Medium viscosity influence on the open states genesis in a DNA molecule.

The studies were carried out by the mathematical modeling of DNA mechanical deformations. Numerical calculations done for the interferon alpha 17 gene, which consists of 980 base pairs. It has been established that the genesis and dynamics of open states in the DNA molecule depends on the magnitude of the external influence (torque) and on the viscosity of the environment. In addition, it is shown that the dynamics of open states zones can have a jump-like character with a small change in the magnitude of the torque. When torque is applied to all 980 base pairs of the gene, the following effect is observed: an increase in the viscosity of the medium leads to an increase in the value of the torque necessary for the occurrence of OS and DNA unwinding, i.e. viscosity plays an important stabilizing role in DNA dynamics. Under the influence of a localized torque on different (by the content of A-T and G-C pairs and location) regions of the interferon alpha 17 gene, it was found that the magnitude of the external torque necessary for the occurrence of open states at all calculated values of viscosity depends on the nucleotide composition. The dependence of the torque magnitude required for the open states occurrence on viscosity is observed when the torque is applied to areas close to the gene boundaries. At the same time, the significance of the end effect, which weakens DNA, decreased with increasing viscosity of the medium.Communicated by Ramaswamy H. Sarma.

8-Oxoguanine-DNA-Glycosylase Gene Polymorphism and the Effects of an Alternating Magnetic Field on the Sensitivity of Peripheral Blood.

BACKGROUND: The production of reactive oxygen species (ROS) in animals and cells often results from exposure to low-intensity factors, including magnetic fields. Much of the discussion about the initiation of oxidative stress and the role of ROS and radicals in the effects of magnetic fields has centered on radical-induced DNA damage. METHODS: The DNA concentration in the final solution was determined spectrophotometrically. Typing of the polymorphic variant rs1052133 of the 8-oxoguanin DNA glycosylase (hOGG1) gene was performed by polymerase chain reaction. An enzyme immunoassay was performed to determine the level of 8-oxyguanine in DNA. To process samples exposed to an alternating magnetic field, the authors developed a device for the automated study of biological fluids in an alternating magnetic field. The content of hydrogen peroxide in aqueous solutions of DNA was determined using the spectrophotometric method. RESULTS: It was experimentally determined that an increase in the concentration of hydrogen peroxide in an aqueous medium by 3-5 times under the action of a low-frequency magnetic field reduces the resistance of the genomic material to oxidative modification and the accumulation of 8-oxyguanine in DNA. A model is proposed for the mechanism of action of a low-frequency magnetic field on aqueous solutions of nucleic acids and proteins, which satisfies the model of a chemical oscillator for the transformations of reactive oxygen species in an aqueous medium. The model illustrates the oscillating nature of the processes occurring in an aqueous solution of DNA and makes it possible to predict changes in the concentration of hydrogen peroxide in an aqueous solution of biopolymers, depending on the frequency of the acting low-intensity magnetic field. CONCLUSIONS: The key element in the mechanisms involved in the effects of low-intensity magnetic field on living systems is the occurrence of ROS generation in the aquatic environment of chemical oscillators, in which the competition of physical and chemical processes (electron transfers, reactions of decay and addition of radicals, spin magnetically induced conversion, synthesis, and decay of the longest-lived form-hydrogen peroxide) is controlled by a magnetic field.

Study of the Magnesium Comenate Structure, Its Neuroprotective and Stress-Protective Activity.

The crystal structure and the biological activity of a new coordination compound of magnesium ions with comenic acid, magnesium comenate, was characterized and studied. Quantitative and qualitative analysis of the compound was investigated in detail using elemental X-ray fluorescent analysis, thermal analysis, IR-Fourier spectrometry, UV spectroscopy, NMR spectroscopy, and X-ray diffraction analysis. Based on experimental analytical data, the empirical formula of magnesium comenate [Mg(HCom)2(H2O)6]·2H2O was established. This complex compound crystallizes with eight water molecules, six of which are the hydration shell of the Mg2+ cation, and two more molecules bind the [Mg(H2O)6]2+ aquacation with ionized ligand molecules by intermolecular hydrogen bonds. The packing of molecules in the crystal lattice is stabilized by a branched system of hydrogen bonds with the participation of solvate water molecules and oxygen atoms of various functional groups of ionized ligand molecules. With regard to the biological activity of magnesium comenate, a neuroprotective, stress-protective, and antioxidant effect was established in in vitro and in vivo models. In in vitro experiments, magnesium comenate protected cerebellar neurons from the toxic effects of glutamate and contributed to the preservation of neurite growth parameters under oxidative stress caused by hydrogen peroxide. In animal studies, magnesium comenate had a stress-protective and antioxidant effect in models of immobilization-cold stress. Oral administration of magnesium comenate at a dose of 2 mg/kg of animal body weight for 3 days before stress exposure and for 3 days during the stress period led to a decrease in oxidative damage and normalization of the antioxidant system of brain tissues against the background of induced stress. The obtained results indicate the advisability of further studies of magnesium comenate as a compound potentially applicable in medicine for the pharmacological correction of conditions associated with oxidative and excitotoxic damage to nerve cells.

