AFM imaging of the transcriptionally active chromatin in mammalian cells' nuclei.

BACKGROUND: Nuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. METHODS: Isolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM). RESULTS: Nuclei of the normal fibroblast cells were completely flattened under mechanical stress, whereas nuclei of the cancerous HeLa were extremely resistant. In the deformed HeLa nuclei, AFM revealed a highly-branched landscape assembled of ~400 nm closed-packed globules and their structure was changing in response to external influence. Normal and cancerous cells' isolated nuclei were strikingly different by DNA resistance to applied mechanical stress. Paradoxically, more transcriptionally active and less optically dense chromatin of the nuclei of the cancerous cells demonstrated higher physical rigidity. A high concentration of the transcription inhibitor actinomycin D led to complete flattening of HeLa nuclei, that might be related to the relaxation of supercoiled DNA tending to deformation. At a low concentration of actinomycin D, we observed the intermediary formation of stochastically distributed nanoloops and nanofilaments with different shapes but constant width ~ 180 nm. We related this phenomenon with partial DNA relaxation, while non-relaxed DNA still remained rigid. CONCLUSIONS: The resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM. GENERAL SIGNIFICANCE: A new outlook to studying internal nuclei structure is proposed.

Observation of nucleic acid and protein correlation in chromatin of HeLa nuclei using small-angle neutron scattering with D_{2}O-H_{2}O contrast variation.

The small-angle neutron scattering (SANS) on HeLa nuclei demonstrates the bifractal nature of the chromatin structural organization. The border line between two fractal structures is detected as a crossover point at Q_{c}≈4×10^{-2}nm^{-1} in the momentum transfer dependence Q^{-D}. The use of contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals clear similarity in the large scale structural organizations of nucleic acids (NA) and proteins. Both NA and protein structures have a mass fractal arrangement with the fractal dimension of D≈2.5 at scales smaller than 150 nm down to 20 nm. Both NA and proteins show a logarithmic fractal behavior with D≈3 at scales larger than 150 nm up to 6000 nm. The combined analysis of the SANS and atomic force microscopy data allows one to conclude that chromatin and its constitutes (DNA and proteins) are characterized as soft, densely packed, logarithmic fractals on the large scale and as rigid, loosely packed, mass fractals on the smaller scale. The comparison of the partial cross sections from NA and proteins with one from chromatin as a whole demonstrates spatial correlation of two chromatin's components in the range up to 900 nm. Thus chromatin in HeLa nuclei is built as the unified structure of the NA and proteins entwined through each other. Correlation between two components is lost upon scale increases toward 6000 nm. The structural features at the large scale, probably, provide nuclei with the flexibility and chromatin-free space to build supercorrelations on the distance of 10^{3} nm resembling cycle cell activity, such as an appearance of nucleoli and a DNA replication.

Switch of fractal properties of DNA in chicken erythrocytes nuclei by mechanical stress.

The small-angle neutron scattering (SANS) on the chicken erythrocyte nuclei demonstrates the bifractal nature of the chromatin structural organization. Use of the contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals the differences in the DNA and protein arrangements inside the chromatin substance. It is the DNA that serves as a framework that constitutes the bifractal behavior showing the mass fractal properties with D=2.22 at a smaller scale and the logarithmic fractal behavior with D≈3 at a larger scale. The protein spatial organization shows the mass fractal properties with D≈2.34 throughout the whole nucleus. The borderline between two fractal levels can be significantly shifted toward smaller scales by centrifugation of the nuclei disposed on the dry substrate, since nuclei suffer from mechanical stress transforming them to a disklike shape. The height of this disk measured by atomic force microscopy (AFM) coincides closely with the fractal borderline, thus characterizing two types of the chromatin with the soft (at larger scale) and rigid (at smaller scale) properties. The combined SANS and AFM measurements demonstrate the stress induced switch of the DNA fractal properties from the rigid, but loosely packed, mass fractal to the soft, but densely packed, logarithmic fractal.

Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase.

CP is a copper-containing ferroxidase of blood plasma, which acts as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by MPO, such as HOCl, HOBr, and HOSCN, on its spectral, enzymatic, and anti-inflammatory properties was studied. We monitored the chemiluminescence of lucigenin and luminol along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by PMA or fMLP. Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr, and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced, along with a drop in the ferroxidase and amino oxidase activities of CP. Likewise, its inhibitory effect on the halogenating activity of MPO was diminished. Sera of either healthy donors or patients with Wilson disease were co-incubated with neutrophils from healthy volunteers. In these experiments, we observed an inverse relationship between the content of CP in sera and the rate of H2O2 production by activated neutrophils. In conclusion, CP is likely to play a role of an anti-inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO-mediated oxidative modifications.

