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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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Related in: MedlinePlus

(Next page) Thyrosine kinase inhibitors (TKI) increases levels of DNA methyltransferases (DNMT). (a) MTT assay. K562 cells were plated in 96-well plates and cultured with dasatinib (10 nM) or nilitinib (100 nM). 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. (d) Western blot analysis. These cells were harvested, and subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) Expression of miRNA. Expression of miRNA of K562 cells treated with dasatinib or nilotinib (0–12 months) was analyzed using an Mir-X miRNA qRT-PCR SYBR Kit to measure the levels of the indicated gene. Results represent mean ± SD of duplicate cultures. (f) Real-time RT-PCR. RNA was extracted from bone marrow mononuclear cells isolated from Ph+ ALL (n = 1) and CML (n = 7) patients before and after treatment with TKI. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated genes. Results represent mean ± SD of duplicate cultures. **P < 0.01; *P < 0.05.
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fig01: (Next page) Thyrosine kinase inhibitors (TKI) increases levels of DNA methyltransferases (DNMT). (a) MTT assay. K562 cells were plated in 96-well plates and cultured with dasatinib (10 nM) or nilitinib (100 nM). 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. (d) Western blot analysis. These cells were harvested, and subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) Expression of miRNA. Expression of miRNA of K562 cells treated with dasatinib or nilotinib (0–12 months) was analyzed using an Mir-X miRNA qRT-PCR SYBR Kit to measure the levels of the indicated gene. Results represent mean ± SD of duplicate cultures. (f) Real-time RT-PCR. RNA was extracted from bone marrow mononuclear cells isolated from Ph+ ALL (n = 1) and CML (n = 7) patients before and after treatment with TKI. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated genes. Results represent mean ± SD of duplicate cultures. **P < 0.01; *P < 0.05.

Mentions: Exposure of CEL cells to low concentration of nilotinib (10 nM, 96 h) or dasatinib (1 nM, 96 h) increased the levels of DNMT proteins.6 Similarly, exposure of K562 cells to low concentration of dasatinib (1 nM, 48–96 h) or nilotinib (10 nM, 48–96 h) increased the levels of DNMT (DNMT1, DNMT3A and DNMT3B) (data not shown). This dose of dasatinib (1 nM, 96 h) or nilotinib (10 nM, 96 h) was not able to inhibit the proliferation of these cells (data not shown). However, exposure of K562 cells to 10-fold higher concentration of dasatinib (10 nM, 96 h) or nilotinib (100 nM, 96 h) inhibited the proliferation of K562 cells and decreased levels of DNMT1 and DNMT3A (Fig.1a,b), but not DNMT3B (data not shown). Intriguingly, when K562 cells were cultured in the presence of this dose of TKI (dasatinib 10 nM, nilotinib 100 nM) for a long period, up to 12 months, these cells lost sensitivity to TKI. In parallel, levels of DNMT1 and DNMT3A were elevated (Fig.1c,d). We next compared the DNA methylation status on the promoter regions of cell cycle regulator and proapoptotic genes between K562 and dasatinib-resistant K562 (K562DR) cells by using methylation-specific PCR. It was found that DNA methylaton on the promoter region of p21waf1 and BAX was increased in K562DR cells as compared with that in K562 cells (Fig. S1).


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)

(Next page) Thyrosine kinase inhibitors (TKI) increases levels of DNA methyltransferases (DNMT). (a) MTT assay. K562 cells were plated in 96-well plates and cultured with dasatinib (10 nM) or nilitinib (100 nM). 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. (d) Western blot analysis. These cells were harvested, and subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) Expression of miRNA. Expression of miRNA of K562 cells treated with dasatinib or nilotinib (0–12 months) was analyzed using an Mir-X miRNA qRT-PCR SYBR Kit to measure the levels of the indicated gene. Results represent mean ± SD of duplicate cultures. (f) Real-time RT-PCR. RNA was extracted from bone marrow mononuclear cells isolated from Ph+ ALL (n = 1) and CML (n = 7) patients before and after treatment with TKI. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated genes. Results represent mean ± SD of duplicate cultures. **P < 0.01; *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4317938&req=5

fig01: (Next page) Thyrosine kinase inhibitors (TKI) increases levels of DNA methyltransferases (DNMT). (a) MTT assay. K562 cells were plated in 96-well plates and cultured with dasatinib (10 nM) or nilitinib (100 nM). 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. (d) Western blot analysis. These cells were harvested, and subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) Expression of miRNA. Expression of miRNA of K562 cells treated with dasatinib or nilotinib (0–12 months) was analyzed using an Mir-X miRNA qRT-PCR SYBR Kit to measure the levels of the indicated gene. Results represent mean ± SD of duplicate cultures. (f) Real-time RT-PCR. RNA was extracted from bone marrow mononuclear cells isolated from Ph+ ALL (n = 1) and CML (n = 7) patients before and after treatment with TKI. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated genes. Results represent mean ± SD of duplicate cultures. **P < 0.01; *P < 0.05.
Mentions: Exposure of CEL cells to low concentration of nilotinib (10 nM, 96 h) or dasatinib (1 nM, 96 h) increased the levels of DNMT proteins.6 Similarly, exposure of K562 cells to low concentration of dasatinib (1 nM, 48–96 h) or nilotinib (10 nM, 48–96 h) increased the levels of DNMT (DNMT1, DNMT3A and DNMT3B) (data not shown). This dose of dasatinib (1 nM, 96 h) or nilotinib (10 nM, 96 h) was not able to inhibit the proliferation of these cells (data not shown). However, exposure of K562 cells to 10-fold higher concentration of dasatinib (10 nM, 96 h) or nilotinib (100 nM, 96 h) inhibited the proliferation of K562 cells and decreased levels of DNMT1 and DNMT3A (Fig.1a,b), but not DNMT3B (data not shown). Intriguingly, when K562 cells were cultured in the presence of this dose of TKI (dasatinib 10 nM, nilotinib 100 nM) for a long period, up to 12 months, these cells lost sensitivity to TKI. In parallel, levels of DNMT1 and DNMT3A were elevated (Fig.1c,d). We next compared the DNA methylation status on the promoter regions of cell cycle regulator and proapoptotic genes between K562 and dasatinib-resistant K562 (K562DR) cells by using methylation-specific PCR. It was found that DNA methylaton on the promoter region of p21waf1 and BAX was increased in K562DR cells as compared with that in K562 cells (Fig. S1).

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