| Background:Acute myeloid leukemia (AML), derived from hematopoietic stem and progenitor cells, characterized by uncontrolled proliferation, differentiation disorders, apoptosis prevention, and inhibition of normal hematopoiesis, is a malignant tumor disease of hematological system, and threatens human health badly. With the breakthroughs in cellular and molecular biology study and the great advance of clinical treatments, the prognosis of AML patients has been improved significantly. But according to the statistics, the five-year survival rate for adult patients under the age of 60 is just 30～40%, and that percentage of patients over 60 even can not reach 10%. Up to now, the pathogenesis of AML is still dim, including factors from living environment and microenvironment in vivo, exact mechanisms of multiple drug-resistance (MDR) in the process of chemotherapy, and the reason for early and late phase relapse of those complete remission (CR) patients, which all need further exploration. Therefore, researching on occurrence and development, MDR, and relapsed/refractory phenomena of AML, detecting the efficient antineoplastic targets, and developing the new medicines against AML become the most important task in the field of hematology.Poly(ADP-ribose) polymerase-1 (PARP-1) is a highly conserved protease, responsible for poly ADP-ribosylation of itself or other proteins in eukaryocytes, and plays an important role in the repair of DNA damage. Activated rapidly when DNA damage as a molecular sensor, PARP-1 recognizes and binds to DNA breakage, catalyzes the cleavage of nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADP-ribose, activates the poly ADP-ribosylation function, and mainly conducts the DNA repair process. Meanwhile, PARP-1 takes part in the regulation of gene transcription, cell cycle progression, and cell proliferation or death, by binding transcriptional factors, changing DNA methylation state, adjusting the chromatin structure, and so on. The current studies show that PARP-1 is over-expressed in breast cancer, hepatic carcinoma, and nasopharyngeal carcinoma, etc. And the overexpression of PARP-1 is found significantly associated with poor prognosis. At present, it is still not clear about the expression state of PARP-1 in AML and the specific mechanisms of it in AML progression. This study aims to detect mRNA level of PARP-1 in bone marrow specimens from AML patients, investigate the effects of PARP-1 in AML progression in vivo by constructing murine model, verify its function in proliferation and apoptosis in AML cell lines, and explore the exact molecular mechanisms involved via microarray analysis. This will provide new ideas for AML fundamental research, and available biological targets for clinical practice.Section IThe role of PARP-1 in development of acute myeloid leukemia Objective:The aim of this part of study is to detect the PARP-1 expression level in AML patients’ bone marrow specimens and analyze its biological effects on clinical outcome firstly; then to investigate the impact of PARP-1 on AML progression via animal experiment. Definitize the role of PARP-1 in AML, which will lay a solid foundation for further clarification on the molecular mechanisms.Materials and methods:1. Patient samples collection:Bone marrow (BM) specimens were taken from AMLpatients and healthy donors, with informed consent and approval from the medical ethics committee of the Qilu Hospital of Shandong University. Mononuclear cells from BM were obtained by density-gradient centrifugation with Ficoll-Hypaque, then stored at -80℃ for subsequent tests.2. Real-time RT-PCR:Total RNA was extracted from BM mononuclear cells with TRIzol. RNA was reversely transcribed into cDNA performed with PrimeScript RT reagent kit. Then SYBR Green assay was used to detect mRNA level of PARP-1 in AML patients and healthy donors. The relationship of PARP-1 expression and clinical treatment was analyzed.3. AML murine model construction:C1498, a cell line of murine AML, was purchased from American Type Culture Collection (ATCC). The cells were injected into mice via caudal vein after amplifying cultured in vitro. Peripheral blood smear, bone marrow smear, and tissue section strain were used to verify the model.4. Effects of PARP-1 on AML mice disease progression.4.1 Virus package and cell infection:Lentivirus carrying the gene of a green fluorescent protein (GFP) was constructed and used to infect C1498 cells. Infection efficiency was tested with a fluorescence microscope after 72 hours. Puromycin was added to cell culture medium to select the cells which can express GFP stably (C1498-GFP). Then these cells were cultured on a large scale, and verified the proportion of GFP-positive cells by flow cytometry (FCM).4.2 Murine model building and experimental grouping:Male C57BL/6 mice aged 6 to 8 weeks were fed with a normal diet. C1498-GFP cells were injected into mice via tail vein, building the AML murine model. The AML mice were divided into two groups:control group, received intraperitoneal injection of normal saline (NS); experimental group, received intraperitoneal injection of PJ34, which is a potent PARP-1 inhibitor.4.3 Mice condition observation:Observe and record the general condition of mice, such as energy, hair smoothness and so on. The body weight was monitored every five days.4.4 Infiltration of AML cells in vivo:(1) Mice were sacrificed by anesthesia, and autopsied to observe organomegaly.