Jiří Pacherník

Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases

The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

Alkaline Phosphatase in Stem Cells

Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells.

Differentiation-specific association of HP1α and HP1β with chromocentres is correlated with clustering of TIF1β at these sites

Mammalian heterochromatin protein 1 (HP1α, HP1β, HP1γ subtypes) and transcriptional intermediary factor TIF1β play an important role in the regulation of chromatin structure and function. Here, we investigated the nuclear arrangement of these proteins during differentiation of embryonal carcinoma P19 cells into primitive endoderm and into the neural pathway. Additionally, the differentiation potential of trichostatin A (TSA) and 5-deoxyazacytidine (5-dAzaC) was studied. In 70% of the cells from the neural pathway and in 20% of TSA-stimulated cells, HP1α and HP1β co-localized and associated with chromocentres (clusters of centromeres), which correlated with clustering of TIF1β at these heterochromatic regions. The cell types that we studied were also characterized by a pronounced focal distribution of HP1γ. The above-mentioned nuclear patterns of HP1 and TIF1β proteins were completely different from the nuclear patterns observed in the remaining cell types investigated, in which HP1α was associated with chromocentres while HP1β and HP1γ were largely localized in distinct nuclear regions. Moreover, a dispersed nuclear distribution of TIF1β was observed. Our findings showed that the nuclear arrangement of HP1 subtypes and TIF1β is differentiation specific, and seems to be more important than changes in the levels of these proteins, which were relatively stable during all the induced differentiation processes.

Lineage specific composition of cyclin D–CDK4/CDK6–p27 complexes reveals distinct functions of CDK4, CDK6 and individual D-type cyclins in differentiating cells of embryonic origin

Abstract.  Objectives: This article is to study the role of G1/S regulators in differentiation of pluripotent embryonic cells. Materials and methods: We established a P19 embryonal carcinoma cell‐based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G1/S regulators were analysed with respect to growth and differentiation parameters of the cells. Results and Conclusions: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E–CDK2 but not of cyclin D–CDK4/6–p27 complexes. In an exponentially growing P19 cell population, the cyclin D1–CDK4 complex is detected, which is replaced by cyclin D2/3–CDK4/6–p27 complex following density arrest. During endodermal differentiation kinase‐inactive cyclin D2/D3–CDK4–p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2–CDK4–p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D–CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G1/S transition‐regulating machinery in early embryonic cells.

Interference of contaminated sediment extracts and environmental pollutants with retinoid signaling

Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis. Some effects, such as malformations in frogs or changes in metabolism of birds, could be related to disruption of the retinoid signaling pathway by exposure to organic contaminants. A new reporter gene assay has been established for evaluation of the modulation of retinoid signaling by individual chemicals or environmental samples. The bioassay is based on the pluripotent embryonic carcinoma cell line P19 stably transfected with the firefly luciferase gene under the control of a retinoic acid-responsive element (clone P19/ A15). The cell line was used to characterize the effects of individual chemicals and sediments extracts on retinoid signaling pathways. The extracts of sediments from the River Kymi, Finland, which contained polychlorinated dioxins and furans and polycyclic aromatic hydrocarbons (PAHs), significantly increased the potency of all-trans retinoic acid (ATRA), while no effect was observed with the extract of the sediment from reference locality. Considerable part of the effect was caused by the labile fraction of the sediment extracts. Also, several individual PAHs potentiated the effect of ATRA; on the other hand, 2,3,7,8-tetrachlorodibenzo-p-dioxin and several phthalates showed slightly inhibiting effect. These results suggest that PAHs could be able to modulate the retinoid signaling pathway and that they could be responsible for a part of the proretinoid activity observed in the sediment extracts. However, the effects of PAHs on the retinoic acid signaling pathways do not seem to be mediated directly by crosstalk with aryl hydrocarbon receptor.

