Study of Abnormal NHEJ Function in Human Myeloid Leukemia.
Authors: Fu Zheng-zheng , Chen Zi-xing , Wang2 Zhi-min , Cen Jian-nong , He Jun , Qiu Qiao-cheng 
Published: April 2020

Abstract
Non-homologous end joining (NHEJ) is a major mechanism by which eukaryote cells can repair the DNA double-strand break (DSB) and protect the cell from further damages. Evidences have suggested that the genomic instability caused by deficient DNA repair function may contribute to the development of solid tumor, while its role in leukemogenesis has not been adequately studied. To study the NHEJ function in myeloid leukemia, an in vitro system was developed for clinical samples by using the linear plasmid pUC18 DNA digested with EcoR I as assay system.. Nuclear protein extracted from leukemic cells and mononuclear cells (MNCs) from normal BM or PB, was incubated with linear plasmid pUC18 DNA under certain conditions. Plasmid DNA was separated by agarose gel electrophoresis and imaged by SYBR greenIstaining. End-ligation efficiency was assessed by dividing the densitometry readings for the sum of all converted plasmid products by the sum of all products. This assay was performed on 7 myeloid leukemia cell lines, 16 samples of normal BM or PB cells, 20 cases of CML cells and 19 cases of de novo AML cells. E. coli strain DH5α was electrotransformed with pUC18 DNA end-joined by nuclear proteins from normal BM or leukemia cells, and was plated on LB agar, containing X-gal and IPTG. Correct ligation of cut plasmid DNA resulted in blue colonies while faulty repair would result in white colonies. The percentage of white colonies over total colonies gave the frequency of misrepair. Primers around the EcoR I site were designed and colony PCR was performed on blue and white colonies. Sequencing of PCR products was performed. Antibodies against the nuclear repair proteins Ku70, Ku86 and DNA-PKcs were used for antibody abrogation studies. We have found that the mean repair efficiency of normal MNCs was 18.6±13.1% (0~46.6%),. The ligation efficiencies in myeloid leukemic cell lines ranged 10.6%~31.0% (mean 22.4%, P&gt;0.05). The mean ligation efficiency was significantly higher in CML cells as compared with normal MNCs (24.8±14.9% v.s 18.6%, p=0.024). The DNA repair capacity of de novo acute myeloid leukemia cells(7.2%~76.9%)was markedly increased as compared with normal MNCs (mean 41.1±15.4% v.s 18.6%, p&lt;0.0001 ). The mean frequency of misrepair from AML and CML cells were considerable higher (AML, 8.17% and CML, 2.10%) than that of normal BM cells (0.91%). Most misrepair were small deletions near EcoR I site. Large deletions (&gt;100bp) were found from assays with AML cells. Three abnormalities of sequence deletion, inversion and insertion were found in a rare misrepair white colony with AML cells. The DNA end-ligation efficiencies of AML cells could be dramatically inhibited by antibodies against proteins Ku70, Ku86 and DNA-PK. We concluded that the Cell-free NHEJ assay system can be used to examine the clinical leukemic samples. NHEJ efficiencies were slightly enhanced in most myeloid leukemia cell lines compared with normal MNCs. The end-ligation efficiency was significantly higher in primary myeloid leukemia cells. The overactive but more error-prone NHEJ function relied on the activity of Ku and DNA-PK proteins for DSB repair may contribute to genomic instability in AML cells.