Effect Of P15INK4b Infection Joint Bcr-abl Specific SiRNA And STI571 On Chronic Myelogenous Leukemia

Chronic myeloid leukemia(CML) is a malignant myeloproloferative clonal disorder of haematopoietic stem cells, which accounts for 15-20% of the newly diagnosed cases of adult leukemias and frequently occurred in old people. Most of CML was characterized by Philadelphia(Ph) chromosome, which was result from a translocation between chromosomes 9 and 22 t(9;22)(q34;q11) or its variants t(V;9;22), the new fusion oncogene is called as BCR-ABL(Breakpoint Cluster Region-Abelson Leukaemia). This oncogene encodes a chimeric 210 kD Bcr-Abl protein that incorporates an activated Abl tyrosine kinase domain, which is a well documented underlying reason for the malignant transformation in CML.The unique presence of BCR-ABL in all CML cells and its absence from normal cells provide a therapeutic opportunity for the treatment of CML. Novartis Pharmaceuticals developed a selective BCR-ABL inhibitor-STI571 for the treatment of CML and approved by FDA in 2001. STI571 is the first target therapeutic agent and was considered as milestone result from its excellent therapeutic effect as explicated increased CML survival incidence to 90% with in 10 years, as compared to 20% survival incidence before STI571 applied in the treatment of CML. However, STI571 is not perfect. With 8 years of follow-up,16% of IRIS patients discontinued treatment for insufficient efficacy and 6% for adverse events. Though more potential TKI was in developing, TKI resistance became more and more apparent, especially with the increased incidence of mutation sites of Bcr-abl, there are more patient needs new treatment strategies to control their condition deteriorated. The center of these strategies was focus on the pharmacological silencing of BCR-ABL combine with simultaneous inhibition of other crucial targets, especially on the Bcr-abl independent targets.P15INK4b is a member of the INK4 family of cyclin-dependent kinase inhibitors, which is also known as CDKN2 b and MTS2. It was located on chromosome 9 and often deleted in co-junction with P16INK4 a and P14 ARF. These changes have been detected in lymphomas as well as carcinomas and sarcomas. P16INK4 a and P15INK4 b could inhibit the activities of CDK4/6, then arrest the cell cycle in G1 stage. In the hematology, the depletion of P16INK4 a and P15INK4 b was always result from the Loss of P16INK4 a and P15INK4 b expression in hematopoietic neoplasms occurred through DNA methylation of the genes encoding these proteins. Moreover, P15INK4 b was the only member of INK4 family in MDS and AML, suggesting that it maybe play a special role in the myeloid lineage. In the clinical studies, about 50-60% of patients with myelodysplastic syndrome and 70-80% of AML take place hypermethylation on P15INK4 b, indicated P15INK4 b was involved into the leukemia processing.It has been demonstrated that P15INK4 b has the following biological activities: 1.P15INK4 b was a tumor suppressor. Retrovirus-induced leukemia of the myelomonocytic phenotype were found to have undergone hypermethylation of the 5’CpG island of the P15INK4 b. When the knockout was redeveloped, the proliferation of myeloid progenitors was inhibited significantly. 2. P15INK4 b promoted the differentiation of hematopoietic stem cells into erythroid and assisting in rapid erythroid replenishment following stress. In other words, P15INK4 b could inhibit their differentiation into granulocytes. 3. P15INK4 b promoted the maturation of dendritic cells, and then enhanced immune supervisory. All of these evidence indicated that the P15INK4 b may be a potential therapeutic target of CML.Based on the above theoretical foundation, the present study was aimed to investigate whether P15INK4 b expression could enhance the anti-CML efficacy of Bcr-abl inhibition in K562 cells. To achieve this object, we firstly build a K562 cell lines with stable expression of P15INK4 b. P15INK4 b was extracted from mononuclear cells cDNA, after its sequences verified, connected to PCDH-CMV-MCS-EF1-COPGFP vector. After that, then P15INK4 b lentiviral plasmid were packaged and infected with K562 cells to stably express P15INK4 b genes. Secondly, Construct Bcr-abl siRNA to inhibit the expression of