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Downregulation of miR-217 correlates with resistance of Ph(+) leukemia cells to ABL tyrosine kinase inhibitors.

Nishioka C, Ikezoe T, Yang J, Nobumoto A, Tsuda M, Yokoyama A - Cancer Sci. (2014)

Bottom Line: Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3'-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI.In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib.Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKI.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Japan.

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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 p21waf1, 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 p21waf1 and BAX. Results represent the mean ± SD of the three experiments performed. **P < 0.01; *P < 0.05.
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fig03: 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 p21waf1, 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 p21waf1 and BAX. Results represent the mean ± SD of the three experiments performed. **P < 0.01; *P < 0.05.

Mentions: We next examined whether DNMT inhibitor 5-AzadC prevented resistance to dasatinib in K562 cells. K562 cells were cultured with either dasatinib (10 nM, 96 h – 2 months) and/or 5-AzadC (0.1 μM, 96 h – 2 months). The culture media were replaced and drugs were added every 5 days. 5-AzadC only slightly inhibited the proliferation of K562 cells. K562 cells lost their sensitivity to dasatinib after 2 months culture in the presence of dasatinib (Fig.3a). Notably, when K562 cells were cultured with a combination of dasatinib (10 nM) and 5-AzadC (0.1 μM), their proliferation was potently inhibited even after 2 months in parallel with upregulation of miR-29b-1, miR-29b-2 and miR-217 and downregulation of DNMT (Fig.3b,c).


Downregulation of miR-217 correlates with resistance of Ph(+) leukemia cells to ABL tyrosine kinase inhibitors.

Nishioka C, Ikezoe T, Yang J, Nobumoto A, Tsuda M, Yokoyama A - Cancer Sci. (2014)

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 p21waf1, 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 p21waf1 and BAX. Results represent the mean ± SD of the three experiments performed. **P < 0.01; *P < 0.05.
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fig03: 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 p21waf1, 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 p21waf1 and BAX. Results represent the mean ± SD of the three experiments performed. **P < 0.01; *P < 0.05.
Mentions: We next examined whether DNMT inhibitor 5-AzadC prevented resistance to dasatinib in K562 cells. K562 cells were cultured with either dasatinib (10 nM, 96 h – 2 months) and/or 5-AzadC (0.1 μM, 96 h – 2 months). The culture media were replaced and drugs were added every 5 days. 5-AzadC only slightly inhibited the proliferation of K562 cells. K562 cells lost their sensitivity to dasatinib after 2 months culture in the presence of dasatinib (Fig.3a). Notably, when K562 cells were cultured with a combination of dasatinib (10 nM) and 5-AzadC (0.1 μM), their proliferation was potently inhibited even after 2 months in parallel with upregulation of miR-29b-1, miR-29b-2 and miR-217 and downregulation of DNMT (Fig.3b,c).

Bottom Line: Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3'-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI.In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib.Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKI.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Japan.

Show MeSH
Related in: MedlinePlus