In vitro Bcr-Abl kinase assays

Bcr-Abl complexed to SHIP2 was immunoprecipitated from cell lysates of K562 cells maintained in log-phase culture conditions. Complexes were collected on protein A-Sepharose, and complexes were washed three times in lysis buffer and then washed twice in abl kinase buffer [50 mM Tris (pH 8.0), 10 mM MgCl2, 1 mM DTT, 2 mM p-nitrophenylphosphate, and 2 μM ATP]. Kinase assays were performed with 10 μM [γ-32P]ATP/sample for 15 ~ 60 mins at 30 ℃ in the presence or absence of the indicated concentrations of drug. The reaction was stopped by the addition of SDS-PAGE sample buffer and heated at 100 ℃ for 10 mins. Proteins were separated on 7.5% SDS-polyacrylamide gels, gels were dried under vacuum, and phosphorylation was visualized by autoradiography on X-ray film.

Cell lines

Bcr-Abl-positive cell lines K562 and RWLeu4

Preparation method

This compound in DMSO is limited. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

10 and 35 nM; 3 days

Applications

In the Bcr-Abl-positive erythroleukemia cell line K562 and the Bcr-Abl-positive myelomonocytic cell line RWLeu4, PD173955 inhibited cell proliferation with the IC50 values of 35 and 10 nM, respectively. At the low nanomolar, PD173955 arrested cell cycle in G1 phase.

PD173955 is a potent inhibitor of Bcr-Abl, Src and Yes with IC50 value of 1-2 nM, 300 nM and 175 nM, respectively [1-3].

Bcr-Abl is a protein tyrosine kinase which has oncogenic potential. Src is an enzyme and plays an important role in a variety of cancer cells survival, angiogenesis, proliferation and invasion pathways. Yes is a proto-oncogene tyrosine-protein kinase smf belongs to the Src kinase family [1-3].

PD173955 is a potent Bcr-Abl, Src and Yes inhibitor. When tested with CML CD34+ GM progenitors, PD173955 inhibited KL-dependent proliferation at an IC50 value of 50 nM and GM-CSF-dependent cell growth at an IC50 value of 1μM and the maximum inhibition was achieved at the dose of 25 nM. It was shown that PD173955 reduced the fractions of cells in G2-M phase and increased the cells in G1 phase with significant difference [2]. In HT29 cells, PD173955 treatment inhibited Src auot-phosphorylation in a dose dependent manner. Further, PD173955 showed inhibition on cell growth with IC50 value of 800 nM without morphologic changes and high concentrations arrested cell cycle at the M phase [1]. When tested with Bcr-Abl-depedent cell lines K562 and RWLeu4, PD173955 showed inhibition on cell proliferation with the IC50 value of 35 and 10 nM, respectively and arrested cell cycle in G1 phase at the low nanomolar [3].

References:
[1].  Windham, T.C., et al., Src activation regulates anoikis in human colon tumor cell lines. Oncogene, 2002. 21(51): p. 7797-807.
[2].  Strife, A., et al., Direct evidence that Bcr-Abl tyrosine kinase activity disrupts normal synergistic interactions between Kit ligand and cytokines in primary primitive progenitor cells. Mol Cancer Res, 2003. 1(3): p. 176-85.
[3].  Wisniewski, D., et al., Characterization of potent inhibitors of the Bcr-Abl and the c-kit receptor tyrosine kinases. Cancer Res, 2002. 62(15): p. 4244-55.