Study On Expression Of Ku70 In CML Cells

ObjectiveThe DNA double strand breaks (DSB) in mammalian cells, caused either by ionizing radiation, radiomimetic drugs, or occurring during gene rearrangements, are predominantly repaired by a process called non-homologous DNA end joining (NHEJ). The NHEJ pathway contains several key proteins to undertake its function, among which Ku70 played a pivotal role. As a common malignant disease of hematological system, chronic myeloid leukemia (CML) can be considered as a paradigm for neoplasias that evolve through a multi-step process. As reported, there were some correlations between the expression of CML special fusion gene BCR/ABL and the high efficiency of the DSB repairs. The present study investigated the expression of the gene Ku70 and the protein Ku70 in leukemia cells of CML patients at different clinical stage. To reveal the correlation among the expression of the gene Ku70, the protein Ku70 and BCR/ABL in cells of CML patients.Methods1. Bone marrow cells were collected from GM-CSF mobilized normal adults and preliminary diagnosed chronic myeloid leukemia (CML) patients, Separated their mononuclear cell by Ficoll, took 2×107 for the experiment. Detect the living cell rate of bone marrow prepare using trypan blue, make sure WBC above 95% by Wright staining.2. Extract RNA of each group by trizol one step, purify and reverse transcripted to cDNA. Detected the expression of gene Ku70 in CML cells and mononuclear cells of normal BM by RT-PCR and RQ-RT-PCR.3. Extract nuclear protein of each group, quantified by Bradford, Investigated the expression of protein Ku70 in nuclear protein extracted from CML cells and mononuclear cells of normal BM by Western Blot, using TBP(TATA Box Binding Protein) as the internal control.Get the result as the ratio of gray scale of Ku70 over TBP. 4. Took tales doses cDNA from each group, detected the expression of the fusion gene BCR/ABL by TaqMan probe Real Time PCR, using ABL as the internal control.5. Investigated the correlation in cells of preliminary diagnosed CML patients using software SPSS version 13.0: (1) between the expression of the gene Ku70 and BCR/ABL;(2) between the expression of the protein Ku70 and BCR/ABL; (3) between the expression of the gene Ku70 and the protein Ku70.Results1. Qualitative analysis by RT-PCR and quantitative analysis by RQ-RT-PCR of gene Ku70 showed: the expression of Ku70 in total CML cells (544.63±185.71Ku70 copies/10000β-Actin copies) was manifestly higher than in normal BM cells (31.08±8.41Ku70 copies/10000β-Actin copies,P=0.039). Meanwhile, its expression in CML cell of acute phase (1103.31±645.62Ku70copies/10000β-Actin copies) was obviously higher than that of chronic phase (97.69±40.73Ku70 copies/10000β-Actin copies, P<0.01). However, there was no significant difference between the expression of gene Ku70 in CML chronic phase and in normal BM cells. The sequencing of Part of the PCR product was completely matched with gene Ku70 in gene bank of NCBI.2. The gray scale of protein Ku70 over TBP of 24 normal BM cells were (0.10±0.09), while which of 27 CML cases were (0.40±0.21,P<0.001). Among these 27 CML cases, 12 were in acute phase, 15 were in chronic phase, the gray scales of Ku70 were significantly different from each other (mean 0.57±0.22 vs. 0.27±0.04, P<0.001).3. The analysis of dependability showed: there were positive correlations of BCR/ABL when compared with the expression of the gene Ku70 (r=0.573 P=0.002) while with it of the protein Ku70 (r=0.705 P<0.001) in cells of CML, meanwhile, the correlations between the expression of the gene Ku70 and the protein Ku70 (r=0. 808, P<0.001).Conclusions1. The gene and protein of Ku70 were expressed significantly higher in CML than in normal BM cells. Meanwhile, its expression level in CML (acute phase) was markedly higher than that in CML (chronic phase). Suggested that Ku70 was higher expressed in CML cells, means DNA in CML cells was mainly damaged by DSB, and this kind of DNA damage was repaired by NHEJ pathway predominantly. This conclusion made us to presume that pathogenesy of CML may be DNA damage and its unfaithful repair. A new strategy of the study on nosogenesis and its gene therapy of CML was in front of us.2. The analysis of dependability showed there were positive correlations among the expression of fusion gene BCR/ABL, the gene Ku70 and the protein Ku70 in cells of CML. Suggest that through the NHEJ pathway repairing DSB played a very important role in the genesis and progression of CML. Meanwhile, it suggested that the DNA damage and its unfaithful repair in CML cells have happened in both gene and protein level. So it provided more accurated localization of the study on the DNA repair in CML cells.3. But there is still no direct and exact evidence of the mechanism about how DSB induced the generation of CML and its fusion gene BCR/ABL, it's meaningful and necessary to study this subject furtherly.