ABC transporters affect the detection of intracellular oxidants by fluorescent probes

Abstract Intracellular production of reactive oxygen species (ROS) plays an important role in the control of cell physiology. For the assessment of intracellular ROS production, a plethora of fluorescent probes is commonly used. Interestingly, chemical structures of these probes imply they could be substrates of plasma membrane efflux pumps, called ABC transporters. This study tested whether the determination of intracellular ROS production and mitochondrial membrane potential by selected fluorescent probes is modulated by the expression and activity of ABC transporters. The sub-clones of the HL-60 cell line over-expressing MDR1, MRP1 and BCRP transporters were employed. ROS production measured by luminol- and L-012-enhaced chemiluminescence and cytochrome c reduction assay showed similar levels of ROS production in all the employed cell lines. It was proved that dihydrorhodamine 123, dihexiloxocarbocyanine iodide, hydroethidine, tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide and tetramethylrhodamine ethyl ester perchlorate are substrates for MDR1; dichlorodihydrofluoresceine, hydroethidine and tetramethylrhodamine ethyl ester perchlorate are substrates for MRP1; dichlorodihydrofluoresceine, dihydrorhodamine 123, hydroethidine and tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide are substrates for BCRP. Thus, the determination of intracellular ROS and mitochondrial potential by the selected probes is significantly altered by ABC transporter activities. The activity of these transporters must be considered when employing fluorescent probes for the assessment of ROS production or mitochondrial membrane potential.

Differentiation-Independent Fluctuation of Pluripotency-Related Transcription Factors and Other Epigenetic Markers in Embryonic Stem Cell Colonies

Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency.

Phosphoinositide 3‐kinase inhibition enables retinoic acid‐induced neurogenesis in monolayer culture of embryonic stem cells

Retinoic acid (RA) is able to induce the differentiation of embryonic stem cells into neuronal lineages. The mechanism of this effect is unknown but it has been evidenced to be dependent on the formation of floating spheroids called embryoid bodies. Results presented here show that the inhibition of phosphoinositide 3‐kinase signaling pre‐determines mouse embryonic stem cells to RA induced neurogenesis in monolayer culture with no need of embryoid bodies formation. J. Cell. Biochem. 113: 563–570, 2012.

Conducting polyaniline based cell culture substrate for embryonic stem cells and embryoid bodies

In this work, thin films consisting of electrically conducting polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonate) (PAMPSA) have been used as cell culture substrates for embryonic stem cells (ESC) and embryoid bodies (EMB). The PANI–PAMPSA films having fibrillar morphology were electrochemically polymerized in a single-step by cyclic voltammetry from an aqueous solution containing aniline and PAMPSA. UV-visible spectroscopy showed that the PANI films were electrically conducting still at pH 10. This makes them suitable for tissue engineering applications operating at physiological pH, in contrast to the commonly used PANI hydrochloride films which loose their electrical conductivity at pH ≥ 4. Our results reveal that the PANI–PAMPSA films allow only for limited ESC adhesion and growth. The inhibition of the EMB growth and adhesion on the PANI–PAMPSA surface in serum-free medium indicates that it can be used as a cell-selective substrate for the growth of only some specific differentiated EMB cell types.

Stem cell differentiation on conducting polyaniline

Polyaniline is a promising conducting polymer with broad application potential in biomedicine. Its medical use, however, requires both biocompatibility and suitable physico-chemical and surface properties. The microstructure, electrical properties, and surface characteristics of polyaniline salt, polyaniline base, and polyaniline deposited with biologically active poly(2-acrylamido-2-methyl-1-propanesulfonic acid) were revealed using atomic force microscopy, contact angle measurements, and Raman spectroscopy. As conducting polymers can be preferentially applied in tissue engineering of heart and nervous tissues, the cardiomyogenesis in pure cardiomyocytes derived from embryonic stem cells and neurogenesis in neural progenitors isolated from embryonal 13 dpc brain were further investigated. The results show that neither cardiomyogenesis nor neurogenesis were influenced by any of the tested polyaniline films. However, the most favorable cell behaviour was observed on pristine polyaniline base; therefore, polyaniline in pristine forms without any further modification can be applied in a variety of biomedical fields.