Study Of Centrosomal Protein 55 In T-Cell Lymphoma Oncogenesis And MCM7 In The Resistance Of Chronic Lymphocytic Leukemia Tofludarabine

T-cell lymphomas(TCLs)and chronic lymphocytic leukemia(CLL)are malignant hematopathy originated from T-lymphocytes and B-lymphocytes,respectively.The most prevalent type of adult non-Hodgkin lymphoma in the western countries,chronic lymphocytic leukemia(CLL),has heterogeneous clinical course and is characterized by accumulating mature B-lymphocytes in peripheral blood,bone marrow,and secondary lymphoid tissues.Although several new drugs emerge as promising potentials to cure CLL,drug resistance is still difficult to be overcome to win the long-term survive.Fludarabine(Flu)-based regimens are clinically used as the frontline treatment for CLL.However,treatment of relapse and refractory patients generally results in an inferior salvage response.Additionally,the development of Flu-resistant disease is indicative of a very poor prognosis,with a median life expectancy of less than 1 year.Therefore,it is necessary to explore new therapeutic strategies to revers Flu resistance.T-cell lymphomas(TCLs)have been heterogeneous diseases of lymphoid malignancies.TCLs appeared to be more aggressive with a worse prognosis compared with that of B-cell lymphomas.However,the etiology and uniformed therapeutic regimen remained to be explored.Therefore,exploring and unveiling specific molecules contributing to signaling pathways and tumorigenesis has become an attractive topic for its significance in the treatment of TCLs.The cell cycle or cell-division cycle is the series of events that take place in a cell leading to its division and duplication of its DNA(DNA replication)to produce two daughter cells.The cell cycle consists of four distinct phases:G1 phase,S phase(synthesis),G2 phase(collectively known as interphase)and M phase(mitosis).M phase is itself composed of two tightly coupled processes:karyokinesis,in which the cell's chromosomes are divided,and cytokinesis,in which the cell's cytoplasm divides forming two daughter cells.Activation of each phase is dependent on the proper progression and completion of the previous one.Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called GO phase.Centrosomal protein 55(CEP55)has been identified and characterized a decade ago.CEP55 was a centrosome-and midbody-associated protein and played a crucial role in cytokinesis,during which two daughter cells were separated physically then finishing the cell division process.CEP55 overexpression has been identified in mRNA microarray expression profiles of many human cancers,resulting in the accumulation of unstable multinucleated cells,which tended to tumor genesis.Previous studies have shown that overexpression of CEP55 was associated with increased cell migration and invasion,whereas apoptosis was significantly increased with the inhibition of CEP55.Minichromosome maintenance(MCM)proteins serve as license components for the S-phase to initiate the cell cycle;they play a crucial role in the processes of DNA replication and cell cycle progression.MCM2-7 assemble into a pre-replication complex(pre-RC)with toroidal structure and ATPase activity;during G1 phase,the pre-RC complex bind to chromatin and initiate DNA replication.The subcomplex MCM4-6-7 exerts additional weak helicase activity to unwind supercoiled DNA at replication forks.When DNA damage is present,to participate in S-phase checkpoint signaling MCM7 functionally interacts with core components of ATM/ATR signaling pathway which plays an important role in CLL genesis and progression.Furthermore,MCM proteins represent better prognostic markers than Ki-67 in myelodysplastic syndromes and mantle cell lymphoma,and MCM7 has been associated with relapse and overall survival in acute myeloid leukemia.Part I.Expression and clinical significance of centrosomal protein 55 in T-cell lymphomaObjectiveThis study was designed to demonstrate the role of CEP55 in the oncogenesis of T cell lymphomas,and to evaluate the correlations between CEP55 and clinical indicators.Materials and Methods1.Patients specimen selection and collection,2.Cell culture,3.Immunohistochemistry,4.Total protein extraction and Western blotting,5.CEP55 suppression using siRNA in TCLs cell lines,respectively,6.Cell Counting Kit-8 assessment of cell viability,7.Detection of cell apoptosis rate using Annexin V-PE/7AAD assay,8.Statistical analysis.Results1.Expression of CEP55 in 46 cases of TCLs was determined by immunohistochemistry,compared with that of in the control of 15 cases of RHL.CEP55 staining was readily observed in most of TCLs samples,but it was hard to be detected in RHL tissues due to weak staining[Figure 1].Samples displaying 30%or more stained cells were categorized as positive cases.The positive cases of CEP55 staining in PTCL-NOS,AITL,TLBL,EATL and NK/TCL were 8(72.73%),5(55.56%),12(85.71%),2(66.67%),and 7(77.78%),respectively.2.The correlation between CEP55 expression