[Quaternary structures of human cytoplasmic and nuclear PCNA are the same]. / Chetvertichnaia struktura iadernogo antigena deliashchikhsia kletok (PCNA) v tsitoplazme i iadre kletok cheloveka organizovana odinakovo.

Properties and mechanisms of PCNA (proliferating cell nuclear antigen) functions have been investigated for a long time and are studied in great detail. As follows from its name, most known PCNA functions (DNA replication, DNA repair, DNA recombination and others) are connected with cell proliferation and localization of this protein in nuclei. In addition, there is good reason to believe that PCNA also performs some functions in the cytoplasm. However, the possible role and mechanisms of PCNA action in the cytoplasm require careful study and clarification. Interestingly, such cells as neutrophils differ in that they are non-dividing on one hand and on the other hand contain a rather large amount of PCNA, which is localized only in the cytoplasm, that is, they are an ideal model for the study of cytoplasmic PCNA. Using cross-linkages with formaldehyde, we showed that this cytoplasmic PCNA is cross-linked in a similar way, that is, organized in the same way as the nuclear PCNA that is present in the proliferating cells. Previously, we showed that PCNA in such cells is organized into a dynamic complex of double trimer on the basis of the back-to-back principle (Naryzhny S.N. et al. (2005) J. Biol. Chem., 280, 13888). Apparently, such organization of this hub-protein allows it to better coordinate the processes taking place in the cytoplasm as well.

Additive scaling law for structural organization of chromatin in chicken erythrocyte nuclei.

Small-angle neutron scattering (SANS) on nuclei of chicken erythrocytes demonstrates the cubic dependence of the scattering intensity Q^{-3} in the range of momentum transfer Q∈10^{-3}-10^{-2}nm^{-1}. Independent spin-echo SANS measurements give the spin-echo function, which is well described by the exponential law in a range of sizes (3×10^{2})-(3×10^{4}) nm. Both experimental dependences reflect the nature of the structural organization of chromatin in the nucleus of a living cell, which corresponds to the correlation function γ(r)=ln(ξ/r) for r<ξ, where ξ=(3.69±0.07)×10^{3} nm, the size of the nucleus. It has the specific scaling property of the logarithmic fractal γ(r/a)=γ(r)+ln(a), i.e., the scaling down by a gives an additive constant to the correlation function, which distinguishes it from the mass fractal, which is characterized by multiplicative constant.

AGGREGATION BY LECTIN-METHODICAL APPROACH FOR EFFECTIVE ISOLATION OF EXOSOMES FROM CELL CULTURE SUPERNATANT FOR PROTEOME PROFILING.

Exosomes are small membrane vesicles secreted by most cell types in vivo and in vitro. Exosomes are found in cell culture media, blood, urine, amniotic fluid, malignant ascite fluids and contain distinct subsets of microRNAs and proteins depending upon the tissue from which they are secreted. Thus exosomes constitute potential biomarkers of human diseases, such as cancer. A major bottleneck in the development of exosome-based diagnostic assays is the challenging purification of these vesicles; this requires time-consuming and instrument-based procedures. Isolation of exosomes can be a tedious, non-specific, and difficult process. Here, we provide a preparative technique for isolation of exosomes based on their ability to aggregate in the presence of lectins. The new method for lectin-based isolation of exosomes from cell culture media was developed as a sample preparation step for exosome-based protein biomarker research.

THE ANALYSIS OF LGI1 GENE EPIGENETIC ALTERATIONS BY MEANS OF POSTTRANSLATIONAL H3 HISTONE MODIFICATIONS IN MALIGNANT GLIOMAS.

