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 -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: Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Yayoi
authors: Y
authors: Nishida
authors: Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Naoki
authors: N
authors: Mizutani
authors: Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Minami
authors: M
authors: Inoue
authors: Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Yukari
authors: Y
authors: Omori
authors: Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Keiko
authors: K
authors: Tamiya-Koizumi
authors: Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Akira
authors: A
authors: Takagi
authors: Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Tetsuhito
authors: T
authors: Kojima
authors: Division of Molecular Carcinogenesis, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Motoshi
authors: M
authors: Suzuki
authors: Tokai Gakuin University, Kakamigahara, Japan.
authors: Yoshinori
authors: Y
authors: Nozawa
authors: Division of Blood Transfusion/Division of Oncology and Hematology, Kobe University Hospital, Kobe, Japan.
authors: Yosuke
authors: Y
authors: Minami
authors: Oncology Center, Hamamatsu University Graduate School of Medicine, Hamamatsu, Japan.
authors: Kazunori
authors: K
authors: Ohnishi
authors: National Hospital Organization, Nagoya Medical Center, Nagoya, Japan.
authors: Tomoki
authors: T
authors: Naoe
authors: Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya, Japan. Electronic address: murate@met.nagoya-u.ac.jp.
authors: Takashi
authors: T
authors: Murate
mesh_terms: D004249:DNA Damage / Q000235:genetics; D059766:DNA End-Joining Repair / Q000235:genetics; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D051747:DNA-Activated Protein Kinase / Q000235:genetics* / Q000378:metabolism; D003630:Daunorubicin / Q000008:administration & dosage; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D007938:Leukemia / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis
chemical_list: D007136:Immunoglobulins; C000607893:LIG4 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; C090698:SP1 antigen; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D003630:Daunorubicin
keywords: DNA ligase IV; DNA-PKcs; Daunorubicin; Multidrug-resistant cell line; NHEJ; Sp1 transcription factor