P210bcr-abl, luciferase gene(gene number M15077, 394 414) were used as a non-specific control. In addition, Combine with the STI571 study, we investigated the anti-CML effect of STI571, P15INK4 b and Bcr-abl in permutations and combinations manner as manifested by K562 cell proliferation, cell cycle and apoptosis. Results and conclusions were as followed: 1.Establishment of P15INK4 b lentiviral vector and K562-P15INK4 b cell line.After package of P15INK4 b lentiviral vector, its viral titer was determined by dilution method(2 × 108U/ml). Infected K562 cells were with P15INK4 b showed slowly proliferation rate, more apoptosis rate and cell cycle rest in G1 stage. 2.Bcr-abl specific siRNA transfection significantly inhibited K562 cell cycle arrest in G0/G1 phase cells, and has a significant induction of apoptosis;In the present study, we found that the expression of P210bcr-abl was significantly inhibited after 48 h of Bcr-abl siRNA transfection. Proliferation study showed that transfection of Bcr-abl siRNA inhibited the K562 cells growth significantly. This study also found that loss of Bcr-abl expression in K562 cell induced more apoptosis and cell cycle rest in G1 stage. 3.P15INK4 b expression combined with inhibition of Bcr-abl or STI571 showed stronger inhibition on the proliferation of K562 cells Cell proliferation assay showed that P15INK4 b lentivirus infection, Bcr-abl specific siRNA transfection or STI571 alone could significantly inhibit the proliferation rate of K562 cells, and showed a significant time-dependent manner. P15INK4 b infection + Bcr-abl inhibition was significantly higher than the alone treatment; P15INK4 b infection + STI571 inhibition was significantly higher than their separate applications, Bcr-abl + STI571 inhibition was significantly higher than lonely application, these results suggested that P15INK4 b joint Bcr-abl inhibition can significantly enhance the inhibition rate of K562 cells. 4. P15INK4 b joint Bcr-abl si RNA or affect STI571 on K562 cell cycle Flow cytometry showed that, P15INK4 b lentivirus infection can significantly increase the percentage of cells in G1 phase, indicating that P15 expression can delay DNA replication. Bcr-abl siRNA transfection and STI571 showed the similar effect with P15INK4 b infection. P15INK4 b and Bcr-abl siRNA or STI571 combination can significantly increase the percentage of cells in G1 phase, suggesting P15INK4 b and Bcr-abl inhibition or STI571 combination can significantly inhibit the replication of DNA, decreased cell proliferation rate. Mechanism study showed that combination treatment could inhibit the expression of CDK4 and Cyclin D1 significantly when compared to that in separate treatment group. 5. Effect of P15INK4 b joint Bcr-abl siRNA or STI571 on apoptosis in K562 cells. Flow cytometry showed that P15INK4 b expression can significantly increase the rate of apoptosis, as explicated by increased Annexin-V positive cell ratio, reducing the ratio of normal cells. Bcr-abl suppression and STI571 showed the similar effect on the cell apoptosis. P15INK4 b infection, Bcr-abl inhibition or STI571 combination can significantly increase the rate of apoptosis, suggesting that inhibition of Bcr-abl P15INK4 b with or STI571 combination can significantly promote apoptosis of K562 cells. Mechanism study showed that combination treatment could increase the ratio between Bax and Bcl-2 when compared to that in separate treatment group.Conclusion: Tyrosine kinase inhibitors played an important role in the treatment of CML, especially the advent of STI571, which greatly improved the survival time and survival rate of CML patients. However, with the extensive use of STI571, its resistance issues grows highlighted, which requires us to find new therapeutic targets to improve the treatment of CML. P15INK4 b as a tumor suppressor protein,which could induce cell cycle rest in the G1 stage. This study showed that P15INK4 b expression combined with Bcr-abl siRNA or STI571 in K562 cells significantly inhibited DNA replication, increased apoptosis rate, slowing the rate of cell proliferation, suggesting that P15INK4 b expression combined with Bcr-abl may have improved cure rates potential, P15INK4 b maybe considerer as a new therapeutic target in the treatment of CML.