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. 2014 Jan 30;105(3):297–307. doi: 10.1111/cas.12339

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© 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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The effect of DNA methyltransferases (DNMT) inhibitor 5-AzadC on BCR/ABL tyrosine kinase inhibitors-resistance. (a) MTT assay. K562 cells were plated in 96-well plates and cultured with either dasatinib (10 nM) and/or 5-AzadC (0.1 μM). At the indicated time point, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. (b, c) Real-time RT-PCR. RNA was extracted from K562 cells. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. The combination of dasatinib and 5-AzadC blocks leukemic growth in the murine xenograft model. (d) When K562 tumors were palpable, mice were randomized into four groups (n = 9) and treatment was initiated. Dasatinib (10 mg/kg) was given to mice i.p. three times a week during a 2-week experimental period. 5-AzadC (0.1 mg/kg) was given to mice i.p. from Monday to Friday for 2 weeks. Each point represents the mean of nine tumors. (e) Tumor size at autopsy. After 2 weeks of treatment, tumors were removed. This experiment was repeated three times. (f, g) Real-time RT-PCR. RNA was extracted from K562 tumors. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. The effects of dasatinib and 5-AzadC in K562 tumors. Methylation-specific PCR. (h) DNA was extracted from K562 tumor cells. DNA with methylated CpG was processed using the EZ DNA Methylatiohn Kit. The recovered DNA was amplified by PCR on methylation of the p21^waf1, and BAX promoter. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). Real time RT-PCR. (i) RNA was extracted from tumor cells. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of p21^waf1 and BAX. Results represent the mean ± SD of the three experiments performed. **P < 0.01; *P < 0.05.