(2) FCM was used to detect infiltration of AML cells in peripheral blood and liver tissue of AML mice.(3) H&E staining of tissue section was for verify infiltration condition further.4.5 Survival curve analysis:Record the survival time of experimental AML mice, and draw a survival curve. The inhibition of PARP-1 on AML progression was analyzed.Results:1. Real-time RT-PCR showed that PARP-1 was significantly up-regulated in AML patients compared with healthy controls, and there was no remarkable difference among different FAB classification. The patients with high level PARP-1 were associated with poor therapeutic outcome.2. AML murine model was successfully constructed by C1498 cells injection. The AML mice suffer significant emaciation, depression, less activity and weight loss. Leukocytes increased or decreased and platelets decreased were tested, and tumor cells were found in peripheral blood and bone marrow. Abdominal organ enlargement was verified in AML mice, with local masses in some organs. The H & E staining of histological sections showed infiltration of a mass of malignant cells. The AML mice died within one month because of disease progression.3. PARP-1 inhibition significantly relieved leukemia progression in AML mice.3.1 Compared with control group, emaciation and weight loss were less severe in experimental group mice.3.2 The enlargement of liver and spleen was alleviative in experimental group mice.3.3 FCM results showed percentage of C1498-GFP cells in peripheral blood and liver were both decreased in experimental group mice.3.4 Infiltration of C1498-GFP cells in liver was also obviously reduced in experimental group mice discovered by H&E staining of tissue section assay.3.5 The median survival of AML mice treated with PARP-1 inhibitor PJ34 was prolonged as compared with control mice (37.5 days vs.23.5 days).Conclusion:1. PARP-1 was over-expressed in AML patients, and high expression level of PARP-1 was related to poor therapeutic outcome. PARP-1 takes part in AML progression, with prognosis significance.2. Animal experiment confirmed that PARP-1 inhibition alleviated the disease progression, including decreasing AML cell infiltration, hepatosplenomegaly, and prolonging survival, providing further evidence of the potentiality of PARP-1 inhibition in tumor therapy.Section ⅡThe study of PARP-1 on AML cells biological behaviour and related mechanismsObjective:To study the effects of PARP-1 inhibition on AML cells biological behavior and molecular pathways; To screen out and verify the downstream molecules that change significantly when PARP-1 inhibition, and conduct gene ontology enrichment analysis and pathway analysis; To define the expression of PARP-1 downstream molecule in AML, relationship with prognosis, and its function on AML cells proliferation and molecular pathways; To further verify relationship between PARP-1 and downstream molecule. In-depth discussion concrete mechanisms of PARP-1 in AML development can provide a strong theory basis for leukemia targeted therapy.Materials and methods:1. Function of PARP-1 inhibition on the proliferation, cell cycle, apoptosis, and molecular pathways of AML cell lines Kasumi-1 and THP-1.1.1 Cell viability assay:The Kasumi-1 and THP-1 cells were cultured with 1640 medium added with serial concentration PJ34 solution. Determine the cell viability and IC50 with CCK8 assay after 48 hours incubation. Infect the two kinds of cells with lentivirus carrying PARP-1 interfering sequence or controls, detect cell viability at 24h,48h and 72h, and portray the growth curve.1.2 Flow cytometry analysis of cell cycle:Cells treated with PJ34 for 48 hours were collected, stained with propidium iodide (PI), and tested by flow cytometry for cell cycle distribution.1.3 Flow cytometry analysis of apoptosis:Cells treated with PJ34 for 48 hours were collected, stained with Annexin V/FITC and PI, then apoptotic cells were detected with flow cytometry.1.4 Western blot analysis:Cells were collected and lysed in RIPA cell lysis solution containing protease inhibitor and phosphatase inhibitor. Total protein was extracted and bicinchoninic acid assay protein reagent kit was used to quantify the protein concentration. Then total cell lysates were separated on SDS-PAGE for gel electrophoresis. Western blot assay analyzed cell cycle and apoptosis related protein cyclin