Proteomic Characterization of Spontaneous Stress-Induced In Vitro Apoptosis of Human Acute Myeloid Leukemia Cells; Focus on Patient Heterogeneity and Endoplasmic Reticulum Stress

Abstract

In vitro culture is widely used for characterization of primary human acute myeloid leukemia (AML) cells, but even when using optimized handling and culture conditions the AML cells show spontaneous in vitro apoptosis with a gradual decrease in cell viability during culture. The extent of this stress-induced apoptosis varies between patients, and a high degree of apoptosis is associated with high pre-culture BCL2 levels together with low levels of BAX and Heat Shock Proteins 30 and 90. We compared the global proteomic profiles during ongoing in vitro apoptosis for patients with high and low AML cell viability (i.e., less extensive versus extensive spontaneous apoptosis) after 48 h of culture. We identified 7902 proteins, but only 276 proteins differed significantly between patients with high (i.e., >25% viable cells; 192 upregulated and 84 downregulated peptides) and low viability after in vitro culture. Protein interaction network analysis based on these 276 protein identified three protein networks that included 18 proteins; most of these proteins were localized to the endoplasmic reticulum and several of them are involved in or are altered during the process of endoplasmic reticulum stress/unfolded protein stress response. To conclude, primary AML cells are heterogeneous with regard to degree of apoptosis in response to cellular stress, and this difference in regulation of apoptosis is associated with differences in the induction of and/or response to the unfolded protein stress response.

Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations

“…All samples were cryopreserved according to the same highly standardized protocol [128] and samples were stored in liquid nitrogen for the whole storage period. The duration of the storage period did not differ significantly for cells with and without NPM1-Ins, and the procedures for thawing and lysate preparation were highly standardized as described previously [25,[129][130][131][132][133][134][135]. Furthermore, in our previous study, the general quality of our proteomic data was confirmed by additional Western blot analyses of two differentially expressed proteins (CDK2, ERK1/2) and three differentially expressed phosphosites (CDK2T160, ERK1/2T202, ERK1/2 Y204).…”

Endocan in Acute Leukemia: Current Knowledge and Future Perspectives

“…As described in a recent review, endocan is expressed mainly in lung and renal vasculature, but it is not restricted to endothelial expression and has been detected in normal serum as well as in several actively proliferative normal and malignant tissues [19,33,34]. A previous study described expression of endocan in AML and ALL blasts [35], but endocan did not reach quantifiable levels in previous proteomic studies of AML secretome, AML cell lysates, MSC secretome, MSC lysates, osteoblast secretome, or osteoblast lysates [36][37][38][39][40][41]. However, acute leukemia is characterized by increased angiogenesis, i.e., endothelial proliferation (see Section 4).…”

Vacuolar ATPase Is a Possible Therapeutic Target in Acute Myeloid Leukemia: Focus on Patient Heterogeneity and Treatment Toxicity

“…Proteomic and phosphoproteomic profiles were available for a consecutive subset of 15 younger AML patients (below 60 years of age), and we compared two contrasting patient groups with an antiproliferative effect corresponding to a relative response (i.e., the proliferation in the presence of bafilomycin A 10 nM being) <0.30 versus >0.60, respectively. These comparisons therefore included eight strong responders (Tables S1 and S2; patients 5,15,55,59,64,69,71) and seven weak responders (Table S1, patients 2, 23,31,34,36,41,51,73).…”

“…Previous studies have demonstrated that cryopreserved AML cells usually include at least 70% viable cells (Annexin V − PI − ) together with minor subsets of early apoptotic (Annexin + PI − ) and late apoptotic/necrotic cells (Annexin V + PI + ) immediately after thawing [40]. During the first days of in vitro culture, cryopreserved AML cells undergo spontaneous or stress-induced in vitro apoptosis mainly due to chaperon-induced apoptosis [40,69]. There is a wide variation between patients with regard to the degree of spontaneous apoptosis during culture.…”