Study Of Abnormal NHEJ DNA Repair Function In Myeloid Leukemia

Objective: An in vitro system for non-homologous end joining (NHEJ) detection was supposed to be developed for clinical samples. The NHEJ efficiencies to repair DNA double-strand break (DSB) and ligation fidelity of DNA broken ends in myeloid leukemic cells and normal bone marrow (BM) or peripheral blood(PB)cells, as a control, were then investigated. To study whether the overactive DNA repair in myeloid leukemia cells relies on the function of principal NHEJ associated proteins, the protein levels and gene expression of Ku and DNA-dependent protein kinase (DNA-PK) were explored. Methods: Nucleic protein extracted from leukemic cells and mononuclear cells of normal BM or PB, was incubated with linear plasmid pUC18 DNA under certain conditions. Plasmid DNA was separated by agarose gel electropherasis and imaged by staining with SYBR greenâ… . End-ligation efficiency was assessed by dividing the densitometry readings for the sum of all converted plasmid products by the sum of all products. Above-mentioned examination was undertaken in 7 myeloid leukemia cell lines, lymphoid leukemia cell line Jurkat, myeloma cell line U266, 5 glioma cell lines as well as in 16 cases of normal BM or PB mononuclear cells, 20 cases of chronic myelocytic leukemia (CML) cells and 19 cases of de novo acute myelocytic leukemia (AML) cells. K562 cell line, normal BM cells and CML cells were each irradiated 8Gy by Co⁶⁰ and incubated for 12¹6h before ligation reaction was done. Irradiated cells were collected immediately for self-control. Ligation efficiencies were detected in CML cells after CML cells were incubated with 10 μmmol/L As₂O₃ for 24h. Escherichia coli strain DH5αwas electrotransformed with pUC18 DNA end-joined by normal BM and leukemia cells, and was plated on LB agar, including X-gal and IPTG. Correct ligation of cut plasmid DNA resulted in blue colonies. 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 to Ku70, Ku86 and DNA-PKcs were used for antibody abrogation studies. Levels of mainly NHEJ associated proteins were detected by Western blot in myeloid leukemia cells and normal BM cells. Gene expression levels of Ku70, Ku86 and DNA-PKcs were detected by RT-PCR. Results: â‘ The repair efficiencies of 16 cases of normal BM or PB mononuclear cells were(0～46.6)%, with a mean level of (18.6±13.1)%. The range of ligation efficiencies showed in 7 myeloid leukemic cell lines were 10.6%³1.0%(mean 22.4%, P>0.05). Among them SHI-1 showed the poorest ligation efficiency lower than the mean level of normal BM or PB. End-joining efficiency in Jurkat was 32.9%, in U266 was 60.6%, in 5 glioma cell lines were 1.9%⁵3.9%(mean 43.2%). The mean ligation efficiency was increased significantly in 20 cases of CML cells(0%⁵4.1%) as compared with normal BM and PB cells[mean(24.8±14.9)% versus 18.6%, P=0.024]. The DNA repair capacity of 19 cases of de novo acute myeloid leukemia cells (7.2%⁷6.9%)was markedly increased as compared with normal BM and PB cells[mean(41.1±15.4)% versus 18.6%, P<0.0001].â‘¡The ligation efficiencies were all increased in normal BM cells and leukemic cells 12¹6h after irradiation: 10.9% versus 25.7% in normal BM,10.0% versus 14.1% in K562,18.5% versus 51.1% in CML cells. After incubated with As2O3 for 24h, the end-ligation efficiencies in CML cells were slightly increased from 23.1% to 27.1%. â‘¢The mean frequency of misrepair from AML and CML cells were increased (AML, 8.17% and CML, 2.10%) compared with normal BM cells(0.91%). Most misrepair were small deletions near EcoR I site to generate mutations with changes in β-galactositase coding sequence. Large deletions (>100bp) were found derived from assays with AML cells and broken ends were joined with microhomology sequences. Three abnormalities of sequence deletion, inversion and insertion were found in a rare misrepair white colony with AML cells. â‘£The DNA end-ligation efficiencies in AML cells were dramatically decreased after interactions with antibodies to Ku70, Ku86 and DNA-PK. ⑤The expression levels of Ku70 were almost equal in myeloid leukemic cells but Ku86 and DNA-PK levels varied obviously. Levels of Ku heterodimer and DNA-PKcs were concomitantly increased in Jurkat. Levels of Ku86 and DNA-PKcs were decreased in SHI-1. The levels of Ku andDNA-PKcs in normal BM cells were very weak. Ku protein levels were increased in some myeloid leukemic cells. â‘¥Gene expression of Ku and DNA-PKcs were found in normal BM cells. The mRNA levels of Ku70, Ku86 and DNA-PKcs varied in different leukemic cell line but were inconsistent with protein levels. Conclusion: â‘ Cell-free non-homologous end joining system was first developed in China to support the detection in clinical leukemic samples. â‘¡NHEJ efficiencies were slightly enhanced in most myeloid leukemia cell lines compared with normal BM or blood cells. The end-ligation efficiencies were dramatically increased in myeloid leukemia cells. Primary acute myeloid leukemia cells showed 2-fold increase and CML cells showed 1.5-fold increase in end-ligation efficiencies as compared with normal BM and PB cells. â‘¢Leukemic cells and normal BM cells would enhance their NHEJ repair ability when more DSB were induced by irradiation or drugs as As2O3. â‘£Myeloid leukemic cells were more error-prone in DNA double strand break repair as compared with normal BM cells. DSB was rejoined by mircrohomologous sequence at broken ends in AML cells, which contribute to large deletions (>100bp). These large deletions were not observed in normal BM cells. Three abnormalities of sequence deletion, inversion and insertion of align genes were found in a rare misrepair white colony with AML cells. An overactive but error prone NHEJ process may contribute to genomic instability observed in myeloid leukemias. ⑤The overactive DNA repair ability found in myeloid cells were through NHEJ pathway and relied on the activity of NHEJ associated Ku proteins and DNA-PK protein. The expression levels of Ku70, Ku86 and DNA-PKcs were enhanced in most leukemia cell lines compared with normal BM cells. Levels of three subunits of DNA-PK were concomitantly increased were one of the mechanisms for enhanced end-joining activity found in leukemia cell lines. The decreased levels of Ku86 and DNA-PKcs may contribute to the decreased NHEJ efficiency found in SHI-1.