Pavel Šimara

Differential effects of insulin and dexamethasone on pulmonary surfactant-associated genes and proteins in A549 and H441 cells and lung tissue

In this study, the effects of insulin and dexamethasone on the expression and mRNA transcription of 4 pulmonary surfactant-associated proteins [surfactant protein (SFTP)A, SFTPB, SFTPC and SFTPD] were examined. The commercially available cell lines, A549 and H441, were used as acceptable models of lung surfactant-producing cells. Subsequently, the effects of insulin on the expression of surfactant-associated proteins were examined in patients with lung adenocarcinoma during lung resection. Our results demonstrated the inhibitory effects of insulin on the transcription of the SFTPB, SFTPC and SFTPD genes in H441 cells and the SFTPB gene in A549 cells. Treatment with insulin significantly decreased the protein expression of SFTPA1 and SFTPA2 in the H441 cells and that of proSFTPB in the A549 cells. Dexamethasone promoted the transcription of the SFTPB, SFTPC and SFTPD genes in the A549 and H441 cells and reduced the transcription of the SFTPA1 and SFTPA2 genes in the H441 cells (SFTPA mRNA expression was not detected in A549 cells). Furthermore, we demonstrated that the mRNA levels of the selected genes were significantly lower in the cell lines compared to the lung tissue. A549 and H441 cells represent similar cell types. Yet, in our experiments, these cells reacted differently to insulin and/or dexamethasone treatment, and the mRNA levels of their main protein products, surfactant-associated proteins, were significantly lower than those in real tissue. Therefore, the results obtained in this study challenge the suitability of A549 and H441 cells as models of type II pneumocytes and Clara cells, respectively. However, we successfully demonstrate the possibility of studying the effects of insulin on pulmonary surfactant-associated genes and proteins in patients with lung adenocarcinoma.

Proliferation and differentiation potential of CD133+ and CD34+populations from the bone marrow and mobilized peripheral blood

CD34 is the most frequently used marker for the selection of cells for bone marrow (BM) transplantation. The use of CD133 as an alternative marker is an open research topic. The goal of this study was to evaluate the proliferation and differentiation potential for hematopoiesis (short and long term) of CD133+ and CD34+ populations from bone marrow and mobilized peripheral blood. Eight cell populations were compared: CD34+ and CD133+ cells from both the BM (CML Ph−, CML Ph+, and healthy volunteers) and mobilized peripheral blood cells. Multicolor flow cytometry and cultivation experiments were used to measure expression and differentiation of the individual populations. It was observed that the CD133+ BM population showed higher cell expansion. Another finding is that during a 6-day cultivation with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), more cells remained in division D0 (non-dividing cells). There was a higher percentage of CD38− cells observed on the CD133+ BM population. It was also observed that the studied populations contained very similar but not the same pools of progenitors: erythroid, lymphoid, and myeloid. This was confirmed by CFU-GM and CFU-E experiments. The VEGFR antigen was used to monitor subpopulations of endothelial sinusoidal progenitors. The CD133+ BM population contained significantly more VEGFR+ cells. Our findings suggest that the CD133+ population from the BM shows better proliferation activity and a higher distribution of primitive progenitors than any other studied population.

Apoptosis in chronic myeloid leukemia cells transiently treated with imatinib or dasatinib is caused by residual BCR-ABL kinase inhibition.

Transient, potent BCR–ABL inhibition with tyrosine kinase inhibitors (TKIs) was recently demonstrated to be sufficient to commit chronic myeloid leukemia (CML) cells to apoptosis irreversibly. This mechanism explains the clinical efficacy of once‐daily dasatinib treatment, despite the rapid clearance of the drug from the plasma. However, our in vitro data suggest that apoptosis induction after transient TKI treatment, observed in the BCR–ABL‐positive cell lines K562, KYO‐1, and LAMA‐84 and progenitor cells from chronic phase CML patients, is instead caused by a residual kinase inhibition that persists in the cells as a consequence of intracellular drug retention. High intracellular concentrations of imatinib and dasatinib residues were measured in transiently treated cells. Furthermore, the apoptosis induced by residual imatinib or dasatinib from transient treatment could be rescued by washing out the intracellularly retained drugs. The residual kinase inhibition was also undetectable by the phospho‐CRKL assay. These findings confirm that continuous target inhibition is required for the optimal efficacy of kinase inhibitors. Am. J. Hematol. 88:385–393, 2013.

The role of the endoplasmic reticulum stress in stemness, pluripotency and development.

The molecular machinery of endoplasmic reticulum (ER) integrates various intracellular and extracellular cues to maintain homeostasis in diverse physiological or pathological scenarios. ER stress and the unfolded protein response (UPR) have been found to mediate molecular and biochemical mechanisms that affect cell proliferation, differentiation, and apoptosis. Although a number of reviews on the ER stress response have been published, comprehensive reviews that broadly summarize ER physiology in the context of pluripotency, embryonic development, and tissue homeostasis are lacking. This review complements the current ER literature and provides a summary of the important findings on the role of the ER stress and UPR in embryonic development and pluripotent stem cells.

The Aberrant DNA Methylation Profile of Human Induced Pluripotent Stem Cells Is Connected to the Reprogramming Process and Is Normalized During In Vitro Culture

The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications.

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.

DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing

BackgroundHuman induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs).MethodsWe measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases.ResultsWe demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages.ConclusionsOur observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.

Cell cycle-dependent changes in H3K56ac in human cells

The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication.

From Endothelial Progenitor Cells to Tissue Engineering: How Far Have We Come?

Cardiovascular disease has become the leading cause of death in high income countries worldwide and the available treatment is not able to provide a complete recovery. Tissue engineering offers a possibility to construct autologous vein replacements for surgery. In this review we summarize approaches leading to artificial vascular graft construction. We discuss biomaterials currently in use, various drug delivery systems and the most appropriate cell cultures for vein engineering. Despite the progress in biomaterials and drug delivery systems, generating a suitable tissue microenvironment and selection of the appropriate cell population for graft seeding remains a major challenge. Here we focus on endothelial progenitor stem cells as the most suitable cell type for vascular graft construction. We discuss its sources, isolation techniques and differentiation procedures.

Flow Cytometry Analysis of Intracellular Protein

The demand for determining protein levels in a continuously decreasing amount of input cells is currently being developed by scientists and medical doctors. Another demanding area is the splitting of active and inactive (phosphorylated and unphosphorylated, respectively) forms of the protein being monitored, e.g., during different phases of the cell cycle, differentiation or carcinogenesis. The Western blot technique has been typically used for this purpose. The utilization of multi color flow cytometry allows for measurements of multiple proteins in parallel, regardless of the protein length. As the technique evolves, it is possible to obtain information on over 13 parameters per cell (Krutzik et al., 2004).