Proteomic Profiling of HL-60 Cells during ATRA-Induced Differentiation

Abstract

Acute promyelocytic leukemia, a form of acute myeloid leukemia, is characterized by cell differentiation arrest at the promyelocyte stage. Current therapeutic options include administration of all trans-retinoic acid (ATRA), but this treatment produces many side effects. ATRA is known to induce differentiation of leukemic cells into granulocytes, but the mechanism of this process is poorly studied. We performed comparative proteomic profiling of HL-60 promyelocytic cells at different stages of ATRA-induced differentiation to identify differentially expressed proteins by high-resolution mass spectrometry and relative quantitative analysis without isotope labels. A total of 1162 proteins identified by at least two unique peptides were analyzed, among them 46 and 172 differentially expressed proteins were identified in the nuclear and cytosol fractions, respectively. These differentially expressed proteins can represent candidate targets for combination therapy of acute promyelocytic leukemia.

Nuclear Proteomics of Induced Leukemia Cell Differentiation

“…This work is a continuation of our previous studies on induced granulocytic differentiation of HL60 cells [ 20 , 21 ]. Applying the optimized protocol of nucleus isolation, alkaline peptide fractionation, together with protein quantitation by TMT, we confidently identified almost a thousand proteins that were not detected in our previous studies ( Figure S6 ).…”

“…Most of them were focused on advanced time points after the ATRA treatment (e.g., 24, 48, and 96 h [ 17 ], 24 h and 72 h [ 18 ], 24 and 48 h [ 19 ]). In our own previous proteomic experiments, we also applied advanced time point schedules (i.e., 24, 48, and 96 h) [ 20 , 21 ], with the addition of only one early time point (i.e., 3 h). At advanced time points, granulocyte-specific surface markers CD11b and CD38 may be detected by cytofluorimetry that reflects the complete maturation of leukemic cells into neutrophils.…”

“…The following supporting information can be downloaded at: . Figure S1: Proteomic experiment design; Figure S2: The heatmap visualization of the results of label-free proteomic profiling of leukemic cell whole lysate (WHL) and nuclear fraction (NF); Figure S3: The Venn diagram shows intersection of differentially expressed transcripts (DETs) and differentially expressed nuclear proteins (DENPs) during ATRA-induced differentiation of HL-60 cell line; Figure S4: The results of the interaction analysis of 42 downregulated TFs by STRING; Figure S5: The results of the interaction analysis of 33 upregulated TFs by STRING; Figure S6: Contribution of mass-spectrometric datasets to the coverage of the proteome of HL60 cell line under ATRA treatment [ 20 , 21 ]; Figure S7: The 1D-heatmap visualization of the functional annotation of 806 nuclear proteins that were uniquely identified in the present study, compared to previously investigated HL60 proteomes; Table S1: TMT-labeling design, MRM transition table and list of SIS peptides; Table S2: Differentially expressed transcripts (DETs) with fold-change equal to or above 2 ( p -value < 0.05) at 0.5 h and 1 h after ATRA treatment; Table S3: The results of label-free proteomic profiling of leukemic cell whole lysate (WHL) and nuclear fraction (NF). Functional annotation of proteins upregulated in leukemic cell NF compared to WHL, according to the GO database Cell Compartment (CC) category; Table S4: Summary of proteomic profiling of HL-60 cells under ATRA treatment.…”