Although there is a progress in understanding the causes and consequences of genetic and epigenetic changes in glioma malignant transformation, many details remain obscure and need further investigation. It is known that process of malignant transformation of gliomas is accompanied by gradual loss of LGI1 gene expression. However, genetic defects causing LGI1 inactivation have not been revealed. In this paper, we have analyzed the LGI1 gene expression in primary cultures of malignant gliomas, and compared these data with epigenetic indicators of transcriptional activity ­ posttranslational H3 histone modifications. We have show the presence of an epigenetic marker of gene repression H3K9me3 near the site of LGI1 transcription initiation in most (5 from 6) studied gliomas. There was not LGI1 expression in these gliomas. Only one glioma showed LGI1 expression, and in this glioma there was no association of LGI1gene with H3K9me3 modification. Thus, we are the first to show a correlation between LGI1 gene expression and the epigenetic indicator H3K9met3 in malignant gliomas. Marker of actively transcribed chromatin Í3K4àñ have not been found in this area of the genome. The data obtained strongly suggest the possibility of gene LGL1 inactivation by epigenetic mechanism: modified «histone code¼.

[Histone deacetylase inhibitors cause the TP53-dependent induction of p21/Waf1 in tumor cells carrying mutations in TP53].

p21/Waf1 protein is one of the main cell cycle arrest regulators and one of the most well-known transcriptional targets of TP53 protein. Here, we demonstrated the activation of expression of the p21/Waf1 gene when the cells were treated to sodium butyrate (NaBu)--one of the natural inhibitors of deacetylase, and investigated whether this phenomenon depends on the presence of functionally active TP53 protein. We compared the effect of the NaBu treatment on the human cell line with different TP53 mutation profile, including: wild-type TP53, single nucleotide substitutions, and the complete absence of TP53 gene. NaBu activated the TP53 protein via hyper acetylation at lysine residue K382, without significant changes in the level of protein expression. Western blotting demonstrated that the addition of NaBu triggers a significant increase in the p21/Waf1 protein level in both the TP53 wild-type cells and in the cells with single nucleotide substitutions in the domain responsible for the binding of TP53 protein to DNA. At the same time, no the p21/Waf1 protein induction was observed in the cells with complete deletion of the TP53 gene. However, NaBu was not able to induce the p2 1/Waf1 production when the expression of TP53 was transiently knocked down by the p53 siRNA. Overall, our results suggest that the NaBu-dependent induction of p21/Waf1 does require the presence of TP53 protein but unexpectedly it can occur regardless of mutational changes in the domain responsible for the TP53 binding to DNA. One of the hypothetical explanations is that NaBu increases the level of TP53 acetylation, and the modified protein is able to establish a new network of protein-protein interactions or trigger some conformational changes affecting the TP53-dependent transcriptional machinery even when its DNA binding ability is impaired.

[Development of barcode and proteome profiling of glioblastoma].

High grade glioma (glioblastoma) is the most common brain tumor. Its malignancy makes it the fourth biggest cause of cancer death. In our experiments we used several glioblastoma cell lines generated in our laboratory to obtain proteomics information specific for this disease. This study starts our developing the complete 2DE map of glioblastoma proteins. 2DE separation with following imaging, immunochemistry, spot picking, and mass-spectrometry allowed us detecting and identifying more than 100 proteins. Several of them have prominent differences in their level between norm and cancer. Among them are alpha-enolase (ENOA_HUMAN), pyruvate kinase isozymes M1/M2 (KPYM_HUMAN), cofilin 1 (COF1_HUMAN), translationally-controlled tumor protein TCTP_HUMAN, annexin 1 (ANXA1_HUMAN), PCNA (PCNA_HUMAN), p53 (TP53_HUMAN) and others. Most interesting results were obtained with protein p53. In all glioblastoma cell lines, its level was dramatically up regulated and enriched by multiple additional isoforms. This distribution is well correlated with presence of these proteins inside of cells themselves. At this initial step we suggest the panel of specific brain tumor markers (signature) to help creating noninvasive techniques to diagnose disease. These preliminary data point to these proteins as promising markers of glioblastoma.

[Exosomal intercellular communication system and its role in the process of metastatic dissemination].

Metabolism and information between cells is the basis of the existence of any multicellular organism. Malfunction of the intercellular communication play an important and sometimes decisive role in the pathogenesis of the most diseases, including cancer. According to traditional views, functional integration of individual cells in tissues, organs and organ systems is mediated by the efficient work of regulatory systems: nervous, immune, endocrine. Over the past few years the attention of scientists is attracted the ability of cells to "communicate" with the help of nanoscale vesicular formations, or so-called exosomes. There are accumulated data that the cells of the most tissues secrete exosomes into the intercellular environment, after which, by means of stream of blood or lymph, exosomes transferred to anatomically distant sites where they are accepted by the other cells. It is showed that the content of exosomes are not random and that vesicular transport may be targeted and to play a significant physiological and even "pathophysiological" role. The aim of this review is the analysis and integration of modern scientific data on the role of exosomes in the process of tumor progression and presentation of possible ways and methods of using these data in the practice of oncology.

