Targeting of Post-Transcriptional Regulation as Treatment Strategy in Acute Leukemia

Post-transcriptional regulation is an important step of gene expression that allows to fine-tune the cellular protein profile (so called proteome) according to the current demands. That mechanism has been developed to aid survival under stress conditions, however it occurs to be hijacked by cancer cells. Adjustment of the protein profile remodels signaling in cancer cells to adapt to therapeutic treatment, thereby enabling persistence despite unfavorable environment or accumulating mutations. The proteome is shaped at the post-transcriptional level by numerous mechanisms such as alternative splicing, mRNA modifications and triage by RNA binding proteins, change of ribosome composition or signaling, which altogether regulate the translation process. This chapter is an overview of the translation disturbances found in leukemia and their role in development of the disease, with special focus on the possible therapeutic strategies tested in acute leukemia which target elements of those regulatory mechanisms

Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry

Cancer cells, including leukemia cells, reside in a complex microenvironment, which influences biology and activity of the cells. The protective role of bone marrow stromal cells is already commonly recognized. Remodeling of stroma cell functions by leukemia cells is also well documented. In this respect, different routes of interactions were defined, such as direct cell-cell interactions or indirect cross talk, by release of soluble factors or vesicular particles containing proteins, RNAs and other molecules. Since intercellular communication seems to play a role in various biological processes, it might be important to conduct studies in co-culture systems, which at least mimic partially more physiological conditions, and enables this intercellular exchange to occur. Thus, it is crucial to improve analytical methods of investigation of co-cultured cells, to study their interactions and so to understand biology of leukemia in order to understand molecular mechanisms and offer novel therapeutic strategies. The present chapter outlines the importance of modern, multiparameter flow cytometry methods, which allow to analyze interactions between different types of cells within the leukemia microenvironment. Importantly, the proposed experimental setups can be easily transformed to study different cell types and different biological systems

Calcium- and proton-dependent relocation of annexin A6 in Jurkat T cells stimulated for interleukin-2 secretion

Annexin A6 (AnxA6) is a Ca2+-dependent membrane-binding protein involved in vesicular traffic. The likely participation of AnxA6 in the response of lymphocytes to Ca2+ signals has not been investigated yet. The present study focuses on intracellular relocation of AnxA6 in human Jurkat T lymphoblasts upon stimulation followed by transient increase of intracellular [Ca2+] and exocytosis of interleukin-2 (IL-2). Stimulation of the cells under different experimental conditions (by lowering pH and/or by rising extracellular [Ca2+] in the presence of ionomycin) induced time-dependent transients of intracellular [Ca2+] and concomitant changes in AnxA6 intracellular localization and in IL-2 secretion, with only minor effects on cell viability and apoptosis. In resting conditions (in the presence of EGTA or with no ionophore) AnxA6 was localized uniformly in the cytosol, whereas it translocated to vesicular structures beneath the plasma membrane within 5 min following stimulation of Jurkat T cells and rise of intracellular [Ca2+] at pH 7.4. Lowering the extracellular pH value from 7.4 to 6.0 significantly enhanced this process. AnxA6 changed its location from the cytosol to the secretory granules and early endosomes which seem to represent membranous targets for annexin. In conclusion, AnxA6 is sensitive to variations in intracellular [Ca2+] upon stimulation of Jurkat T cells, as manifested by a switch in its intracellular localization from the cytosol to vesicular structures located in close proximity to the plasma membrane, suggestive of participation of AnxA6 in calcium- and proton-dependent secretion of cytokines by lymphocytes

Stimulators of mineralization limit the invasive phenotype of human osteosarcoma cells by a mechanism involving impaired invadopodia formation.

BackgroundOsteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis).ResultsIn this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization.ConclusionsOur study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma

NFAT1 and NFAT3 Cooperate with HDAC4 during Regulation of Alternative Splicing of PMCA Isoforms in PC12 Cells

<div><p>Background</p><p>The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca²⁺-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca²⁺-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (<i>Atp2b1</i>, <i>Atp2b2</i>, <i>Atp2b3, Atp2b4),</i> whose transcript products undergo alternative splicing giving almost 30 variants.</p><p>Results</p><p>In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation.</p><p>Conclusions</p><p>We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.</p></div

Alternative splicing of PMCA in PMCA2- or PMCA3-deficient PC12 cells upon NFAT inhibition.

<p>Alternative splicing pattern at sites A and C of mRNA transcripts of PMCAs: <i>Atp21b1</i> (PMCA1) (<b>A</b>), <i>Atp21b2</i> (PMCA2) (<b>B</b>), <i>Atp21b3</i> (PMCA3) (<b>C</b>), <i>Atp21b4</i> (PMCA4) (<b>D</b>) was determined by RT-PCR in non-treated and 1 µM 11R-VIVIT-treated PC12 cells. RT-PCR product bands were quantified densitometrically, standardized to <i>Gapdh</i> and normalized to control cells, expressed as y = 1, both for non-treated (<b>E</b>) and 11R-VIVIT-treated cells (<b>F</b>). Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3-reduced cells (n = 3). Bars represent mean values ± SEM. *P≤0.05. Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3). Black arrows indicate the PCR product bands for PMCA2 site A and PMCA3 site C. White asterisks on the images of gels indicate the PCR product bands generated by alternative splicing that underwent a significant change upon NFAT inhibition with 11R-VIVIT.</p

NFAT activation in PC12 cells with reduce PMCAs content.

<p>PC12 cells were transfected with plasmids encoding firefly luciferase under NFAT-dependent promoter (pGL3-NFAT-luc) and reference plasmids with <i>Renilla</i> luciferase (pRL-SV40). Negative controls were wild type PC12 cells transfected with promoterless pGL3-luc plasmids and positive controls were wild type PC12 transfected with plasmids overexpressing NFAT together with the pGL3-NFAT-luc (pGL3-NFAT-luc-NFAT+/+). NFAT activity was determined with a luciferase reporter dual assay (Thermo Scientific Pierce) and showed as the ratio of the luminescence signals derived from Firefly and <i>Renilla</i> luciferases. Bars represent mean values ± SEM. Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3) and pGL3-NFAT-luc+/+NFAT – wild type cells overexpressing NFAT. Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3-deficient cells. *P≤0.05, n = 5 (<b>A</b>). Nuclear content of dephosphorylated NFAT1 and NFAT3 was analyzed by immunoblotting. Protein bands were quantified densitometrically, standardized to PARP (nuclear marker) and normalized to control cells, expressed as y = 1. Bars represent mean values ± SEM. Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3-deficient cells. *P≤0.05, n = 6 (<b>B</b>). Representative immunoblots of nuclear content of dephosphorylated NFAT1 (<b>C</b>) and NFAT3 (<b>D</b>) are demonstrated. Symbols correspond to: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3).</p

Primers for PMCA alternative splicing analysis.

<p>Primers for PMCA alternative splicing analysis.</p