Early Cell Cultures from Prostate Cancer Tissue Express Tissue Specific Epithelial and Cancer Markers.

Prostate cancer (PCa) is a widespread oncological disease that proceeds in the indolent form in most patients. However, in some cases, the indolent form can transform into aggressive metastatic incurable cancer. The most important task of PCa diagnostics is to search for early markers that can be used for predicting the transition of indolent cancer into its aggressive form. Currently, there are two effective preclinical models to study PCa pathogenesis: patients derived xenografts (PDXs) and patients derived organoids (PDOs). Both models have limitations that restrict their use in research. In this work, we investigated the ability of the primary 2D prostate cell cultures (PCCs) from PCa patients to express epithelial and cancer markers. Early PCCs were formed by epithelial cells that were progressively replaced with the fibroblast-like cells. Early PCCs contained tissue-specific stem cells that could grow in a 3D culture and form PDOs similar to those produced from the prostate tissue. Early PCCs and PDOs derived from the tissues of PCa patients expressed prostate basal and luminal epithelial markers, as well as cancer markers AMACR, TMPRSS2-ERG, and EZH2, the latter being a promising candidate to mark the transition from the indolent to aggressive PCa. We also identified various TMPRSS2-ERG fusion transcripts in PCCs and PDOs, including new chimeric variants resulting from the intra- and interchromosomal translocations. The results suggest that early PCCs derived from cancerous and normal prostate tissues sustain the phenotype of prostate cells and can be used as a preclinical model to study the pathogenesis of PCa.

Structure and Neuroprotector Properties of a Complex Compound of Lithium with Comenic Acid.

The structure, antioxidant and neuroprotective properties of lithium comenate (lithium 5-hydroxy-4-oxo-4H-pyran-2-carboxylate) were studied. Lithium comenate was obtained by reacting comenic acid (H2Com) with lithium hydroxide in an aqueous solution. The structure of lithium comenate was confirmed via thermal analysis, mass spectrometry, IR, NMR and UV spectroscopy. The crystal structure was studied in detail via X-ray diffraction. The compound crystallized in a non-centrosymmetric space group of symmetry of the orthorhombic system Pna21 in the form of a hydrate, with three water molecules entering the first coordination sphere of the cation Li+ and one molecule forming a second environment through non-valent contacts. The gross formula of the complex compound was established [Li(HCom)(H2O)3]·H2O. It has been established that lithium comenate has a pronounced neuroprotective activity under the excitotoxic effect of glutamate, increasing the survival rate of cultured rat cerebellar neurons more than two-fold. It has also been found that the pre-stress use of lithium comenate at doses of 1 and 2 mg/kg has an antioxidant effect, which is manifested in a decrease in oxidative damage to the brain tissues of mice subjected to immobilization stress. Based on the data available in the literature, we believe that the high neuroprotective and antioxidant efficacy of lithium comenate is a consequence of the mutual potentiation of the pharmacological effects of lithium and comenic acid.

Synthesis and Study of Palladium Mono- and Bimetallic (with Ag and Pt) Nanoparticles in Catalytic and Membrane Hydrogen Processes.

A controlled strategy for the electrochemical synthesis of mono- and bimetallic nanoparticles with a unique and complex morphology has been developed. The investigation of the effect of changing the surfactant concentration and current density regulating the medium pH has revealed the fundamental patterns of nanoparticle growth. The developed method has allowed to synthesis of nanoparticles with a controlled pentabranched structure for the monometallic palladium as well as for favorable combinations of metals-Pd-Ag and Pd-Pt. The obtained nanoparticles were investigated in alkaline methanol oxidation. The results demonstrated quite high catalytic activity up to 83.51 mA cm-2 and long-term stability, which are caused by the increase in electrochemically active surface area by increasing the active center's number. This was made possible due to the creation of unusual nanoparticle morphology, namely the presence of high-energy high-index facets. The developed nanoparticles were also studied as a modifying coating for hydrogen-permeable membranes in the processes of hydrogen transport. The membranes coated with the nanoparticles demonstrated sufficiently high hydrogen flux up to 11.33 mmol s-1 m-2 and high H2/N2 selectivity up to 2254. Such results can be explained by the obvious acceleration of surface processes through the application of the developed nanoparticles. The novel synthesis strategy can potentially be extended to other metal nanoparticle systems. Thus it can be an effective way to solve relevant problems of design of controlled synthetic methods allowing the nanoparticle morphology tuning according to the required functional properties.

