Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib‑resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients.
- MeSH
- bcr-abl fúzové proteiny metabolismus MeSH
- chemorezistence účinky léků MeSH
- chronická myeloidní leukemie farmakoterapie metabolismus MeSH
- homeostáza účinky léků MeSH
- imatinib mesylát farmakologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- lidé MeSH
- mutace účinky léků MeSH
- nádorové buněčné linie MeSH
- proteom metabolismus MeSH
- proteomika metody MeSH
- signální transdukce účinky léků MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
AIMS: To find out membrane tolerance strategy to ethanol in Bacillus subtilis that possesses a powerful system of protection against environmental stresses. METHODS AND RESULTS: Cytoplasmic membranes of B. subtilis were severely affected by even short-term exposure to 3% (v/v) ethanol: the growth rate and membrane protein synthesis were markedly reduced, and no adaptive alterations in phospholipids were detected. Simultaneously, steady-state DPH fluorescence anisotropy (r(ss)) showed that the membrane rigidity increased substantially. Analysis of the membrane phosphoproteome using in vitro labelling with [γ-(32) P]ATP revealed the association of DnaK and GroEL chaperones with membrane, indicating a stress induction process. Upon a long-term 3% (v/v) ethanol stress, the cell growth accelerated slightly and the composition of polar head groups and fatty acids of membrane phospholipids underwent an extensive reconstruction. Correspondingly, membrane fluidity turned back to the original r(ss) values of the control cells. CONCLUSIONS: In B. subtilis, the adaptive response to short-term ethanol stress comprises the recruitment of molecular chaperones on the impaired membrane structure; consequently, the phospholipid synthesis is restored and membrane fluidity adapts properly to the continuing ethanol stress. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings underline the role of membrane lipids in establishing tolerance towards ethanol and also suggest the contribution of molecular chaperones to the membrane and cell recovery.
- MeSH
- Bacillus subtilis účinky léků růst a vývoj fyziologie MeSH
- bakteriální proteiny metabolismus MeSH
- buněčná membrána chemie metabolismus MeSH
- ethanol metabolismus farmakologie MeSH
- fluidita membrány účinky léků MeSH
- fluorescenční polarizace MeSH
- fosfolipidy metabolismus MeSH
- fyziologický stres MeSH
- mastné kyseliny analýza MeSH
- membránové lipidy chemie metabolismus MeSH
- molekulární chaperony metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
