Comparative proteomics analysis on differentiation of human promyelocytic leukemia HL-60 cells into granulocyte and monocyte lineages.
OBJECTIVE
Human promyelocytic leukemia cell line HL-60 has potential to differentiate into granulocytes and monocytes by different inducers, such as NSC67657 and all-trans retinoic acid (ATRA). However, the mechanism is not clear yet. This study was to induce differentiation of HL-60 cells using ATRA and NSC67657, and compare the protein expression patterns using two-dimensional electrophoresis (2-DE).


METHODS
HL-60 cells were cultured with ATRA and NSC67657 respectively. Cell proliferation was analyzed by MTT assay. Cellular surface specific antigen CD11b/CD14 was detected using flow cytometry (FCM) to assess cell differentiation. The alterations of cell morphology were observed with cellular chemical staining under electron microscope. Total protein was separated by modified 2-DE. The differentially expressed proteins were identified using PD-Quest software and analyzed by MOLDI-TOF MS. ICAT protein with differential expression was verified by reverse transcription-polymerase chain reaction (RT-PCR) and western blot.


RESULTS
The granulocytic and monocytic differentiation of HL-60 cells was induced by ATRA and NSC67657, respectively. The positive rates of both CD11b and CD14 in HL-60 cells were over 90% after 5-day treatment (2 micromol/L ATRA or 10 micromol/L NSC67657); cell morphology also represented characteristics of differentiation. Proteomic analysis showed that 25 proteins were differentially expressed with the same pattern in both differentiation groups, ten were differentially expressed only in monocytic differentiation group and 15 only in granulocytic differentiation group. Among them, ICAT expression was upregulated during NSC67657-induced differentiation of HL-60 cells.


CONCLUSIONS
A batch of differentially expressed proteins has be found by 2-DE in HL-60 cells with granulocytic and monocytic differentiation, which would contribute to the following functional research on related proteins.
Wang
Wei-Jia
WJ
Key Laboratory of Chongqing, Chongqing Medical University, Chongqing, PR China.
Tang
Wei
W
Qiu
Zong-Yin
ZY
D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D039481:CD11b Antigen / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D018922:HL-60 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D008854:Microscopy, Electron; D009000:Monocytes / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D040901:Proteomics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013256:Steroids / Q000494:pharmacology; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology
C544140:(4-(10, 13-dimethyl-3-methylsulfonyloxy-2,3,4,5,6,7,8,9,11,12,14,15,16, 17-tetradecahydro-1H-cyclopenta(a)phenanthren-17-yl)pentyl) methanesulfonate; D048868:Adaptor Proteins, Signal Transducing; D039481:CD11b Antigen; C411982:CTNNBIP1 protein, human; C509295:ITGAM protein, human; D047908:Intracellular Signaling Peptides and Proteins; D018950:Lipopolysaccharide Receptors; D008698:Mesylates; D013256:Steroids; D014212:Tretinoin