Influence of Single Deuterium Replacement on Frequency of Hydrogen Bond Dissociation in IFNA17 under the Highest Critical Energy Range.

The effect of single substitutions of protium for deuterium in hydrogen bonds between pairs of nitrogenous bases on the open states occurrence probability at high critical breaking energies of these bonds has been studied. The study was carried out using numerical methods based on the angular mathematical model of DNA. The IFNA17 gene was divided into three approximately equal parts. A comparison of the open states occurrence probability in these parts of the gene was done. To improve the accuracy of the results, a special data processing algorithm was developed. The developed methods have shown their suitability for taking into account the occurrence of open states in the entire range of high critical energies. It has been established that single 2H/1H substitutions in certain nitrogenous bases can be a mechanism for maintaining the vital activity of IFNA17 under critical conditions. In general, the developed method of the mathematical modeling provide unprecedented insight into the DNA behavior under the highest critical energy range, which greatly expands scientific understanding of nucleobases interaction.

Genesis of Open States Zones in a DNA Molecule Depends on the Localization and Value of the Torque.

The formation and dynamics of the open states in a double-stranded DNA molecule are largely determined by its mechanical parameters. The main one is the torque. However, the experimental study of DNA dynamics and the occurrence of open states is limited by the spatial resolution of available biophysical instruments. Therefore, in this work, on the basis of a mechanical mathematical model of DNA, calculations of the torque effect on the process of occurrence and dynamics of open states were carried out for the interferon alpha 17 gene. It was shown that torsion action leads to the occurrence of rotational movements of nitrogenous bases. This influence is nonlinear, and an increase in the amplitude of the torsion action does not lead to an automatic increase in the amplitude of rotational movements and an increase in the zones' open states. Calculations with a constant torsion moment demonstrate that open states zones are more often formed at the boundaries of the gen and in regions with a predominance of A-T pairs. It is shown, that for the occurrence of open states in the part of the gene that contains a small number of A-T pairs, a large amount of torque is required. When the torque is applied to a certain region of the gene, the probability of the formation of the open state depends on the content of A-T pairs in this region, the size of this region, and on the exposure time. For this mathematical model, open states zones can be closed when the torsion action stops. The simulation results showed that the values of the torsion moment required for the appearance of open states zones, in some cases, are close to experimentally measured (13-15 pN·nm).

Changes in Number and Antibacterial Activity of Silver Nanoparticles on the Surface of Suture Materials during Cyclic Freezing.

This article presents the results of the 10-fold cyclic freezing (-37.0 °C) and thawing (0.0 °C) effect on the number and size range of silver nanoparticles (AgNPs). AgNPs were obtained by the cavitation-diffusion photochemical reduction method and their sorption on the fiber surface of various suture materials, perlon, silk, and catgut, was studied. The distribution of nanoparticles of different diameters before and after the application of the cyclic freezing/thawing processes for each type of fibers studied was determined using electron microscopy. In general, the present study demonstrates the effectiveness of using the technique of 10-fold cyclic freezing. It is applicable to increase the absolute amount of AgNPs on the surface of the suture material with a simultaneous decrease in the size dispersion. It was also found that the application of the developed technique leads to the overwhelming predominance of nanoparticles with 1 to 15 nm diameter on all the investigated fibers. In addition, it was shown that after the application of the freeze/thaw method, the antibacterial activity of silk and catgut suture materials with AgNPs was significantly higher than before their treatment by cyclic freezing.

Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter.