and clinical characteristics was shown.The results of Pearson's ?2 test or Fisher's exact test when necessary indicated that CEP55 expression was significantly associated with Ki-67(P = 0.0171).No other clinical parameter was detected to be associated with CEP55 overexpression.3.CEP55 expression was downregulated.Significant decrease of cells viability and increase in apoptosis rate was observed in siCEP55-interfered cells,compared with that of in cells treated with siNC.ConclusionThis study indicated that CEP55 overexpressed in TCLs and cell lines and its expression is significantly associated with Ki-67.Suppression of CEP55 impaired biological behavior in TCL cell lines,which provided a novel target to demonstrate the mechanism of TCL tumor genesis.Part ?.Suppression of minichromosome maintenance 7 sensitizes chronic lymphocytic leukemia cells to fludarabineObjectiveThis study was designed to illustrate the role of MCM7 in the resistance to Fludarabine in CLL,and to evaluate the influence in CLL cells after suppression of MCM7.Materials and Methods1.Patients specimen selection and collection,2.Isolation of peripheral blood mononuclear cells(PBMCs),3.Detection of CD 19+ B cells and ?-H2A.X,4.Cell culture,5.CLL primary cells stimulation with anti-BCR and anti-CD40,6.RNA extraction and Real-time quantitative polymerase chain reaction,7.Total protein extraction and Western blotting,8.MCM7 suppression using siRNA and shRNA in CLL primary cells and cell lines,respectively,9.Cell Counting Kit-8 assessment of cell viability,10.EDU assay for cell proliferation,11 Detection of cell apoptosis rate using Annexin V-PE/7AAD assay,12.Cell cycle analysis by PI assay,13.Statistical analysis.Results1.MCM7 is upregulated in primary CLL cells and in CLL cell linesTo identify changes in MCM7 expression,we examined the MCM7 mRNA levels in primary CLL cells from patients(n=28)and in normal B lymphocytes from healthy donors(n=8)via qRT-PCR.MCM7 exhibited an increase in expression in samples from CLL patients compared with samples from normal control subjects.The higher levels of MCM7 protein expression in CLL patients were further confirmed by western blotting.Similarly,elevated MCM7 mRNA levels were observed in the two CLL-derived cell lines MEC-1 and EHEB.We then confirmed this upregulation using western blotting.2.MCM7 alleviates Flu-induced DNA damage and limits the response of primary CLL cells to FluTo determine whether downregulating MCM7 could sensitize primary CLL cells isolated from 11 CLL patients to Flu,we used siRNA to knock down MCM7,which was confirmed by western blotting.Analysis of apoptosis activation via flow cytometry using annexin V-PE and 7-AAD staining showed that there was a significant increasing trend in apoptosis rate after MCM7 silencing in CD40-stimulated primary CLL cells.After CD40-activation and incubation with Flu for 24 hours,Flu-induced apoptosis was significantly increase in primary CLL cells with MCM7 supression.Flu triggered gamma-H2A.X phosphorylation accumulation as analyzed by flow cytometry,which was consistent with increases in DNA double-strand breaks(DSBs)and replication stress.3.Reduction of MCM7 impairs the biological behavior of MEC-1 and EHEB cellsIn both the MEC-1 and EHEB cell lines,greater than 80%of cells were successfully transduced with lentivirus.MCM7 expression was stably downregulated by approximately 85%of either the endogenous mRNA or protein levels,which was confirmed by western blotting.A decrease in cell viability and S-phase arrest were observed in cells transduced with shMCM7 compared with cells transduced with shNC.Correspondingly,the mean apoptosis rate increased MEC-1 and EHEB cells.4.MCM7 inhibition in CLL cell lines increases the response to Flu.To further determine whether the effectiveness of Flu was altered by MCM7 inhibition,we tested the survival rate of cells treated with different concentrations of Flu for 24 hours and used cells incubated with the corresponding concentration of DMSO as controls.With increasing doses of Flu,cells showed decreasing survival rates;at relatively higher concentrations,shMCM7-transduced cells exhibited a significantly better response to Flu.5.Reinforced Flu-induced replication stress after knockdown of MCM7We measured the cell replication rates using an EdU assay.After cells were incubated with 2 ?M Flu,EdU was added to the medium for another 24 hours.MCM7 reduction led to significant increase of EdU-positive cells,which indicates more incorporation of EdU during replication.As more DNA was synthesized,we then examined the phosphorylated gamma-H2A.X levels.Gamma-H2A.X phosphorylation accumulated as the Flu concentration increased.ConclusionThis study indicated that MCM7 overexpressed in CLL primary cells and cell lines.Suppression of MCM7 impaired biological behavior in CLL cell lines,and sensitized CLL primary cells and cell lines to fludarabine,which provided a novel therapeutic strategy to reverse fludarabine resistance.