| Background and ObjectiveAcute leukemia (AL) is one of the most common hematologic Malignancies.The pathogenesis is the malignant clonal proliferation of bone marrow hematopoieticstem cells, which restrained the normal hematopoiesis. At present, part of patients canbe cured by chemotherapy and/or hematopoietic stem cell transplantation, butrefractory or appeared relapse is still exist in a large portion of the patients, which isassociated with leukemic multi-drug resistance. Looking for new therapeutic targetsand clarifying the drug resistant mechanism of leukemia are becoming importanttopics in this area. At the same time, for allogeneic hematopoietic stem celltransplantation, the graft versus host disease (GVHD) is one of the important factorsthat affecting the patients’ life quality, and restricts the clinical application ofallogeneic hematopoietic stem cell transplantation to some extent. The susceptibilityof GVHD is becoming a research hotspot at present.Heat shock protein90(Hsp90) widely exists in many kinds of cells, and highlyexpressed in a variety of malignant tumors. Hsp90is probably related to theoccurrence and development of tumor. Hsp90was also found participating in theprocess of immunoloregulation and antigen presenting of the body. Some scholarsfound that Hsp90alpha, one of Hsp90subtypes, was upregulated in patients with acute leukemia. After T cells activation, the other subtype of the Hsp90--Hsp90betaobviously rose, and which was associated with p-glycoprotein. All these resultssuggest that Hsp90alpha may participate in the occurrence of acute leukemia, andHsp90beta may be involved in the occurrence of GVHD and the process of drugresistance in refractory and relapsed acute leukemia.This research is to investigate the expression of heat shock protein90(Hsp90)and its subtypes (Hsp90alpha and beta) in different disease stages of acute leukemiaas well as leukemia cell line, analyze the differences of expression, and observe therelationship of clinical prognosis and the expression of Hsp90, including the subtypesalpha and beta. Through dealing with leukemia cell lines by Hsp90inhibitors-17-[2-(Dimethylamino)ethyl]amino-17-desmethoxygeldanamycin (17-DMAG), the role ofHsp90in proliferation and apoptosis of leukemia cells can be observed. By celltransfection, the role of Hsp90α and Hsp90β in proliferation and apoptosis ofleukemia cells and the effects of17-DMAG on the expression of Hsp90α andHsp90β can be observed.Materials and methodsThe first part:(1) the cells of leukemia cell lines were collected; bone marrowmononuclear cells and serum were extracted from different stages of patients withacute leukemia. Through Semi-quantitative RT-PCR analysis, the gene expressionlevel of Hsp90in cell lines and bone marrow mononuclear cells was detectedrespectively. The expression of Hsp90in serum was detected by using ELISA.(2) Byclinical data statistics, the relationship between the Hsp90expression and the clinicaloutcome in patients with acute leukemia was observed.(3) Jurkat and K562cellswere collected, after dealing with the two kinds of cell lines by17-DMAG, theexpression of Hsp90was examined by Semi-quantitative RT-PCR analysis, the effectsof17-DMAG on cell proliferation were detected by using WST, and cell apoptosiswere detected by Annexin V flow cytometry.The second part:(1) the cells of leukemia cell lines were collected; bone marrowmononuclear cells and serum were extracted from different disease stages of patients with acute leukemia. By real-time quantitative PCR analysis and western blot, thegene and protein expression level of Hsp90in cell lines and bone marrowmononuclear cells was detected respectively. Hsp90α protein expression level inserum was detected by using ELISA.(2) By clinical data statistics, the relationshipbetween the Hsp90α expression and the clinical outcome in patients with acuteleukemia was observed.(3) Jurkat and K562cells were collected and transfected withthe Hsp90α interferential plasmid and Lipofectamine2000Reagent by using RNAinterference. After that, gene expression of Hsp90α was examined by real-timequantitative PCR analysis, protein expression of Hsp90α was examined by westernblot, the effects of cell proliferation were detected using WST, and cell apoptosis wasdetected by using Annexin V flow cytometry.(4) Jurkat and K562cells werecollected, after dealt with17-DMAG, the gene and protein expression of Hsp90α inthe two cell lines were examined by real-time quantitative PCR analysis and westernblot.The third part:(1) the cells of leukemia cell lines were collected; bone marrowmononuclear cells and serum were extracted from different disease stages of patientswith acute leukemia. By real-time quantitative PCR analysis and western blot, thegene and protein expression level of Hsp90in cell lines and bone marrowmononuclear cells were detected. Hsp90β protein expression level in serum wasdetected by using ELISA.(2) By clinical data statistics, the relationship between theHsp90β expression and the clinical outcome in patients with acute leukemia wasobserved.(3) Jurkat and K562cells were collected and transfected with the Hsp90βinterferential plasmid and Lipofectamine2000Reagent by using RNA interference.After that, gene expression of Hsp90β was examined by real-time quantitative PCRanalysis, protein expression of Hsp90β was examined by western blot, the effects ofcell proliferation were detected using WST, cell apoptosis were detected by usingAnnexin V flow cytometry.