The retinoblastoma gene product (pRb) is a chromatin-associated protein that can either suppress or promote activity of key regulators of tissue-specific differentiation. We found that twelve weeks after transfection of the exogenous active (ΔB/X and Δр34) or inactive (ΔS/N) forms of RB into the 10T1/2 mesenchymal stem cells and clonal selection not a single cell line did contain exogenous RB, despite being G-418 resistant. However, the consequences of the transient production of exogenous RB had different effects on the cell fate. The ΔB/X and Δр34 cells transfected with active form of RB showed elevated levels of inducible adipocyte differentiation (AD). On the contrary, the ΔS/N cells transfected with inactive RB mutant were insensitive to induction of AD associated with abolishing of expression of the PPARγ2. Additionally, the PPARγ2 promoter in undifferentiated ΔS/N cells was hypermethylated, but all except -60 position CpG became mostly demethylated after cells exposure to AD. We conclude that while transient expression of inactive exogenous RB induces long term epigenetic alterations that prevent adipogenesis, production of active exogenous RBs results in an AD-promoting epigenetic state. These results indicate that pRb is involved in the establishment of hereditary epigenetic memory at least by creating a methylation pattern of PPARγ2.

Influence of Environmental Parameters on the Stability of the DNA Molecule.

Fluctuations in viscosity within the cell nucleus have wide limits. When a DNA molecule passes from the region of high viscosity values to the region of low values, open states, denaturation bubbles, and unweaving of DNA strands can occur. Stabilization of the molecule is provided by energy dissipation-dissipation due to interaction with the environment. Separate sections of a DNA molecule in a twisted state can experience supercoiling stress, which, among other things, is due to complex entropic effects caused by interaction with a solvent. In this work, based on the numerical solution of a mechanical mathematical model for the interferon alpha 17 gene and a fragment of the Drosophila gene, an analysis of the external environment viscosity influence on the dynamics of the DNA molecule and its stability was carried out. It has been shown that an increase in viscosity leads to a rapid stabilization of the angular vibrations of nitrogenous bases, while a decrease in viscosity changes the dynamics of DNA: the rate of change in the angular deviations of nitrogenous bases increases and the angular deformations of the DNA strands increase at each moment of time. These processes lead to DNA instability, which increases with time. Thus, the paper considers the influence of the external environment viscosity on the dissipation of the DNA nitrogenous bases' vibrational motion energy. Additionally, the study on the basis of the described model of the molecular dynamics of physiological processes at different indicators of the rheological behavior of nucleoplasm will allow a deeper understanding of the processes of nonequilibrium physics of an active substance in a living cell to be obtained.

DNA Dynamics under Periodic Force Effects.

The sensitivity of DNA to electromagnetic radiation in different ranges differs depending on various factors. The aim of this study was to examine the molecular dynamics of DNA under the influence of external periodic influences with different frequencies. In the present paper, within the framework of a mechanical model without simplifications, we investigated the effect of various frequencies of external periodic action in the range from 1011 s-1 to 108 s-1 on the dynamics of a DNA molecule. It was shown that under the influence of an external periodic force, a DNA molecule can perform oscillatory movements with a specific frequency characteristic of this molecule, which differs from the frequency of the external influence ω. It was found that the frequency of such specific vibrations of a DNA molecule depends on the sequence of nucleotides. Using the developed mathematical model describing the rotational motion of the nitrogenous bases around the sugar-phosphate chain, it is possible to calculate the frequency and amplitude of the oscillations of an individual DNA area. Such calculations can find application in the field of molecular nanotechnology.

Electrophysiological Activity and Survival Rate of Rats Nervous Tissue Cells Depends on D/H Isotopic Composition of Medium.

The deuterium content modification in an organism has a neuroprotective effect during the hypoxia model, affecting anxiety, memory and stress resistance. The aim of this work was to elucidate the possible mechanisms of the medium D/H composition modification on nerve cells. We studied the effect of an incubation medium with a 50 ppm deuterium content compared to a medium with 150 ppm on: (1) the activity of Wistar rats' hippocampus CA1 field neurons, (2) the level of cultured cerebellar neuron death during glucose deprivation and temperature stress, (3) mitochondrial membrane potential (MMP) and the generation of reactive oxygen species in cultures of cerebellar neurons. The results of the analysis showed that the incubation of hippocampal sections in a medium with a 50 ppm deuterium reduced the amplitude of the pop-spike. The restoration of neuron activity was observed when sections were returned to the incubation medium with a 150 ppm deuterium content. An environment with a 50 ppm deuterium did not significantly affect the level of reactive oxygen species in neuron cultures, while MMP decreased by 16-20%. In experiments with glucose deprivation and temperature stress, the medium with 50 ppm increased the death of neurons. Thus, a short exposure of nerve cells in the medium with 50 ppm deuterium acts as an additional stressful factor, which is possibly associated with the violation of the cell energy balance. The decrease in the mitochondrial membrane potential, which is known to be associated with ATP synthesis, indicates that this effect may be associated with the cell energy imbalance. The decrease in the activity of the CA1 field hippocampal neurons may reflect reversible adaptive changes in the operation of fast-reacting ion channels.

