Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and apoptosis. These results provided a potential management by which ACM might have a crucial impact on increasing sensitivity of CML cells to imatinib in the differentiation therapeutic approaches.

The p38 mitogen-activated protein kinase (p38) is involved in multiple cellular functions such as cell proliferation and differentiation. Previously, we found that activin A mediated hemoglobin synthesis and cell growth inhibition through p38, whereas, basic fibroblast growth factor (bFGF) inactivated p38 to antagonize the activin A effects. In this study, we selected three structurally different histone deacetylase (HDAC) inhibitors, apicidin, MS275, and sodium butyrate that activate p38, to probe the signal pathway from activin A to p38 in chronic myeloid leukemia (CML)-derived K562 cells. HDAC inhibitors and activin A showed additive p38 phosphorylation. The enhanced phosphorylation of p38 was correlated with increased cell differentiation and decreased cell proliferation. The use of p38 inhibitor SB203580 in conjunction with activin A or with the HDAC inhibitors inhibited cell differentiation and restored cell proliferation, indicating that activin A and the HDAC inhibitors exert their effects through p38 activation. However, bFGF did not affect HDAC inhibitors-induced cell differentiation or growth inhibition. Western blots showed that p38 phosphorylation remained at similar levels with or without bFGF in the presence of HDAC inhibitors. Thus, the HDAC inhibitors activate p38 in a manner different from the activin A pathway. Furthermore, mRNA expressions for activin type I, IB, II, and IIB receptors remained constant in the presence of activin A, bFGF, or both activin A and bFGF. These results indicate that bFGF does not directly act on p38 nor on the mRNA expression levels of activin receptors but inhibit activin A activation of p38 upstream of p38 in K562 cells.

Interferon alpha (IFN-alpha) modulates the proliferation of different human tumour cell lines. It has been shown that IFN-alpha induces the growth inhibition of T-cell acute lymphoblastic leukaemia (T-ALL). However, its intracellular signalling mechanisms remain unknown. This study found that IFN-alpha inhibited the cell proliferation of human T-ALL cell line Jurkat in a dose- and time-dependent manner. A p38 inhibitor (SB203580), but not an extracellular signal-regulated kinase 1/2 inhibitor (PD98059) or c-Jun N-terminal kinase inhibitor (SP600125), eliminated IFN-alpha inhibition of Jurkat cell proliferation, indicating that p38 pathway is crucial for IFN-alpha-mediated growth inhibition. SB203580 targeted two p38 isoforms, p38alpha and p38beta. The expression of p38alpha and p38beta mRNA in Jurkat cells was examined by reverse transcriptase-polymerase chain reaction. The kinase activity of p38alpha and p38beta was activated by IFN-alpha in Jurkat cells. To investigate the role of p38alpha and p38beta isoforms in IFN-alpha-mediated growth inhibition, we generated stable clones that overexpressed the dominant-negative p38 isoform, p38alpha(AF) or p38beta(AF), in Jurkat cells. Overexpression of p38alpha(AF) or p38beta(AF) inhibited IFN-alpha-mediated p38 kinase activity and growth inhibition in Jurkat cells. Similarly, down-regulation of either p38alpha or p38beta by isoform-specific small interference RNAs also reduced IFN-alpha-mediated growth inhibition. These results demonstrate that IFN-alpha can regulate growth inhibition of Jurkat cells through p38alpha and p38beta.

Interleukin-5 (IL-5) induction of c-Myc expression is associated with IL-5 inhibition of apoptosis in hematopoietic cells. In this study, TFalpha1 and TFalpha8 cells with stable overexpression of IL-5 receptor alpha (IL-5Ralpha) subunit in TF-1 cells, a human hematopoietic progenitor cell line which expressed low levels of IL-5Ralpha, were used to explored how IL-5 up-regulate c-Myc and the role of c-Myc in IL-5 signaling. First, we demonstrate that IL-5 induced c-Myc RNA and protein expressions, as well as activated Janus kinases (JAK1 and JAK2) and signal transducer and activator of transcription-5b (STAT5b). JAK inhibitor AG490 and c-Myc inhibitor 10058-F4, both, reduced IL-5-mediated cell proliferation in a dose- and time-dependent manner. Both, AG490 and 10058-F4, also reduced IL-5-mediated anti-apoptotic activity. Furthermore, AG490 inhibited IL-5-mediated c-Myc induction and promoter activity. We further examined the role of JAK1 and JAK2 in the induction of c-Myc expression using the CDJAK fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1) or JAK2 (CDJAK2) as intracellular domains. Simultaneous activation of JAK1 and JAK2 by anti-CD16 antibody crosslinking of CDJAK1 and CDJAK2 could induced c-Myc expression and promoter activity; AG490 inhibited CDJAK1 and CDJAK2-mediated effects. These results suggest that IL-5 induces cell proliferation and anti-apoptosis through the JAK/c-Myc pathway, and that JAK1 and JAK2 activation participate in IL-5-induced up-regulation of c-Myc.

Chronic myeloid leukemia (CML) is a clonal disorder of hematopoietic stem/progenitor cells that is caused by the Bcr-Abl oncoprotein. Clinical resistance to the Bcr-Abl inhibitor imatinib is a critical problem in treating CML. This study investigated the antitumor effect and mechanism of MPT0B169, a new antitubulin agent, in K562 CML cells and their derived imatinib-resistant cells, IMR2 and IMR3. IMR2 and IMR3 cells showed complete resistance to imatinib-induced growth inhibition and apoptosis. Resistance involved ERK1/2 overactivation and MDR1 overexpression. MPT0B169 inhibited the growth of K562, IMR2, and IMR3 cells in a dose- and time-dependent manner. MPT0B169 substantially inhibited the mRNA and protein levels of Bcr-Abl, followed by its downstream pathways including Akt, ERK1/2, and STAT3 in these cells. MPT0B169 treatment resulted in a decrease in the polymer form of tubulin according to Western blot analysis. It triggered cell cycle arrest at the G2/M phase before apoptosis, which was related to the upregulation of the mitotic marker MPM2 and the cyclin B1 level, and a change in the phosphorylation of Cdk1. MPT0B169 induced apoptosis in nonresistant and imatinib-resistant cells via a mitochondrion-mediated caspase pathway. Further study showed that the agent led to a decrease in the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 and an increase in the apoptotic protein Bax. Taken together, our results suggest that MPT0B169 might be a promising agent for overcoming imatinib resistance in CML cells.