyrosine kinase activity and a previously undescribed microfilament-binding function. Two regions of bcr which activate these functions to different degrees have been mapped: amino acids 1 to 63 were strongly activating and amino acids 64 to 509 were weakly activating. The tyrosine kinase and microfilament-binding functions were not interdependent, as a kinase defective bcr/abl mutant still associated with actin filaments and a bcr/abl mutant lacking actin association still had deregulated kinase activity. Modification of actin filament functions by the bcr/abl tyrosine kinase may be an important event in leukemogenesis.

scholarly journals Morphological Differentiation of Oligodendrocytes Requires Activation of Fyn Tyrosine Kinase

1999 ◽  
Vol 145 (6) ◽  
pp. 1209-1218 ◽  
Author(s):  
Donna J. Osterhout ◽  
Amy Wolven ◽  
Rebecca M. Wolf ◽  
Marilyn D. Resh ◽  
Moses V. Chao
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Morphological Differentiation ◽  
Dominant Negative ◽  
Tyrosine Kinase Activity ◽  
Oligodendrocyte Progenitors ◽  
Differentiated Cells ◽  
Process Formation ◽  
Fyn Tyrosine Kinase

In the central nervous system, myelination of axons occurs when oligodendrocyte progenitors undergo terminal differentiation and initiate process formation and axonal ensheathment. Although it is hypothesized that neuron-oligodendrocyte contact initiates this process, the molecular signals are not known. Here we find that Fyn tyrosine kinase activity is upregulated very early during oligodendrocyte progenitor cell differentiation. Concomitant with this increase is the appearance of several tyrosine phosphorylated proteins present only in differentiated cells. The increased tyrosine kinase activity is specific to Fyn, as other Src family members are not active in oligodendrocytes. To investigate the function of Fyn activation on differentiation, we used Src family tyrosine kinase inhibitors, PP1 and PP2, in cultures of differentiating oligodendrocyte progenitors. Treatment of progenitors with these compounds prevented activation of Fyn and reduced process extension and myelin membrane formation. This inhibition was reversible and not observed with related inactive analogues. A similar effect was observed when a dominant negative Fyn was introduced in progenitor cells. These findings strongly suggest that activation of Fyn is an essential signaling component for the morphological differentiation of oligodendrocytes.

scholarly journals Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system

2021 ◽  
Author(s):  
Naoki Yamamoto ◽  
Jiro Kikuchi ◽  
Yusuke Furukawa ◽  
Naoya Shibayama
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Expression System ◽  
Structural Studies ◽  
Expression And Purification ◽  
Tyrosine Kinase Activity ◽  
Fast Screening ◽  
Baculovirus System

We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 potential kinase inhibitors, and succeeded in identifying two potent inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies.

Tyrosine kinase regulates epithelial sodium transport in A6 cells

1993 ◽  
Vol 264 (1) ◽  
pp. C246-C250 ◽  
Author(s):  
P. S. Matsumoto ◽  
A. Ohara ◽  
P. Duchatelle ◽  
D. C. Eaton
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Na Transport ◽  
Tyrosine Kinase Activity ◽  
Open Probability ◽  
Cyclic Monophosphate ◽  
Tyrphostin 23

Insulin increases epithelial Na+ reabsorption, and many of its actions involve tyrosine kinase. We used tyrosine kinase inhibitors to examine the role of tyrosine kinase in the action of insulin. Pretreatment of Na+ transporting cells with tyrosine kinase inhibitors attenuates the subsequent action of insulin, suggesting that the action of insulin on epithelial Na+ transport involves tyrosine kinase activity. In addition to their effect on insulin-induced Na+ transport, the tyrosine kinase inhibitors also significantly reduce Na+ transport in Na(+)-transporting epithelial cells, suggesting that there is a significant tonic tyrosine kinase activity that modulates epithelial Na+ transport. Using patch-clamp methods, we found that one inhibitor, genistein, reduces the number of active Na+ channels in cell-attached patches without significantly affecting the open probability of any remaining channels. The effects of the tyrosine kinase inhibitors are not due to inhibition of protein kinase A (PKA), since H89, a PKA inhibitor, does not affect Na+ transport of control cells (as the tyrosine kinase inhibitors do), and the tyrosine kinase inhibitor, genistein or tyrphostin 23, does not alter the stimulation of ion transport by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue (as H89 does).

Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products

1985 ◽  
Vol 5 (11) ◽  
pp. 3116-3123
Author(s):  
J B Konopka ◽  
O N Witte
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Myelogenous Leukemia ◽  
Gene Products ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Abl Tyrosine Kinase ◽  
Assay Conditions

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

Sign in / Sign up

Export Citation Format

Share Document