Reduction of Deuterium Level Supports Resistance of Neurons to Glucose Deprivation and Hypoxia: Study in Cultures of Neurons and on Animals.

The effect of a reduced deuterium (D) content in the incubation medium on the survival of cultured neurons in vitro and under glucose deprivation was studied. In addition, we studied the effect of a decrease in the deuterium content in the rat brain on oxidative processes in the nervous tissue, its antioxidant protection, and training of rats in the T-shaped maze test under hypoxic conditions. For experiments with cultures of neurons, 7-8-day cultures of cerebellar neurons were used. Determination of the rate of neuronal death in cultures was carried out using propidium iodide. Acute hypoxia with hypercapnia was simulated in rats by placing them in sealed vessels with a capacity of 1 L. The effect on oxidative processes in brain tissues was assessed by changes in the level of free radical oxidation and malondialdehyde. The effect on the antioxidant system of the brain was assessed by the activity of catalase. The study in the T-maze was carried out in accordance with the generally accepted methodology, the skill of alternating right-sided and left-sided loops on positive reinforcement was developed. This work has shown that a decrease in the deuterium content in the incubation medium to a level of -357‱ has a neuroprotective effect, increasing the survival rate of cultured neurons under glucose deprivation. When exposed to hypoxia, a preliminary decrease in the deuterium content in the rat brain to -261‱ prevents the development of oxidative stress in their nervous tissue and preserves the learning ability of animals in the T-shaped maze test at the level of the control group. A similar protective effect during the modification of the 2H/1H internal environment of the body by the consumption of DDW can potentially be used for the prevention of pathological conditions associated with the development of oxidative stress with damage to the central nervous system.

Gas-Transport Characteristics of PdCu-Nb-PdCu Membranes Modified with Nanostructured Palladium Coating.

A method for obtaining composite gas-diffusion PdCu-Nb-PdCu membranes modified with a nanostructured crystalline coating was developed to increase the performance of Nb-based membranes. A modifying functional layer with a controlled size and composition was synthesized by electrochemical deposition, which made it possible to determine a certain geometric shape for palladium nanocrystallites. Developed PdCu-Nb-PdCu membranes have demonstrated flux values up to 0.232 mmol s-1 m-2 in the processes of diffusion purification of hydrogen at 400 °C. A very significant difference in the hydrogen fluxes through the modified and non-modified composite PdCu-Nb-PdCu membranes reached 1.73 times at the lower threshold temperature of 300 °C. Cu doping of protective layer did not affect the selective properties of the membranes, which was confirmed by the obtained high selectivity values up to 1323, and made it possible to reduce the noble metal content. The research data indicate that the modification of the membrane surface significantly accelerates the hydrogen transfer process at sufficiently low temperatures due to the acceleration of dissociative-associative processes on the surface. The reported approach demonstrates new possibilities for creating productive and cost-efficient membranes based on niobium.

Synthesis, Electrocatalytic and Gas Transport Characteristics of Pentagonally Structured Star-Shaped Nanocrystallites of Pd-Ag.

The method of synthesis of bimetallic Pd-Ag pentagonally structured catalyst "nanostar" on the surface of Pd-23%Ag alloy films has been developed. The resulting catalyst was studied as a highly active functional layer for methanol oxidation reaction (MOR) in alkaline media and the intensification of hydrogen transport through the Pd-23%Ag membrane in the processes of hydrogen diffusion purification. A modifying layer with a controlled size, composition and excellent electrocatalytic activity was synthesized by electrochemical deposition at a reduced current density compared to classical methods. The low deposition rate affects the formation of pentagonally structured nanocrystallites, allowing Pd and Ag particles to form well-defined structures due to the properties of the surfactant used. Electrochemical studies have demonstrated that the catalyst synthesized by the "nanostar" method shows better electrocatalytic activity in relation to MOR and demonstrates a higher peak current (up to 17.82 µA cm-2) in comparison with one for the catalyst synthesized by the "nanoparticle" method (up to 10.66 µA cm-2) in a cyclic voltammetric study. The nanostar catalyst electrode releases the highest current density (0.25 µA cm-2) for MOR and demonstrates higher catalytic activity for the oxidation of possible intermediates such as sodium formate in MOR. In the processes of diffusion membrane purification of hydrogen, a multiple increase in the density of the penetrating flux of hydrogen through the membranes modified by the "nanostar" catalyst (up to 10.6 mmol s-1 m-2) was demonstrated in comparison with the membranes modified by the "nanoparticles" method (up to 4.49 mmol s-1 m-2). Research data may indicate that the properties of the developed pentagonally structured catalyst "nanostar" and its enhanced activity with respect to reactions involving hydrogen increase the desorption activity of the membrane, which ultimately accelerates the overall stepwise transfer of hydrogen across the membrane.

