Nuclear Proteomics of Induced Leukemia Cell Differentiation

Abstract

Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all-trans-retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3–12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear proteome in DNA damage recognition in the RUNX1-triggered pathway, and in the p53-regulation pathway. By applying scheduled multiple reaction monitoring using stable isotopically labeled peptide standards (MRM/SIS), we found a persistent increase in the content of the following proteins: PRAM1, CEPBP, RBPJ, and HIC1 in the HL-60 cell nuclear fraction during ATRA-induced granulocytic differentiation. In the case of STAT1, CASP3, PARP1, and PRKDC proteins, a transient increase in their content was observed at early time points (3–12 h) after the ATRA treatment. Obtained data on nuclear proteome composition and dynamics during granulocytic differentiation could be beneficial for the development of new treatment approaches for leukemias with the mutated p53 gene.

Systems Biology for Drug Target Discovery in Acute Myeloid Leukemia

“…CDK6 blocks myeloid differentiation by preventing RUNX1 DNA binding and RUNX1-C/EBPα interaction [35]. In our previous study of the HL-60 nuclear proteome changes induced by the ATRA treatment, we found the TFs belonging to the SWI1/SNF1 and ARID families involved in the RUNX1-triggered pathway [36]. The pharmacological inhibition of CDK6 by palbociclib is one approach for the treatment of breast cancer [37].…”

“…In our previous studies, we applied different time point schedules to the time-course proteomic (i.e., 0, 3, 24, 48, and 96 h, and 0, 3, 6, 9, 12, and 72 h) and transcriptomic (0, 3, 24, and 96 h) [11,36] investigation of leukemic cells under the ATRA treatment. In the current study, we used unified schedules for both transcriptomic and proteomic analysis.…”

“…A 3 µL sample containing 15 µg of native peptides and SIS peptides was separated on a ZORBAX SB-C18 analytical column (150 × 0.5 mm; particle size, 5 µm; Agilent Technologies, Santa Clara, CA, USA) in a linear gradient of acetonitrile concentration at a flow rate of 20 µL/min. The LC-SRM analysis was performed as described previously [36]. All transitions with recorded signals were used for quantification with the Skyline software (version 4.1.0, University of Washington, Seattle, WA, USA).…”

The Proteomic Landscape of Monocytes in Response to Colorectal Cancer Cells

“…A persistent increase has been identified in the HL-60 cell line (acute myeloid leukemia) under all-trans-retinoid acid treatment, indicating participation in granulocytic differentiation. 61 In the realm of tumor prognosis, limited research has been conducted on these two proteins. Through our cocultivation experiments in conjunction with public databases, we initially observed their distinctive overexpression on monocytes/ macrophages within colorectal cancer patient tissues.…”

ESPRESSO: Spatiotemporal omics based on organelle phenotyping

“…Techniques like the Cell Painting 17 assay leverage organelle properties using fluorescent dyes to target various components like the nucleus, endoplasmic reticulum, mitochondria, cytoskeleton, Golgi apparatus, and RNA, and demonstrate applications in drug discovery. Proteomics has also been applied in organelle-specific contexts with techniques like nuclear proteomics 18 , where nuclear isolation followed by mass spectrometry analysis unveils protein composition and interactions within the nucleus. Overall, a clear link exists between the morphological and functional characteristics of the organelle network (hence, the "organelle landscape") and cell state.…”