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 -49bp 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.

• Authors: 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
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1839 Issue 4 Pg. 265-74 ( 2014) ISSN: 0006-3002 [Print] Netherlands
• PMID: 24530422 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier B.V. All rights reserved.

Chemical References

• Immunoglobulins
• LIG4 protein, human
• Nuclear Proteins
• RNA, Messenger
• SP1 antigen
• DNA-Activated Protein Kinase
• PRKDC protein, human
• DNA Ligases
• DNA Ligase ATP
• Daunorubicin

Topics

• DNA Damage (genetics)
• DNA End-Joining Repair (genetics)
• DNA Ligase ATP
• DNA Ligases (genetics)
• DNA Repair (genetics)
• DNA-Activated Protein Kinase (genetics, metabolism)
• Daunorubicin (administration & dosage)
• Drug Resistance, Neoplasm (genetics)
• Gene Expression Regulation, Leukemic (drug effects)
• HL-60 Cells
• Humans
• Immunoglobulins (genetics, metabolism)
• K562 Cells
• Leukemia (drug therapy, genetics, pathology)
• Nuclear Proteins (genetics, metabolism)
• Phosphorylation
• RNA, Messenger (biosynthesis)