Inequality in the Frequency of the Open States Occurrence Depends on Single 2H/1H Replacement in DNA.

In the present study, the effect of 2H/1H isotopic exchange in hydrogen bonds between nitrogenous base pairs on occurrence and open states zones dynamics is investigated. These processes are studied using mathematical modeling, taking into account the number of open states between base pairs. The calculations of the probability of occurrence of open states in different parts of the gene were done depending on the localization of the deuterium atom. The mathematical modeling study demonstrated significant inequality (dependent on single 2H/1H replacement in DNA) among three parts of the gene similar in length of the frequency of occurrence of the open states. In this paper, the new convenient approach of the analysis of the abnormal frequency of open states in different parts of the gene encoding interferon alpha 17 was presented, which took into account both rising and decreasing of them that allowed to make a prediction of the functional instability of the specific DNA regions. One advantage of the new algorithm is diminishing the number of both false positive and false negative results in data filtered by this approach compared to the pure fractile methods, such as deciles or quartiles.

Deuterium-Depleted Water Influence on the Isotope 2H/1H Regulation in Body and Individual Adaptation.

This review article presents data about the influence of deuterium-depleted water (DDW) on biological systems. It is known that the isotope abundances of natural and bottled waters are variable worldwide. That is why different drinking rations lead to changes of stable isotopes content in body water fluxes in human and animal organisms. Also, intracellular water isotope ratios in living systems depends on metabolic activity and food consumption. We found the 2H/1H gradient in human fluids (δ2H saliva >> δ2H blood plasma > δ2Hbreast milk), which decreases significantly during DDW intake. Moreover, DDW induces several important biological effects in organism (antioxidant, metabolic detoxification, anticancer, rejuvenation, behavior, etc.). Changing the isotope 2H/1H gradient from "2H blood plasma > δ2H visceral organs" to "δ2H blood plasma << δ2H visceral organs" via DDW drinking increases individual adaptation by isotopic shock. The other possible mechanisms of long-term adaptation is DDW influence on the growth rate of cells, enzyme activity and cellular energetics (e.g., stimulation of the mitochondrion activity). In addition, DDW reduces the number of single-stranded DNA breaks and modifies the miRNA profile.

Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program.

CCR2 is the cognate receptor to the chemokine CCL2. CCR2⁻CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2⁻CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments.

DNA methylation of the Oct4A enhancers in embryonal carcinoma cells after etoposide treatment is associated with alternative splicing and altered pluripotency in reversibly senescent cells.

The epigenetic mechanisms underlying chemoresistance in cancer cells resulting from drug-induced reversible senescence are poorly understood. Chemoresistant ESC-like embryonal carcinoma PA1 cells treated with etoposide (ETO) were previously found to undergo prolonged G2 arrest with transient p53-dependent upregulation of opposing fate regulators, p21CIP1 (senescence) and OCT4A (self-renewal). Here we report on the analysis of the DNA methylation state of the distal enhancer (DE) and proximal enhancer (PE) of the Oct4A gene during this dual response. When compared to non-treated controls the methylation level increased from 1.3% to 12.5% and from 3% to 19.4%, in the DE and PE respectively. It included CpG and non-CpG methylation, which was not chaotic but presented two patterns in each enhancer. Discorrelating with methylation of enhancers, the transcription of Oct4A increased, however, a strong expression of the splicing form Oct4B was also induced, along with down-regulation of the Oct4A partners of in the pluripotency/self-renewal network Sox2 and Lin28. WB demonstrated disjoining of the OCT4A protein from the chromatin-bound fraction. In survival clones, methylation of the DE was considerably erased, while some remnant of methylation of the PE was still observed. The alternative splicing for Oct4B was reduced, Oct4A level insignificantly decreased, while the expression of Sox2 and Lin28 recovered, all three became proportionally above the control. These findings indicate the involvement of the transient patterned methylation of the Oct4A enhancers and alternative splicing in the adaptive regulation of cell fate choice during the p53-dependant dual state of reversible senescence in ESC-like cancer stem cells.