[Aberrant expression of sox2 gene in malignant gliomas].

Both genetic and epigenetic changes underlite the mechanisms of tumor initiation and progression. In the present study we analyze sox2 gene expression and its epigenetic regulation in primary cultures of malignant gliomas. The sox2 expression was detected in the vast majority (74%) of the investigated gliomas and absent in morphologically normal brain tissue. This indicates the process of glioma malignant transformation. We have also shown that the association of different areas of the sox2 gene with important epigenetic markers, posttranslational modifications of H3 histone H3K4ac and H3K9met3, does not correlate with the sox2 expression. However, this may indicate the stochastic nature of the regulation of sox2 gene expression in malignant gliomas.

[Genetic and epigenetic markers of gliomas].

Malignant gliomas, aggressive and highly invasive tumors with a great number of genetic and epigenetic alterations in genes involved in the cell cycle regulation, the apoptotic pathways, cell invasion ability, and angiogenesis, are considered to be among the deadliest of human cancers. The role of epigenetic mechanisms in the pathogenesis of malignant transformation despite recent progress is not yet clear elucidated and remains under intensive study. This review describes the mechanisms of epigenetic regulation of gene expression, including post-translational modification of histones, DNA methylation in the promoter regions, and microRNA regulation. The genetic and epigenetic factors driving the pathogenesis of gliomas in their possible mutual influence and the potential epigenetic targets that can be used in the development of diagnostics and new therapeutic approaches are also discussed.

[Current insights into chromatin structure organization].

This review summarizes current insights into organization of chromatin structure at different levels of DNA compaction. Analysis of available experimental data allowed concluding that only nucleosomal level of structural organization was sufficiently investigated, whereas structure of a 30-nm chromatin fiber remains an open issue. The data on the chromatin structure obtained at the level of the nucleus speak in favor of a biphasic fractal organization of chromatin.

[Possibilities of epigenetic anti-tumor therapy in in-vitro models].

Research during the past decade has shown that epigenetic events have a key role in carcinogenesis and tumour progression. Histone deacetylase inhibitors (HDACi) comprise structurally diverse compounds that are a group of targeted epigenetic anticancer agents. Here we explored the in vitro efficacy of HDACi such as sodium butyrate (BuNa), valproic acid (VaNa) and several novel HDAC inhibitors for the treatment of cancer. Both BuNa and VaNa inhibited cancer cell proliferation in a time--and dose-dependent fashion. In the present study we demonstrated the significant effect of two novel HDACi, Adipo or BuNHOH, able to induce apoptosis of cancer cells, but not of normal line. Since HDAC inhibitors have been proposed as radio--or chemosensitizers in cancer therapy, we have studied the radiosensitizing effect of sodium butyrate on cancer cells. The combination of BuNa and radiation significantly inhibited tumor cell growth. Besides, combining Cisplatin or Gemzar with HDAC inhibitors results in synergistic antiproliferative activity that could be therapeutically exploited. These results suggest that HDACi acts as an antitumor agent and that combining HDAC inhibitors with radio or--chemotherapeutic strategy may provide a novel chemotherapeutic treatment of cancers insensitive to traditional antitumor agents.

[P73 gene expression in gliomas].

We propose an RT-PCR primer system allowing a definite mRNA identification for different p73 (p53 homologue) isoforms identification. Using the system proposed the p73 expression was studied in 18 glioma samples (cell lines, biopsy samples, primary cultures). Most of the samples reveal no p73 expression or it is expressed simultaneously with its isoforms shown to suppress its activity. This expression pattern predisposes to cancerogenesis.

[Absorption of iodofolic acids by the cells of malignant tumors].

