Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines

Yayoi Nishida; Naoki Mizutani; Minami Inoue; Yukari Omori; Keiko Tamiya-Koizumi; Akira Takagi; Tetsuhito Kojima; Motoshi Suzuki; Yoshinori Nozawa; Yosuke Minami; Kazunori Ohnishi; Tomoki Naoe; Takashi Murate

doi:10.1016/j.bbagrm.2014.02.004

Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines

Yayoi Nishida, Naoki Mizutani, Minami Inoue, Yukari Omori, Keiko Tamiya-Koizumi, Akira Takagi, Tetsuhito Kojima, Motoshi Suzuki, Yoshinori Nozawa, Yosuke Minami, Kazunori Ohnishi, Tomoki Naoe, Takashi Murate

Molecular Oncology

Research output: Contribution to journal › Article

14 Citations (Scopus)

Abstract

Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between - 49. bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

Original language	English
Pages (from-to)	265-274
Number of pages	10
Journal	Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
Volume	1839
Issue number	4
DOIs	https://doi.org/10.1016/j.bbagrm.2014.02.004
Publication status	Published - 04-2014

All Science Journal Classification (ASJC) codes

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics

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Nishida, Y., Mizutani, N., Inoue, M., Omori, Y., Tamiya-Koizumi, K., Takagi, A., Kojima, T., Suzuki, M., Nozawa, Y., Minami, Y., Ohnishi, K., Naoe, T., & Murate, T. (2014). Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines. Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, 1839(4), 265-274. https://doi.org/10.1016/j.bbagrm.2014.02.004

Nishida, Yayoi ; Mizutani, Naoki ; Inoue, Minami ; Omori, Yukari ; Tamiya-Koizumi, Keiko ; Takagi, Akira ; Kojima, Tetsuhito ; Suzuki, Motoshi ; Nozawa, Yoshinori ; Minami, Yosuke ; Ohnishi, Kazunori ; Naoe, Tomoki ; Murate, Takashi. / Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines. In: Biochimica et Biophysica Acta - Gene Regulatory Mechanisms. 2014 ; Vol. 1839, No. 4. pp. 265-274.

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title = "Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines",

abstract = "Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between - 49. bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.",

author = "Yayoi Nishida and Naoki Mizutani and Minami Inoue and Yukari Omori and Keiko Tamiya-Koizumi and Akira Takagi and Tetsuhito Kojima and Motoshi Suzuki and Yoshinori Nozawa and Yosuke Minami and Kazunori Ohnishi and Tomoki Naoe and Takashi Murate",

year = "2014",

month = apr,

doi = "10.1016/j.bbagrm.2014.02.004",

language = "English",

volume = "1839",

pages = "265--274",

journal = "Biochimica et Biophysica Acta - Gene Regulatory Mechanisms",

issn = "1874-9399",

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number = "4",

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Nishida, Y, Mizutani, N, Inoue, M, Omori, Y, Tamiya-Koizumi, K, Takagi, A, Kojima, T, Suzuki, M, Nozawa, Y, Minami, Y, Ohnishi, K, Naoe, T & Murate, T 2014, 'Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines', Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, vol. 1839, no. 4, pp. 265-274. https://doi.org/10.1016/j.bbagrm.2014.02.004

Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines. / Nishida, Yayoi; Mizutani, Naoki; Inoue, Minami; Omori, Yukari; Tamiya-Koizumi, Keiko; Takagi, Akira; Kojima, Tetsuhito; Suzuki, Motoshi; Nozawa, Yoshinori; Minami, Yosuke; Ohnishi, Kazunori; Naoe, Tomoki; Murate, Takashi.

In: Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, Vol. 1839, No. 4, 04.2014, p. 265-274.

Research output: Contribution to journal › Article

TY - JOUR

T1 - Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines

AU - Nishida, Yayoi

AU - Mizutani, Naoki

AU - Inoue, Minami

AU - Omori, Yukari

AU - Tamiya-Koizumi, Keiko

AU - Takagi, Akira

AU - Kojima, Tetsuhito

AU - Suzuki, Motoshi

AU - Nozawa, Yoshinori

AU - Minami, Yosuke

AU - Ohnishi, Kazunori

AU - Naoe, Tomoki

AU - Murate, Takashi

PY - 2014/4

Y1 - 2014/4

N2 - Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between - 49. bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

AB - Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between - 49. bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

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