Figure 1.
Downregulation of MDM2 by nilotinib in ALL.
A, western blot assay for the expression of BCR-ABL, MDM2, p53 and XIAP in six cultured ALL cell lines. The p53 status of these lines (EU-1, UOC-B1 and Sup-B15: wt-p53; EU-6: mutant-p53; EU-5 and EU-9: p53-null) was previously characterized [28]. B, changes of the expression levels of MDM2 and XIAP by nilotinib treatment. Representative cell lines, as indicated, were treated (T) with 2 µM nilotinib for 24 h. Untreated cells served as a control (C). The expression of proteins was detected by western blot, as indicated. C and D, western blot assays showed the dose-response and time-course of MDM2 and XIAP inhibition by nilotinib, in both BCR-ABL positive (SUP-B15) and negative (EU-1) ALL cell lines.
Figure 2.
Evaluation of the mechanism by which nilotinib inhibits MDM2.
A, RT-PCR for MDM2 mRNA expression in SUP-B15 cells, treated with 2 µM nilotinib and 0.2 µM triptolide (as a control) for the different times, as indicated. B, SUP-B15 cells were treated with or without 2 µM nilotinib for 4 h; then their cytoplasmic lysates were fractionated on a sucrose gradient. RNA was extracted from each of the fractions and subjected to quantitative RT-PCR for analysis of the distribution of MDM2 and actin mRNAs. Data represent the percentage of the total amount of corresponding mRNA in each fraction. C, CHX pulse-chase assay for detection of MDM2 turnover in SUP-B15 cells treated with or without 2 µM nilotinib for 4 h. Numerical labels under each protein band represent protein expression levels after normalization for GAPDH, compared with the untreated (0) samples (defined as 1 unit). D, SUP-B15 cells with or without nilotinib exposure were treated with 10 µM MG32 for 4 h and then the expression of proteins as indicated was assessed by Western blot. E, co-IP and western blot assay to detect the effect of nilotinib on the interaction between MDM2 and MDM4. Cell lysates from SUP-B15 treated with or without nilotinib were immunoprecipitated with the indicated antibodies. Normal mouse IgG served as a control. Proteins in immune complexes were separated on denaturing gels, transferred to filters, and then detected by western blotting with antibodies, as indicated. Antibodies for western blotting were from different species than those used in the IP. F, IP-western blot assay for detection of MDM2 ubiquitination after nilotinib treatment. SUP-B15 cells were treated with 2 µM nilotinib for the indicated length of time. Cellular lysates were immunoprecipitated with anti-MDM2 antibody, and then the MDM2 ubiquitination (MDM2-Ub) was detected by Western blot using anti-ubiquitin antibody.
Figure 3.
The effect of nilotinib-mediated downregulation of MDM2 and XIAP on activation of p53 and caspases.
A, SUP-B15 (BCR-ABL+) and EU-1 (BCR-ABL–) were treated with 2 µM nilotinib for different times and different doses for 24 h, respectively. The expression of p53, p21 and PARP was detected by western blotting. B, cell-cycle analysis in SUP-B15 cells performed 4 h post-treatment with 2 µM nilotinib, as compared with untreated cells. C and D, activation of caspase-9 (C) and -3 (D) in nilotinib-treated SUP-B15 and EU-1 cells was detected by enzyme-linked immunosorbent assay (ELISA), *p>0.05.
Figure 4.
Cytotoxic and apoptotic effects of nilotinib on ALL cells.
A, dose-dependent cytotoxic response to nilotinib in the six ALL cell lines, as tested for Fig. 1A. The viability of cells incubated with different concentrations of nilotinib for 24 h was determined by WST assay. Data from three independent experiments is represented by the mean percentage ± SD of surviving cells, as compared to the untreated controls. *p<0.01; **p<0.05 (as compared with SUP-B15) B, dose-response of apoptosis of the six cell lines treated with the nilotinib doses indicated for 24 h, as quatitatively detected by flow cytometry. *p<0.01; **p<0.05 (as compared with SUP-B15). C, the effect of p53 knockdown on sensitivity of BCR-ABL+ cells to nilotinib. SUP-B15 cells, transfected with 200 nM of either p53 siRNA or control siRNA, were treated with different dose of nilotinib for 24 h, and cell viability was detected by WST assay, *p>0.5. The expression of p53 in SUP-B15 cells transfected with p53 siRNA was determined by Western blot assay (insert). D, the effect of enforced overexpression of XIAP on sensitivity of ALL cells to nilotinib. SUP-B15 cells, transfected with pCDNA3-6myc-XIAP plasmid and vehicle control, were treated with different dose of nilotinib for 24 h, and cell viability was detected by WST assay, *p<0.05. Western blot showing the expression of ectopic XIAP (as detected by Myc antibody) in SUP-B15 cells (insert).