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 | |
| Publication status | Published - 04-2014 |
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All Science Journal Classification (ASJC) codes
- Biophysics
- Structural Biology
- Biochemistry
- Molecular Biology
- Genetics
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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.
UR - http://www.scopus.com/inward/record.url?scp=84896999685&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84896999685&partnerID=8YFLogxK
U2 - 10.1016/j.bbagrm.2014.02.004
DO - 10.1016/j.bbagrm.2014.02.004
M3 - Article
C2 - 24530422
AN - SCOPUS:84896999685
VL - 1839
SP - 265
EP - 274
JO - Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
JF - Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
SN - 1874-9399
IS - 4
ER -