| Leukemia is the disorder of hematopoietic stem cells. Leukemia cells lose the capacity of differentiation to mature cells and stop at different stage of cell growth, and also, leukemia cells are highly proliferated and accumulated in bone marrow and hematopoietic tissues in order to inhibite normal hematopoietic function. Based on the level of maturation of leukemia cells and courses of the disease, leukemia is divided into two big groups:acute leukemia and chronic leukemia. And acute leukemia also have two categories, acute myeloid leukemia (AML, also known as acute myelogenous leukemia) and acute lymphoma leukemia. The incidence of AML increases with the increase with age. Although AML is a relatively rare disease, it accounts for approximately1.2%of cancer deaths in the United States. In China, the incidence rate of leukemia is2.76/100000, accounting about2.6%of cancer patients, and most of them are AML, taking up about1.5%of the2.6%.Adhesion molecules have originally been thought to be essential for the formation of multicellular organisms and to tether cells to the extracellular matrix or to neighbouring cells. The CD44surface antigen is one of the most improtant cell surface receptors, on leukemic blasts and also some other type of malignant tumors from most of cancer patients, is involved in cell growth, differentiation and apoptosis, and highly variant isoform CD44(CD44v) is essential of invasion and matastasis in maligant tumors. Since the end of last century, CD44is becoming a brand new targeting of novel therapy of tumors. In breast cancer progression and recurrency, expression level of standard form CD44(CD44s) is upregulated, and other maligant tumor, such as, melanoma and prostate cancer, also highly express CD44. In AML, different isoforms of CD44expressed on different subtype AML. So far, the most popular tool to study CD44is monoclonal antibodies (mAbs). Studies showed that, CD44mAbs could induce differentiation or apoptosis of AML cells, and low concentration of CD44mAbs could also inhibit cell growth and arrest cell cycle, so CD44mAbs could be used as a way to cure AML. There are also other reports showed that small molecular weight hyaluronic acid (HA) or soluble protein also could interrupt the cell signaling cascade controlled by CD44and inhibit multidrug resistance (MDR). Furthermore, CD44is also highly expressed on the surface of tumor stem cells, making it more and more important for modern cancer research. It is reported that AML stem cells could be eradicated by anti-CD44monoclonal antibody H90. This evidence shows that CD44is very important in the occurance, progression, treatment of AML and other tumors.Besides bone marrow transplantation, cell differentiation is the most common and effective method used in AML therapy in recent years. But, only AML M3can be treated by using retinoic acid via degradation of PML-rara as AML M3express PML-rara, other AML patients, lacking of PML-rara, can not be treated with retinoic acid and have to be treated with bone marrow transplant chemotherapy, and radiotherapy. So it is very important to find other methods for the treatment of AML, and eradicating AML cells or leukemia stem cells is the research direction. Many groups found that CD44is highly expressed on AML cell surface, and different subtypes of AML express different types of CD44, so targeting CD44might be an ideal therapy to AML. CD44mAb A3D8has many function in eradicating AML cells or reversing AML cells to normal cells, including undergoing apoptosis or differentiation, and meanwhile, inhibiting cell proliferation. But at present, except cell differentiation, other anti-tumor activities of A3D8are unclear and the application of A3D8is still at its initial stages. So it is critical to study the anti-tumor mechanisms and clinial application of A3D8in AML therapy.Furthermore, cytarabine (Ara-c), a preferred chemotherapeutic agent for AML, is already widely used in clinic, but it causes severe excruciation for the patientis. So enhancing targeting to AML cells and reducing drug concentration and administration time of Ara-c via A3D8targeting CD44might be an ideal way to solve the problem.In this thesis, the mechanisms of A3D8in influencing AML cell cycle distriution and apoptosis were studied, and the mechanisms of synergistic effect of A3D8and Ara-c were explored, which would provide a new thread for AML treatment.1Study on cell cycle arrest for anti-CD44monoclonal antibody A3D8treatmentIn order to understand the cell growth inhibition effect and cytotoxicity of A3D8or HA, the treated AML cells were stained with trypan blue and then inspected under microscope. The results showed that A3D8could induce cell growth inhibition in different AML subtypes, but under the same condition, HA did not inhibit AML cell growth.After staining AML cells by propidium iodide (PI), cell flow cytometry was used to detect the cell cycle distribution, and differences of cell cycle distribution between treated and untreated AML cells were compared. It was found that after the treatment of A3D8, AML cell cycle was arrested in S phase, but AML cell cycle was not effected by HA.By using Western blot, the differences of the relating proteins for cell cycle before and after treatment can inspected. In our experiment, A3D8treatment caused upregulation of p27and Bid expression at48h compared with isotype treatment in NB4cells. By using siRNA, specific genes such as Bid and P27can be silenced to understand the function of these genes. After p27and Bid were silenced, the cell cycle distribution is dramatically changed with A3D8treatment compared with vector-transfected