A Systematic Study On The Regulation Of Chronic Myeloid Leukemia Cell Proliferation Related Molecules

Background and purpose: Chronic myeloid leukemia(CML) is a myeloproliferative neoplasm. Imatinib, a type of tyrosine kinase inhibitors, has been regarded as a standard treatment for CML since it was approved by the Food And Drug Administration of USA after 2001. CML cells may not response to imatinib as it was widely used. In order to improve the prognosis of CML patients, a new strategy was exploited to reverse imatinib resistance.Currently, tumor cell signal pathway and molecular mechanisms has been a new research focus. p14ARF�'Mdm2�'p53 cell signal pathway, namely p53 signal pathway, has been extensively discussed in recently years, which plays an important role in regulating tumor cell proliferation. P14 ARF can play an effect through the p53 dependent pathway or p53 independent pathway. But,the role of p53 and p14 ARFin CML cell proliferation is still poorly understood now.In order to investigate the role of p14ARF�'Mdm2�'p53 in CML cell proliferation progress, we acquired the model of p53 cell signal pathway from Models Database, and at the same time, we systematically analyzed the role of some related molecular of CML cell proliferation with COPASI software. We verified the role of some related molecular in CML cell proliferation progress from theory aspects. We further discussed the impacts of p14 ARF in CML cell proliferation and apoptosis with the p14 ARFgene transduction of K562 and CBL-BC cell lines. Cell tests can afford some ideas for further studying CML cell signal pathway and molecular mechanisms, which benefits for the development and exploitation of new treatment. Ultimately, a new treatment can enhance clinical efficacy of CML and improves the prognosis and survivalof CML patients.Methods: p14 ARF, Mdm2 and p53 were searched as keywords from Bil Models Database, and the article, Explaining oscillations and variability in the p53-Mdm2 system(Proctor CJ, Gray DA. BMC Syst Biol, 2008; 2: 75),was found. The model of p53 cell signal pathway was obtained from this literature, and we downloaded related contents of the model of p53 cell signal pathway using SBML L2V3(.xml). The horizontal axis of the model was defined as time, and the concentration was regarded as vertical axis, meanwhile,the total time, time interval and total interval was set. The oscillation curve of concentrations of different signal molecular was analyzed with COPASI-4.16.104 software.p14ARF suppressor gene was transduced into K562 cell line and primary leukemia cells of 4 cases of CML patients with lentiviral vector, and these cells with pure vector were considered as control. The colony formation of leukemia cells in the experimental group and the control group were compared by the semi solid culture method, and the difference was statistically significant(P <0.05). Counting cell colony count by fluorescence microscope. Cell apoptosis was examined by flow cytometry tabbed with Annexin V-FITC/PI.Results: The concentration of ARF and Mdm2 was low in CML patients,and the slight change of ARF can cause a remarkable change of p53. The overall trend of cell signal pathway was p14ARF↑�'Mdm2↓�'p53↑. The transcription of Mdm2-m RNA was slower than p53 expression.p14ARF�'Mdm2�'p53 cell signal pathway can stabilize the level of p53 by inhibiting p53 degradation not promoting p53 synthesis. The concentration change of p53 was not consistent with the concentration change of ARF.Cell test: CML cells infected with HIV-hr GFP, when multiplicity of infection(MOI) was 50, the rate of transduction of K562 cell line was nearly100%, and the rate of transduction of CML-BC1, CML-BC2, CML-BC3 andCML-BC4 was(98.00±1.73)%,(77.00±1.63)%,(89.00±2.65)% and(87.00±1.41)%, respectively. Cell test revealed that the inhibition rate of K562 cell line was 43% when K562 cell line was transduced with p14 ARF, and the K562 cell line was significantly inhibited. The rate of CML cell apoptosis transduced with p14 ARF gene was analyzed with Annexin V-FITC/PI. The results showed that the rate of p14ARF-positive K562 cell apoptosis was only20%, however, the rate of 4 primary CML leukemia cell apoptosis was obviously higher than those in control, and the P value had significance(P<0.05).Conclusion: Both systems biology and cell tests displayed that p14 ARF was a suppressor gene. In p14ARF�'Mdm2�'p53 cell signal pathway, p14 ARF could regulate the level of p53 by inhibiting Mdm2 expression, and the slight change of p14 ARF level can cause the significant change of p53. The total trend of p53 cell signal pathway was p14ARF↑�'Mdm2↓�'p53↑. But, there was a degradation-trans activate circulation path of p53 and Mdm2.p14ARF�'Mdm2�'p53 cell signal pathway can stabilize the level of p53 by inhibiting p53 degradation not promoting p53 synthesis. Cell proliferation was inhibited and cell apoptosis was induced when p14 ARF gene was transduced into CML cells. p14 ARFmay be a new target for the treatment of CML.