Comparative Proteome Analysis Of The Influence Of BCR/ABL Fusion Protein On Cell Repair Proteins After DNA Damage

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by a cytogenetic aberration consisting of a reciprocal translocation between the chromosomes 22 and 9:t (9; 22) (q34;q11). The translocation generates a novel bcr/abl fusion gene, which encodes BCR/ABL fusion protein with deregulated tyrosine kinase activity. Multiple different signaling pathways are activated by BCR/ABL tyrosine kinase, ultimately lead to neoplastic transformation of cell. Amounting evidences have suggested that the deregulation of cell repair function after DNA damage plays an important role in the leukemogenesis, malignant progression and the resistance to genotoxic treatment in CML.To study the influence of BCR/ABL fusion protein on cell repair proteins after DNA damage, the cellular comparative proteomic approach was used for the analysis of the transformant BaF3-P210 cells stably expressing BCR/ABL fusion protein and the control cells after DNA damage induced by hydrogen peroxide. This dissertation was composed of three part described as follows: 1. The construction of BaF3-P210 cell line stably expressing BCR/ABL protein and the investigation of its biological activityThe retroviral vector with the bcr/abl gene was transfected into the packaging cell line. The BaF3 cells were infected with the collected viral supernatant. The transgenic BaF3-P210 cell line stably expressing BCR/ABL were screened and sub-cloned. The integration of the bcr/abl gene in the genome of the target cell was determined by PCR and DNA sequencing. Trypan blue staining assay, flow cytometry and MTT assay.The results showed that the bcr/abl gene was integrated into the BaF3 cell genome; RT-PCR and Western blot verified the stable expression of the bcr/abl gene and protein in the screened sub-clone cell line BaF3-P210.When compared with the control group, the cell proliferation rate was promoted (P <0.05) ,and the cell proportion in the G0/G1 phase was decreased with the increase of the cell proportion in both S and G2/M phase(P <0.05);the cell proliferation inhibition rate and the early apoptosis rate of the BaF3-P210 cells were respectively(54.08±1.90)%(P <0.05) and (12.35±2.04)%(P <0.05) after the treatment of STI571.2. The establishment of the DNA damage model for the BaF3-P210 cells and BaF3-MIGR1 cells and a dynamic observation on the DNA repairThe comet assay was employed to detect the DNA damage in BaF3-P210 cells induced by hydrogen peroxide at various concentrations to construct the DNA damage model. The BaF3-MIGR1 cell line transfected by the empty vector MIGR1 without the BCR/ABL fusion gene was used as a control. A dynamic observation on the DNA repair after cell DNA damage was performed by the comet assay.The results suggested that the cell DNA damage models for both BaF3-P210 and BaF3-MIGR1 cells were established with hydrogen peroxide treatment at a final concentration of 50μmol/L for 10 min at 4℃as confirmed by the comet assay results. When compared with the control BaF3-MIGR1 cell group, the time duration required for the DNA repair of the BaF3-P210 cell group was significantly decreased—with 60 min for BaF3-P210 cells and 120 min for BaF3-MIGR1 cells (P <0.05).3. The comparative proteome analysis of BaF3-P210 cells and BaF3-MIGR1 cells after DNA damage induced by hydrogen peroxideAnalysis of differently expressed proteins in BaF3-MIGR1 and BaF3-P210 cells after DNA damage induced by hydrogen peroxide with comparative proteome: First, suitable conditions for two-dimensional electrophoresis were ascertained, including suitable loading quantity of sample and volt-hours of isoelectrofocusing. Image analysis was performed using PDQuest (7.0) image analysis software. Matrix assisted laser desorption/isonization time of flying mass spectrometry (MALDI-TOF-MS) was adopted to identify differentia protein spots and then mascot software was used to search for proteins in SWISS-PROT database. The results showed that the loading quantity of sample and volt-hours of isoelectrofocusing were selected as 150μg and 70,000 volt-hours respectively via the optimizing conditions. Compared with control, 41 protein spots showed differentia expression in BaF3-P210 cells after DNA damage. Among them, 29 protein spots were up-regulated while 12 were down-regulated.13 differentia proteins were successfully identified by MALDI-TOF-MS.Conclusion :The transformed mouse BaF3-P210 cell line stably expressing BCR/ABL was successfully constructed. The expression of BCR/ABL not only promoted the proliferation, but also accelerated the cell cycle progression of the BaF3 cells; what's more, it endowed the BaF3-P210 transformed cell line with the sensitivity to STI571. The cell DNA damage model for the transformed mouse BaF3-P210 cell line stably expressing BCR/ABL is successfully established and BCR/ABL fusion protein strengthens the DNA repair capacity of the BaF3-P210 cell line after DNA damage. When compared with the control, BaF3-P210 cells after DNA damage showed comprehensive and complex alterations in protein expressions both at a quantitative and quanlitative level. These differentially expressed proteins were found out to be involved in cellular processes such as oxidative damage resistance, cell cycle regulation, apoptosis, proliferation, metabolism and signal transduction. These differentially expressing proteins lay a basis for the further elucidation of the signal transduction pathways regulated by the BCR/ABL fusion protein and the exploration of the influence of BCR/ABL fusion protein on cell repair proteins after DNA damage. In adidition, these proteins may serve as potential targets for cancer therapy.