Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.

Overcoming the UCB HSCs –Derived NK cells Dysfunction through Harnessing RAS/MAPK, IGF-1R and TGF-β Signaling Pathways

Background The natural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to the NK cells from adult donors. This is due to the possibility to choose alloreactive donors and potentially more robust in vivo expansion. However, the cytotoxicity of UCB-HSC-derived NK cells against cancer cells might be suboptimal. To overcome this obstacle, we attempted to generate NK cells with potent antitumor activity by targeting RAS/MAPK, IGF-1R and TGF-β signaling pathways using IL-15, IGF-1 and SIS3 respectively. Methods The CD34 + cells were isolated from human UCB mononuclear cells through magnetic activation cell sorting (MACS) with purity of (≥ 90%) and were subjected to differentiate into NK cells. After 21 days of induction with SFTG36 (SCF, FLt-3L, TPO, GM-CSF, IL-3 and IL-6), IS721 (IGF-1, SIS3, IL-7 and IL-21) and IL-15/Hsp70 media, NK cells phenotypes were studied and their cytotoxicity against K562 human erythroleukemia cells and SKOV3 ovarian carcinoma cells was analyzed. Results The NK cells induced in SFTG36/IS721 medium were selected for activation due to their higher expression of CD56 + 16 + CD3 − (93.23% ± 0.75) and mean fluorescence intensity (MFI) of NKG2D + (168.66 ± 20.00) and also a higher fold expansion potential (11.893 ± 1.712) compared to the other groups. These cells once activated with IL-15, demonstrated a higher cytotoxicity against K562 (≥ 90%; P ≤ 0.001) and SKOV3 tumor cells (≥ 65%; P ≤ 0.001) compared to IL-15/Hsp70-activated NK cells. Conclusions The differentiation of ex vivo expanded CD34 + cells through manipulation of RAS/MAPK, IGF-1R and TGF-β signaling pathways is an efficient approach for generating functional NK cells that can be used for cancer immunotherapy.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Construction of Recombinant Human GM-CSF and GM-CSF-ApoA-I Fusion Protein and Evaluation of Their Biological Activity

In this study, two strains of the yeast P. pastoris were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins’ yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units—erythroid) at a concentration 1.7 fold less than non-glycosylated E. coli-derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.

miR-16-5p Promotes Erythroid Maturation of Erythroleukemia Cells by Regulating Ribosome Biogenesis

miRNAs constitute a class of non-coding RNA that act as powerful epigenetic regulators in animal and plant cells. In order to identify putative tumor-suppressor miRNAs we profiled the expression of various miRNAs during differentiation of erythroleukemia cells. RNA was purified before and after differentiation induction and subjected to quantitative RT-PCR. The majority of the miRNAs tested were found upregulated in differentiated cells with miR-16-5p showing the most significant increase. Functional studies using gain- and loss-of-function constructs proposed that miR-16-5p has a role in promoting the erythroid differentiation program of murine erythroleukemia (MEL) cells. In order to identify the underlying mechanism of action, we utilized bioinformatic in-silico platforms that incorporate predictions for the genes targeted by miR-16-5p. Interestingly, ribosome constituents, as well as ribosome biogenesis factors, were overrepresented among the miR-16-5p predicted gene targets. Accordingly, biochemical experiments showed that, indeed, miR-16-5p could modulate the levels of independent ribosomal proteins, and the overall ribosomal levels in cultured cells. In conclusion, miR-16-5p is identified as a differentiation-promoting agent in erythroleukemia cells, demonstrating antiproliferative activity, likely as a result of its ability to target the ribosomal machinery and restore any imbalanced activity imposed by the malignancy and the blockade of differentiation.