NB4cells and non-transfected NB4cells. After the genes of Bid and P27were knocked down, the expressions of the genes were no longer upregulated with A3D8treatment compared with vector-transfected NB4cells. We hypothesize that the cell cycle arrest in S phase is regulated by Bid stimulation and followed by P27stimulation with A3D8treatment.Isolation of cytoplasm and cell nuclear is one of the way to test protein translocation. The experiment showed that the portion of the Bid protein was localized in the nucleus, which means the importance of Bid translocation from cytosol to nucleus.2Study on the mechanisms of A3D8in inducing cell apoptosis (A3D8treatment alone or in combination with Ara-c)Using Annexin V staining and DNA fragment assay, the apoptotic rate of AML cells after A3D8treatment was tested. The result showed that A3D8could induce some subtype AML cells undergoing apoptosis, such as AML M2, M3, about30%and50%apoptosis in NB4and SKNO-1cells respectively.,The differences of cell apoptosis relating proteins before and after treatment were analyzed by using Western blot. The results indicated that the apoptosis induced by A3D8treatment in NB4cells was mediated principally through caspase-8activation, which means death receptor signaling pathway plays a critical role in A3D8induced apoptosis. Furthermore, it was found that CD44s expression level was very important for apoptotic effect, the higher the CD44s expression, the more the apoptotic cells. The caspase-8inhibitor Z-IETD-fmk, but not the caspase-9inhibitor Z-LEHD-fmk, significantly decreased the level of apoptotic cells treated with A3D8at2.5μg/ml for3days.In order to study interaction of lipid rafts and Fas, the translocation of proteins inside and outside AML cells was detected by using confocal microscopy. The results showed that after A3D8treatment, the formation of lipid rafts occurred on the surface of AML cells, and meanwhile, Fas was translocated on the lipid rafts to activate caspase-8. In order to study the function of lipid rafts, the lipid rafts were treated with cholesterol damaging agent methylated-β-cyclodextrin (MCD) for30min under37℃and observed with confocal microscopy or analyzed using Western blot. The results showed that the A3D8-induced lipid raft formation was abrogated with MCD treatment, the A3D8-induced apoptosis was significantly inhibited by MCD, and the cleavage of caspase-8in NB4cells treated with A3D8was blocked by MCD transient treatment. These data suggest that lipid raft formation by A3D8treatment leads to apoptosis induction and caspase-8activation.Furthermore, our experiments also found that Ara-c could increase the expression level of CD44s, via activation of Mnk and eIF4E. HL-60cells, which does not have CD44s expression, could express CD44s after Ara-c treatment. Combination of Ara-c and A3D8could strongly induce HL-60apoptosis compared with single treatment alone. By using confocal microscopy, it was found that the combination of Ara-c and A3D8could cluster a "cap" like structure with stronger fluorescence at one pole of the cells, suggesting that the combination could apply to those AML subtypes which do not have CD44expression. Apoptosis induction in HL-60cells by A3D8plus Ara-C is blocked by silencing Fas-Associated protein with Death Domain (FADD). Silencing FADD decreased A3D8/Ara-C-induced apoptosis from37%to19%. Cleavage of caspase-8induced by A3D8/Ara-C was inhibited following FADD knock-down. The co-localization of CD44s with ezrin was determined by confocal microscopy in HL-60cells treated with A3D8/Ara-C. After treatment with A3D8/Ara-C both CD44s and ezrin localized in structures similar to lipid rafts as determined with FITC-CTx B. To prove the bridging effect of ezrin in the A3D8/Ara-C-induced caspase activation, Ezrin was silenced using siRNA. Ezrin siRNA blocked A3D8/Ara-C-induced apoptosis in HL-60cells. These data suggest that ezrin plays an important role in A3D8/Ara-C-induced apoptosis in leukemia cells with clustering lipid rafts into the "cap" like structure.Our conclusion and achievements are as follow:Cell apoptosis and cell growth inhibition are two important methods to cure maligant tumor in clinic. Although AML cells respond to chemotherapeutic agents which induce apoptosis (mainly through the mitochondrial-mediated pathway), resistance and relapse due to failure of targeting leukemic stem cells are barriers to the cure of this disease. CD44is not only used as a marker to isolate leukemic stem cells but also is found to be one reason for resistance to chemotherapy. Targeting CD44should be helpful for the eradication of leukemic stem cells and cure of leukemia. Utilizing the positive signaling of CD44s in apoptosis induction to target CD44should benefit AML therapy.The findings and conclusions of this thesis are as follows:(1) Cell cycle redistribution and cell growth inhibition induced by A3D8are regulated by Bid, and the translocation of Bid from cytosolic to nucleus for P27activation is required. (2) A3D8induced cell apoptosis is caused by the redistrubution of lipid rafts into "cap" like structure to activate FasL independent death receptor signaling pathway.(3) A3D8induced apoptosis is related to CD44s protein expression level. The more CD44s expression, the more apoptotic cells in AML.(4) Upregulation of CD44s by Ara-c is via the activation of Mnk and eIF4E.(5) The targeting of cell surface receptor CD44, using combination of A3D8and Ara-c, synergistically promote apoptosis through death recepter pathway in AML due to lipid rafts redistribution, and it is related to ezrin. |
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