A Study Of Screening, Identifying And Functional Investigating Critical Proteins In HL60 Cells Induced By A New Steroidal Drug NSC67657

ObjectiveTo construct the models of promyelocytic leukemia HL60 cells'granulocytic and monocytic differentiation, and use comparative proteome method to separate and identify differentially expressed proteins in HL60 cells which are induced either by NSC67657 or ATRA. In addition, to further the functional research of ICAT which is screened out from these differentially expressed proteins.Method1. The verification of the activity of NSC67657 on the expression of CEBPα,and the construction of differentiating models: HL60 cells were treated by NSC67657 and ATRA respectively. Then the differentiating level, direction could be observed, and the most suitable drug concentration for introduction could be screened out by light microscope observation, MTT assay, ultramicrostucture analysis and cellular surface antigen detection. What's more, the analysis of apoptosis was also performed as the cooperated detection by flow cytometry analysis and ultracmicrostructure observation. Only in this way, the proteins we screened out from differentiating HL60 cells were associated with cellular differentiation, but not apoptosis.2. The modification of two dimensional electrophoretic separation of total proteins in HL60 cells: Three methods for protein extraction were employed: standard protein extraction, TCA/Ac precipitation and transonic quassation to extract total protein of HL60 cells with traditional and modified procedure. Then the protein spots could be counted and repeatation could be analyzed, which were the prerequisites for screening out the most suitable way for two dimensional electrophoresis.3. Analysis differentially expressed proteins in HL60 cells when they were induced into granulocyte and monocyte in the most suitable way for separation: the total protein in differenting HL60 cells was separated using former mentioned suitable method, and then the 2DE gel image could be outlined and quantified using PDQuest 7.0 software. Matrix assisted laser desorption/isonization time of flying mass spectrometry (MALDI-TOF-MS) was adopted to identify differentia protein spots and then mascot software was used to search for proteins in Swissprot database.4. Verify the expressing level of ICAT which was only differentially expressed in HL60 cells when they were treated by NSC67657, then locate the protein ICAT in HL60 cells: HL60 cells differentiation were induced by NSC67657 and ATRA respectively. Then RT-PCR and Western blot method were employed to verify the expressing level of ICAT mRNA and protein when cells were treated by the drugs. Furthermore, ICAT protein could be located using fluorescent microscope by fluorescent staining method.5. Constructing eukaryotic expressing vector and verifying mRNA and protein overexpression after the transfection: The CDS fragment of ICAT gene which was inserted two incision enzyme recognization sites was cloned using RT-PCR method in pDsRed-C1 that was labeled by a red fluorescence tag. Then the transfection of recombinent ICAT into HL60 and THP-1 cells was performed using a modified electroporating procedure. RT-PCR and Western blot methods were employed to verificate the overexpression of ICAT mRAN and protein.6. Analysis of biological character of sucessfully transfected HL60 cells: The proliferation of NSC67657 treated HL60 cells could be detected by MTT assay when recombinant ICAT was successfully transfected. Then the differentiation of drug treated or untreated, transfectd or untransfected HL60 cells was detected by flow cytometry analysis, chemical staining and ultramicrostructure observation.7. An initial functional research about ICAT protein in other leukemic cells: THP-1 cell line was selected and subjected to NSC67657 treatment before or after the transfection of ICAT eukaryotic expressing vector. Results1. The expression of CEBPαmRNA and protein were elevated firstly and then down-regulated when HL60 cells were treated by NSC67657. NSC67657 and ATRA could noticeably inhibit the proliferation of HL60 cells and the IC50(50% inhibitory concentration)dose was about 10μM and 2μM respectively; The percentage of CD14 and CD11b positive cells could up to over 90% when HL60 cells were treated by these two drugs. At the same time, the kidney like and anisotrophy like caryomorphism which were frequently appeared in monocyte, and rhabditiform like and sublobe like caryomorphism which were frequently appeared in granulocyte, could be easily found with many azurophil granules in endochylema . The apoptosis could not be found in drug treated HL60 cells.2. Under traditional focusing process with high voltage, chiastic stripe and lamellar shadow could be found everywhere on 2DE gels, by which protein spots were not that easy to investigate. However, the separation of proteins which were extracted by all the three methods was significantly up graded. But, the quantity of protein lost much using TCA/Ac precipitation, and the repeatation of 2DE gel images was low using transonic quassation. Inversely, not only protein quantity, but also 2DE gel images repeatation could be siginificantly improved using standard protein extraction under modified focusing procedure. Most of protein spots arrange from pH 4 to 7, so pH3~10 NL IPG was the best choice for further experiment.3. Through the analysis of 2DE gel images, more than 70 protin spots showed differentially expressed, among which 63 protein spots were identified by MALDI-TOF-MS, and then 50 protein spots were found to be potential candidates as differentially expression during cellular differentiation. 25 of these 50 protein spots were either elevated or down regulated both in NSC67657 and ATRA treated HL60 cells, and 10 protein spots were only differentially expressed in NSC67657 treated HL60 cells, 15 protein spots were only differentially expressed in ATRA treated HL60 cells.4. ICAT protein was one of 10 protein spots which were only differentially expressed in NSC67657 treated HL60 cells. The expressing levels of ICAT mRNA and protein were significantly up-regulated in NSC67657 treated group compared to untreated and ATRA treated group. The subcellular location of ICAT protein is cellular nucleus and endochylema.5. The pDsRed-ICAT eucaryotic expressing vector was successfully constructed. And the expressing level of ICAT mRNA and protein was promoted after transfection.6. The expression of CD14 did not change significantly between pDsRed-ICAT transfected and untransfected HL60 cells. However, the expression of CD14 in pDsRed-ICAT was greatly enhanced by the treatment of NSC67657, and the cellular proliferation was significantly inhibited.7. THP-1 cell line could be induced into monocytic differentiation after the treatment of NSC67657. However, cellular differentiation and proliferation of pDsRed-ICAT transfected THP-1 cells did not show marked differences compared to untransfected THP-1 cells when they were treated by NSC67657.Conclusions1. NSC67657 was a potential activator of CEBPα. HL60 cells could be induced into monocytic and granulocytic differentiation without the heppening of apoptosis under 10μM NSC67657 and 2μM ATRA.2. Focusing with low voltage was a new effective, reliable, time saved method for the eukaryocyte proteome analysis.3. 50 differentially expressed proteins which had been identified might play critical roles in the differentiation of HL60 cells induced by NSC67657 and ATRA.4. The expressing level of ICAT protein was elevated only in NSC67657 treated HL60 cells.5. The overexpression of ICAT gene in HL60 cells and THP-1 cells had been successfully constructed.6. The overexpression of ICAT protein could enhance the monocytic differentiation of NSC67657 treated HL60 cells, however, ICAT over-expression could not sensitize THP-1 cells under NSC67657 treatment. We concluded that ICAT might exert its effect only on granulo-monocytic progenitor cell stage, but not in monocytic progenitor cell stage.