B1, CDK1, P27, Bcl-2, Bcl-xL and AKT, ERK1/2 pathways.2. Downstream molecules and pathways screening:Differentially expressed genes that changed significantly by PARP-1 inhibition were found out by a genome-wide microarray assay. Gene ontology and KEGG analysis were applied to analyze gene function and molecular pathways that affected obviously by PARP-1.3. Study on the effects of MPL in AML.3.1 Real-time RT-PCR assay was used to validate the expression level of MPL in AML patients and control donors BM samples. The relationship of MPL expression and clinical treatment was analyzed.3.2 Kasumi-1 and THP-1 cells were up-regulated or down-regulated expression of MPL by lentivirus infection. Then verify the efficiency via western blot assay.3.3 The AML cells that had been over-expressed or interfered MPL expression were conventionally cultured, and the cell viability was examined by CCK8 assay.3.4 Western blot method was used to determine the change of molecular pathways, including AKT, ERK1/2, JNK, and P38 pathway.4. Verify the interaction effects between PARP-1 and MPL.4.1 The mRNA and protein level change of MPL were evaluated with real-time RT-PCR and western blot assay when PARP-1 was inhibited by gene intervention or function inhibition.4.2 The MPL in AML was up-regulated by lentivirus infection or activated by ligand thrombopoietin. Then CCK8 and FCM were preformed to assess the function of PARP-1 inhibition on proliferation and apoptosis of these AML cells.Results:1. Effects of PARP-1 on AML cells proliferation, cell cycle, apoptosis, and involved molecular pathways.1.1 The proliferation of AML cells was suppressed by PARP-1 inhibition. Serial dilution concentration PJ34 could all significantly inhibit Kasumi-1 and THP-1 proliferation in a dose-dependent manner. The IC50 values were 23.5±3.9 μM and 35.6±5.5 μM, respectively. And viral interference of PARP-1 expression got the same effect.1.2 The cell cycle was arrested at G2/M phase when PARP-1 was inhibited. FCM results showed that cells staying at G0/G1 and S phase were decreased, but cells at G2/M were increased obviously. Western blot assay confirmed the cell cycle protein cyclin B1 and CDK1 were down-regulated, while P27 was up-regulated, which supported the FCM results.1.3 Apoptotic cells raised when PARP-1 interference. The number of apoptotic AML cells increased when treated with PJ34. And western blot assay confirmed the level of anti-apoptotic protein Bcl-2 and Bcl-xL diminished.1.4 The AKT and ERK1/2 pathway activity were suppressed, because p-AKT/t-AKT and p-ERK/t-ERK level were lessened when treated with PJ34.2. The microarrays identified 18 genes with>2.0-fold up-regulation and 9 genes with≥0.5-fold down-regulation in 3 independent experiments. GO analysis revealed PARP-1’s roles in cell proliferation, apoptosis, cell cycle, differentiation, adhesion, migration, angiogenesis, immune response and transcriptional regulation. And pathway analysis based on the KEGG database further defined PARP-1 in various molecular pathways, such as TCR signal, MAPK pathway, Toll-like receptor signal, NF-κB signal and JAK/STAT pathway.3. The role of MPL in AML disease.3.1 The expression level of MPL was detected up-regulated in AML patients, and involved in bad therapeutic effect.3.2 Cells proliferation could be enhanced by MPL. Lentivirus infection efficiency of the Kasumi-1 and THP-1 cells to heighten or lessen the expression of MPL was confirmed by western blot. CCK8 assay revealed MPL accelerated AML cell growth, with MPL interference associated with diminished cell growth rate.3.3 MPL acted on AKT and ERK1/2 pathways. When MPL expression was suppressed, p-AKT/t-AKT and p-ERK/t-ERK level distinctly reduced, but p-JNK/t-JNK and p-P38/t-P38 level changed with no statistical significance.4. Study on the interaction effects between PARP-1 and MPL.4.1 Both PARP-1 function inhibition and gene interference could down-regulate the expression of MPL level.4.2 Activation of MPL signal partial overturned the effects of PARP-1 inhibition. The suppression of cell proliferation and promotion of cell apoptosis by PJ34 significantly weakened by thrombopoietin stimulation or MPL over-expression.Conclusion:1. Inhibition of PARP-1 could reduce AML cell proliferation, block the cell cycle progression, and induce cell apoptosis, which was associated with AKT and ERK1/2 pathways.2. MPL was remarkably high expressed in AML patients, involved in poor prognosis. And MPL could activate AKT and ERK1/2 pathways, promoting AML cells multiplication.3. PARP-1 could regulate MPL expression, and meanwhile MPL could reverse the effects of PARP-1 inhibition.4. PARP-1 and downstream molecular pathways played important parts in AML development and progression. It may be a new way to conduct targeted therapy against over-expression or over-activation of associated molecules. |
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