(4) Jurkat and K562cells were collected, after dealt withthe two kinds of cell lines by17-DMAG, gene and protein expression of Hsp90β wasexamined by real-time quantitative PCR analysis and western blot. ResultThe first part1The detection of Hsp90expressionThe results of gene and protein testing showed that, Hsp90expression in alltested cell lines was increased obviously compared with the healthy controls.Likewise, the expression of Hsp90in the untreated AL patients, remission patientsand the patients who had a previous allogeneic hematopoietic stem celltransplantation was also increased obviously. Especially in refractory and relapsedpatients, the expression of Hsp90is the highest compared with other patients. Nosignificant difference in the expression of Hsp90was found in the untreated AML andALL patients. Transplanted patients suffering with GVHD showed higher Hsp90expression compared with transplanted patients without GVHD.2Clinical observation related to Hsp90The untreated AL patients with higher expression levels of Hsp90α had lowercomplete remission rates. Regarding the remission of untreated patients, theexpression of hsp90α decreased and the expression of hsp90α increased again beforerelapse and reached a higher level compared with the level prior to treatment.3Treat leukemia cells with17-DMAGAfter treating Jurkat and K562cells with17-DMAG, the gene expression ofHsp90reduced, cell proliferation was inhibited, and the apoptosis was promoted.The second part1The detection of Hsp90α expressionThe expression of Hsp90α in the untreated AL patients increased compared withthe healthy controls, and there was a similar level compared with remission patients,There was a significantly higher level in all of the tested leukemia cell lines and in the refractory and relapsed patients. No significant difference in the expression ofHsp90α was found in the untreated acute myeloid leukemia and acute lymphoblasticleukemia patients. No significant differences were observed in the Hsp90α expressionbetween the healthy controls and the patients who had a previous allogeneichematopoietic stem cell transplantation.2Clinical observation related to Hsp90αThe untreated AL patients with higher expression levels of Hsp90α had lowercomplete remission rates. Regarding the remission of untreated patients, theexpression of Hsp90α decreased and reached the lowest level after transplantation,but the expression of Hsp90α increased again before relapse and reached a higherlevel compared with the level prior to treatment.3Deal leukemia cells with transfectionAfter transfecting Jurkat and K562cells with Hsp90α interferential plasmid, thegene and protein expression of Hsp90α reduced, cell proliferation were inhibited, andthe apoptosis was promoted by17-DMAG.4Treat leukemia cells with17-DMAGAfter treating Jurkat and K562cells with17-DMAG, the gene and proteinexpression of Hsp90α reduced.The third part1The detection of Hsp90β expressionThe results of Polymerase Chain Reaction and Western Blotting test showed thatthe gene and protein expression of Hsp90β in all tested leukemia cell lines increasedsignificantly compared with the healthy control. The untreated AL patients andremission patients also expressed higher Hsp90β and the expression of Hsp90β washighest in refractory and relapsed patients. Transplanted patients suffering with graftversus host disease (GVHD) expressed higher Hsp90β compared with transplantedpatients without GVHD. There was no significant difference between untreated acute myeloid leukemia and untreated acute lymphoblastic leukemia patients in theexpression of Hsp90β.2Clinical observation related to Hsp90βUntreated AL patients with higher expression of Hsp90β had lower completeremission rates. As for the remission of untreated patients, the expression of Hsp90βdecreased but it increased again before relapse and even reached the higher level thanthe level prior to treatment. But the results of enzyme-linked immunosorbent assayshowed that the protein expression of Hsp90β in each cohort of patients was preciouslittle, and there was no significant difference among all cohorts.3Deal leukemia cells with transfectionAfter transfecting Jurkat and K562cells with Hsp90β interferential fragment, thegene and protein expression of Hsp90β reduced, and cell proliferation was weaklyinhibited and apoptosis of Jurkat and K562cells could not be promoted significantlyby17-DMAG.4Treat leukemia cells with17-DMAGAfter treating Jurkat and K562cells with17-DMAG, the gene and proteinexpression of Hsp90β reduced.Conclusion1Hsp90may play an important role in the occurence and development of acuteleukemia and can be used as a predictor of clinical outcomes.17-DMAG can inhibitcell proliferation and17-DMAG lead the Jurkat and K562cells to apoptosis. Thestudy of ours provides a theoretical basis on clinical treatment of acute leukemiausing17-DMAG.2Hsp90α may play an important role in the occurence and development of acuteleukemia and can be used as a predictor of clinical outcomes. Hsp90α selectiveinhibitor can be used as new agents for targeted therapy of acute leukemia patients.17-DMAG inhibits the expression of Hsp90α both in gene and protein levels. 3The expression of Hsp90β is associated with the occurrence of GVHD, andHsp90β can be used for predicting the occurrence of GVHD after transplantation, andalso it can be used as a new therapeutic target of GVHD. The expression of Hsp90βsignificantly increased in refractory and relapsed patients with acute leukemia, and itmay be associated with drug resistance in the process of treatment. |
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