Quantitative proteomic analysis and comparison of two bone marrow stromal cell lines using the SILAC method

Abstract

Two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells, have frequently been used in studies of cross talk between cells in the bone marrow microenvironment and hematopoietic cells. Altered expression of proteins is typically associated with cell-cell signal transduction and regulation of cellular functions. Many studies have focused on key proteins that contribute to functional differences in cell co-culture models, but global quantitative proteome analysis of HS5 and HS27a has not been performed. We employed the stable isotope labeling by amino acids in cell culture (SILAC) method using two stable isotopes each of arginine and lysine to label proteins in the two cell lines. Labeled proteins were analyzed by 2-D ultrahigh-resolution liquid chromatography- LTQ/Orbitrap mass spectrometry. Among 4,213 unique identified and annotated proteins in the cell lines, 1,462 were detected in two independent experiments. Of these, 69 exhibited significant upregulation and 48 significant downregulation (>95% confidence) in HS27a relative to HS5 cells. Gene ontology term and pathway analysis indicated that the differentially regulated proteins were involved in cellular movement, cell-to-cell signaling and interaction, and hematologic system development and function. A total of 55 items were identified in both genomic and proteomic databases. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed on 7 proteins randomly selected from 28 differentially expressed proteins that were identified in both databases and were involved in the top networks/pathways. We observed a decrease in apoptosis in co-cultured KG1a cells when integrin αV was inhibited in HS27a cells, which suggested the functional role of integrin αV in the co-culture system. The integrated genomic/proteomic approach described here, and the identified proteins, will provide a useful basis for further elucidation of molecular mechanisms in the bone marrow microenvironment and for ongoing studies of cross talk among stromal cells and myeloma cells in co-culture systems.

Screening differentially expressed proteins from co-cultured hematopoietic cells and bone marrow-derived stromal cells by quantitative proteomics (SILAC) method

“…Based on our in vitro observations, we intravenously co-injected HS5 cells and MDS marrow-derived hematopoietic cells into Nod.cg-Prkdc scid Il2rg tm1wjll (NSG) mice, and observed that differential stromal contact had structural and functional effects on proliferation of MDS precursor cells [40]. Also, we used glycomics (lectin microarray) and proteomics (SILAC method) approaches to compare two bone marrow stromal cell lines, HS5 and HS27a, and observed differential expression of several glycans and proteins [18, 41]. These and many similar findings indicate that microenvironmental factors play key roles in both normal and pathological hematopoiesis, and that hematopoiesis is modulated by bidirectional signals between hematopoietic cells and the microenvironment.…”

“…HS27a supports “cobblestone area” formation by early hematopoietic progenitor cells, whereas HS5 secretes multiple cytokines that support proliferation of committed progenitor cells [17]. In our previous studies, we integrated information on quantitative proteomic data with genomic data to discover several differentially expressed proteins in two bone marrow stromal cells HS5 and HS27a [18]. The SILAC method has many advantages to reduce sample to sample variability therefore is highly suitable for protein quantification [19, 20].…”

Altered susceptibility to apoptosis and N‑glycan profiles of hematopoietic KG1a cells following co‑culture with bone marrow‑derived stromal cells under hypoxic conditions

“…KG1a cells (derived from acute myeloid leukemia) and bone marrow-derived stromal cell line HS27A are generous gifts from Professor H. Joachim Deeg (Fred-Hutchinson Cancer Research Center, Seattle, WA, USA). KG1a and HS27A were grown, propagated and subjected to experiments between passages 8 and 24, as previously described (27). Multiple aliquots from early passages were cryopreserved for later use.…”

SILAC–based quantitative MS approach for real-time recording protein-mediated cell-cell interactions

“…The secretory pattern of tumor cells and their neighboring stromal cells is dynamically changed in tumor microenvironment. The secreted proteins are often present with high numbers and low abundance, and interfered by serum proteins in culture medium and contaminations of intracellular proteins released by cell lysis during sample preparation 7 – 9 . In order to sensitively detect active cell-cell interactions mediated by secreted proteins in tumor microenvironment, the SILAC (stable isotope labeling of amino acids in cell culture) based quantitative proteomic strategy showed us the good confidence due to its sensitivity and accuracy 6 , 10 .…”

“…In order to sensitively detect active cell-cell interactions mediated by secreted proteins in tumor microenvironment, the SILAC (stable isotope labeling of amino acids in cell culture) based quantitative proteomic strategy showed us the good confidence due to its sensitivity and accuracy 6 , 10 . The SILAC-combined mass spectrometry (MS) has been widely used in varied studies, including screening disease biomarkers 9 – 13 , drug targets 14 – 16 , monitoring changes in post-translational modifications 17 – 19 and finding key factors in the complex signal pathway 10 , 20 , 21 .…”