| Part I The expression of Lc3in bone marrow samples of acutemyeloid leukemia patients.Objective: The major glycosphingolipids expression profiles in the human bonemarrow will be analyzed. We will also compare the differences between bone marrowsamples of acute myeloid leukemia patients and bone marrow samples of healthycontrols, and will be looking for characteristic glycosphingolipids associated with acutemyeloid leukemia. The study aims to elucidate the function and important roles ofglycosphingolipids in acute myeloid leukemia.Methods:(1) Glycosphingolipids were extracted by sonication four times withorganic solvents from bone marrow samples both in acute myeloid leukemia patientsand healthy controls;(2) A modification of the method presented by Ciucanu andCostello was employed for per-N,O-methylation of glycosphingolipids;(3) Using massspectrometry to characterize the structure of major glycosphingolipids from bonemarrow samples, and the major kinds of glycosphingolipids from acute myeloidleukemia patients and healthy controls were obtained;(4) The relative quantitative ofobtained glycosphingolipids from bone marrow samples were performed with massspectrometry. We used differential isotope labeling to compare the amount of LacCer inbone marrow cells of healthy donors with that in the bone marrow cells of acutemyeloid leukemia patients. In bone marrow cells from acute myeloid leukemia patients,glycosphingolipids were permethylated with deuterated iodomethane and mixed withglycosphingolipids from the same number of iodomethane-permethylated bone marrowcells (as determined by hemocytometer cell count) from healthy donors. We found thelevels of LacCer in the bone marrow samples from acute myeloid leukemia patients tobe similar to those in the bone marrow samples from healthy donors, so we choseLacCer as the internal standard. We used a method of relative quantification to determine the ratios of Gb3, Lc3, and GM3to LacCer, and the differences werecompared between acute myeloid leukemia patients and healthy controls.Results:(1) Various glycosphingolipids in the bone marrow samples of acutemyeloid leukemia patients and healthy controls were qualitatively analyzed usedelectrospray ionization tandem mass spectrometry, containing major glycosphingolipids:LacCer, Gb3, Lc3, and GM3. Gb4and nLc4are isomers, Gb4mainly presents in thebone marrow samples of healthy controls, nLc4mainly in the bone marrow samples ofacute myeloid leukemia patients;(2) The relative quantitative of glycosphingolipids inthe bone marrow samples was analyzed. LacCer was taken as the internal standardamount in the MS1spectrum, the ratio of the abundance of Gb3, Lc3, and GM3toLacCer represent the relative amount of Gb3, Lc3and GM3, we detected Lc3and GM3content in bone marrow samples of acute myeloid leukemia patients are higher than inbone marrow samples of healthy controls, but no significant difference of Gb3contentbetween acute myeloid leukemia patients and healthy controls. In the bone marrowsamples of acute myeloid leukemia patients, the ratio of nLc4to Gb4and nLc4-mixturewas significantly higher than in healthy controls;(3) In accordance with the clinical data,we analyzed Glycosphingolipids in the bone marrow samples of differentFrench-American-Britain (FAB) subtype, found that the content of glycosphingolipidsin the bone marrow samples is associated with FAB classification, In the M1subtype(acute myeloblastic leukemia without maturation), Lc3was more highly expressed thanin the control group. However, in the M2a subtype (acute myeloblastic leukemia withgranulocytic maturation), Lc3expression did not differ significantly from the control, soLc3content in bone marrow is closely associated with cell differentiation;(4) Theexpression of Lc3synthase β3Gn-T5in bone marrow samples of acute myeloidleukemia patients was significant higher than that of healthy controls.Conclusion: The glycosphingolipids content in bone marrow samples of acutemyeloid leukemia patients is different from that of healthy controls, and differentiationassociated Lc3content in bone marrow samples of acute myeloid leukemia patients wassignificantly higher than that of healthy controls. Part II The high expression of Lc3in acute myeloid leukemia isassociated with cell differentiation.Objective: The relationship between the high expression of Lc3and acute myeloidleukemia will be studied. We will observe the mutual influence between the Lc3expression changes and cell differentiation, and explore the potential roles of Lc3inprocess and treatment of acute myeloid leukemia.Methods:(1) The comprehensive analysis of the glycosphingolipids in leukemiacell lines to determine the HL-60and NB4cell lines as a tool for functional experiment;(2) HL-60and NB4cell lines were treated with drug ATRA and PMA to induce celldifferentiation, the differentiated cells were then underwent glycosphingolipids analysisto observe the changes;(3) RNA interference technology was used to silence the Lc3synthase β3Gn-T5gene. Glycosphingolipids analysis of cells after the gene silence, andthe cells differentiation condition were detectedResults:(1) In a variety of leukemia cell lines, similar to clinical samples, onlyacute myeloid leukemia cells HL-60and NB4have high expression of Lc3, while innon-acute myeloid leukemia cell lines, Lc3content is relatively low;(2) HL-60andNB4cells can be induced to enter differentiation along the neutrophil lineage uponstimulation with ATRA or along the monocyte lineage after PMA treatment, and Lc3content decreased;(3) RNA interference reduced expression of the Lc3synthaseβ3Gn-T5, thus reduced the GSL Lc3content in HL-60and NB4cells and showeddifferentiation phenomenon.Conclusion: In acute myeloid leukemia cell line HL-60and NB4, Lc3content isclosely associated with cell differentiation. When drug effects lead to cell differentiation,Lc3content will be decreased. Similarly, when reduce Lc3content, the cells also appeardifferentiation phenomenon. |
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