The uptake of 125-iodine labeled 3' iodofolic acid (I*F) and 3' iodo, 5 formyl tetrahydrofolic acid (I*FT) by the cells HeLa, ECV, L-41, human glioma, and rat glioma was studied. Human Embrionic Lung Fibroblasts (HELF) were taken for comparison as healthy cells. It was shown for *IF that its long-term uptake by cells L-41 and ECV is hundreds oftimes higher than those of HELF cells. The short-term uptake phase was studied for *IFT uptake. The dissociation constant was determined for a complex formed by *IFT and an acceptor in the HeLa cells, which is supposed to cause concentrative uptake of *IFT in cells. The dissociation constants of this acceptor complexes with folic acid, 3' iodofolic acid and 3',5'-diiodofolic acid were determined by competition with I*FT. The distribution ratio of *IF and *IFT in tissues of different organs of healthy mice and rats and rats with a sarcoma grafted on his thigh and glioma grafted into the brain was studied. As was shown there are large differences in the concentration of *IF and *IFT in the tumor and in the healthy tissue, *IF concentration in thigh muscle of healthy being 5 times lower than those in tumor grafted to the thigh, and *IFT concentration in healthy brain being 10 times lower than in brain tumor.

[Study of chromatin decondensation factors in human spermatozoids by flow cytometry].

To date, the mechanisms responsible for radical change of chromatin structure in male germ cells during fertilization are unclear. Evidence suggesting the existence of proteolytic nuclear enzymes in mature human spermatozoids are presented in this work. The possible role of these previously unknown proteases in decondensation of chromatin of spermatozoids in a fertilized ovum is discussed. Application of the flow cytometry technique has shown that treatment of human spermatozoid nuclei with SH-reagents leads not only to destruction of disulfide bonds between protamine molecules that is necessary for their effective utilization but also induces specific endogenous proteolytic activity that consequently results in rather fast decondensation of chromatin followed by proteolytic cleavage of nuclear proteins. A chromatin decondensation process can be almost totally blocked by serine protease inhibitors and components of seminal fluid. An original cytochemical approach of binding of fluorescently labeled protease inhibitor to the target of investigation has been used in order to visualize the localization of proteases in male germ cell nuclei. The results of our study suggest that one of the factors of chromatin reorganization involved in the formation of male pronucleus is endogenous nuclear protease of spermatozoids, which is activated by glutathione or other SH-components of ovum cytoplasm.

[Immunological similarity of diphtheria toxin and EGF receptor].

Cardiomyopathy and neuropathy are the two commonly observed complications in diphtheria patients and in, some instances, individuals vaccinated against diphtheria. The nature of these complications remains not well understood. It was suggested that autoimmunity may play a role in the development of these afflictions. Based on functional similarities between diphtheria toxin (DT) and epidermal growth factor receptor (EGFR), which both can bind to the heparin-binding EGF-like growth factor (HB-EGF) precursors, we suggested that antibodies developed against DT can cross react with EGFR. Here, using serum from healthy donors (n = 10) and diphtheria patients (n = 15), we demonstrated that B-subunit of DT has the antigenic epitopes similar to those of EGFR. Diphtheria toxin as well as EGFR could be recognized by antibodies raised against EGFR and by serum antibodies from diphtheria patients. Moreover serum of diphtheria patients competitively inhibits binding of anti-EGFR antibodies to the receptor. The truncated diphtheria toxin without B-subunit could be detected by serum antibodies of diphtheria patients, but not by anti-EGFR antibodies. Collectively, these studies demonstrate cross-reactivity of antibodies raised against B-subunit of DT and extracellular domain of EGFR and suggest that clinically observed post-diphtheria complications may result from autoimmune inhibition of EGFR function and possible destruction of receptor-positive tissues.

[Investigation of exosomes secreted by different normal and malignant cells in vitro and in vivo].

Laser correlation spectroscopy, atomic force microscopy, and immunoaffinity chromatography were used to characterize exosomes produced by different human cells. The exosomes secreted into a culture medium by normal fibroblasts, dendritic cells, lymphocytes, as well as malignant cells obtained from tumors of various tissue origins. The similar investigations were made for exosomes detectable in plasma and cerebrospinal fluid. The dynamic light scattering technique has demonstrated that the exosomes from different sources are homogenous and similar in size of the order of 20 and 90 nm. The exceptional homogeneity of exosomes was confirmed by atomic force microscopy. The immunoaffinity method has shown that all the exosomes under study carry antigenic determinants recognizable by antibodies to the major histocompatibility complex of type 1 (HLA-ABC). A method is proposed for evidence-based detection of exosomes in various biological fluids. For this, dynamic light scattering detects 20- and 90-nm particles and whether they can be removed by immunoaffinity chromatography with HLA-ABC antibodies is checked.