Small-angle neutron and X-ray scattering data for HeLa nuclei with the normal and suppressed transcription activities are reported. Small-angle neutron scattering data demonstrate the presence of a bifractal structure inside a nucleus. The logarithmic fractal structure is observed in the range from the size of the nucleus to several hundreds of nanometer, whereas the volume fractal structure exists at smaller scales down to a nucleosome structure. Small-angle X-ray scattering data show that the presence of the volume fractal structure correlates with the transcription activity of a cell. In view of the successful description of chromatin by the fractal globule model (Hi–C method data), a scenario for the formation of the bifractal structure inside the nucleus has been proposed. A system of transport channels (logarithmic fractal) is located inside close-packed chromatin, whereas active chromatin is localized near transport channels or inside them and forms volume fractal structures due to the transcription activity. Thus, the logarithmic fractal structure ensures the uniform distribution of voids at various scales, which is potentially necessary for the transcription and transport of substances inside the nucleus, whereas the volume fractal structure is due to the transcription activity of the cell.

Background: The gene expression differs in the nuclei of normal and malignant mammalian cells, and transcription is a critical initial step, which defines the difference. The mechanical properties of transcriptionally active chromatin are still poorly understood. Recently we have probed transcriptionally active chromatin of the nuclei subjected to mechanical stress, by Atomic Force Microscopy (AFM) [1]. Nonetheless, a systematic study of the phenomenon is needed. Methods: Nuclei were deformed and studied by AFM. Non-deformed nuclei were studied by fluorescence confocal microscopy. Their transcriptional activity was studied by RNA electrophoresis. Results: The malignant nuclei under the study were stable to deformation and assembled of 100-300 nm beadslike units, while normal cell nuclei were prone to deformation. The difference in stability to deformation of the nuclei correlated with DNA supercoiling, and transcription-depended units were responsive to supercoils breakage. The inhibitors of the topoisomerases I and II disrupted supercoiling and made the malignant nucleus prone to deformation. Cell nuclei treatment with histone deacetylase inhibitors (HDACIs) preserved the mechanical stability of deformed malignant nuclei and, at the same time, made it possible to observe chromatin decondensation up to 20-60 nm units. The AFM results were supplemented with confocal microscopy and RNA electrophoresis data. Conclusions: Self-assembly of transcriptionally active chromatin and its decondensation, driven by DNA supercoiling-dependent rigidity, was visualized by AFM in the mechanically deformed nuclei. General significance: We demonstrated that supercoiled DNA defines the transcription mechanics, and hypothesized the nuclear mechanics in vivo should depend on the chromatin architecture.

The characteristics of six new glioblastoma cell lines obtained from tumor material from patients are presented. The studied glioblastomas do not have mutations in the genes IDH1 and IDH2, which indicated a poor prognosis for their therapy. Two glioblastomas carry the pathogenic mutation p.Arg110Pro in the gene TP53. All cell lines studied express RNA of tumor suppressor and oncogenic isoforms of the p73 protein. The glioblastomas responded differently to radiotherapy, with five of them being more resistant to γ irradiation than the standard A172 glioma line. All six cell lines express RNA genes for vascular endothelial growth factor and its receptor (VEGFR-1) in different ratios. Testing of an immunotherapeutic regimen with monoclonal antibodies to VEGFR-1 on one of the cell lines confirms that the studied glioblastomas are sensitive to blocking vascular growth factor and its receptor. Thus, these glioblastomas can become a promising model for studying the formation of tumor-cell resistance to radiotherapy and the effectiveness of immunotherapy that blocks growth factors and their receptors.

It has been generally accepted that heterochromatin is represented by a regular, dense and closed structure, while euchromatin is open and sparse. Recent evidence indicates that chromatin is comprised of irregular nucleosome clutches compacted within the nucleus. Transcriptional events transform the chromatin architecture, resulting in appearance of 100-300 nm nucleosomal aggregates. Meanwhile, the current paradigm of chromatin architecture is largely fragmented. In this communication, we unraveled chromatin ultrastructure of normal and malignant cell nuclei through mechanical deformation of the nuclei and Atomic Force Microscopy (AFM) analysis of the resulting landscape. In human skin fibroblasts cell nuclei, nanodomains of about 16.5-33.5 nm were revealed. Hierarchical folding of the chromatin of normal nuclei was observed: the nanodomains formed irregular fiber-like structures that coalesced into the macroscale chromatin compartments. In fibrosarcoma cell nuclei DNA supercoiling domains (SDs) of about 66.3-113.0 nm, uniformly distributed within the nuclei, were revealed. Transformation of the morphology of the condensed chromatin domains through up- and downregulation of supercoiling was demonstrated.

The HeLa cell’s nuclei surface landscape, which appeared after the mechanical deformation, was studied by atomic-force microscopy. Nuclei were isolated from HeLa-line cells, and deformed under centrifugal forces, followed by fixation with glutaraldehyde. The resulting landscape was shown to be mainly due to the high chromatin resistance to deformation. This stability correlates with DNA supercoiling. Cells’ treatment with topoisomerase inhibitors I and II leads to the relaxation of supercoiling and significant flattening of the nuclei. On the contrary, cells’ treatment with a DNA intercalator leads to an increase in the DNA rigidity and, as a result, the resistance of chromatin to mechanical deformation. Thus, the observed changes in morphology reflect the functional features of the cell nucleus.

Stochastic gene expression can play an important role in the fate of both each specific cell and the cell population as a whole. In this work, we demonstrate significant variability of p53 expression, up to its complete absence, in human primary glioblastoma cells, which is not observed in cells of normal FLEH fibroblasts and transferable tumour cell lines, such as HeLa and HT1080. At the same time, the variability of p53 expression in cells depends on the density of seeding. We show that when the transcription is suppressed with actinomycin D or the p53 gene is knocked down by RNA interference, the restoration of its expression level in cells proceeds stochastically.

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 similar to 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 similar to 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.

The small-angle neutron scattering (SANS) on the rat lymphocyte nuclei demonstrates the bifractal nature of the chromatin structural organization. The scattering intensity from rat lymphocyte nuclei is described by power law Q^{-D} with fractal dimension approximately 2.3 on smaller scales and 3 on larger scales. The crossover between two fractal structures is detected at momentum transfer near 10^{-1}nm^{-1}. The use of contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals clear similarity in the structural organizations of nucleic acids (NA) and proteins. Both chromatin components show bifractal behavior with logarithmic fractal structure on the large scale and volume fractal with slightly smaller than 2.5 structure on the small scale. Scattering intensities from chromatin, protein component, and NA component demonstrate an extremely extensive range of logarithmic fractal behavior (from 10^{-3} to approximately 10^{-1}nm^{-1}). We compare the fractal arrangement of rat lymphocyte nuclei with that of chicken erythrocytes and the immortal HeLa cell line. We conclude that the bifractal nature of the chromatin arrangement is inherent in the nuclei of all these cells. The details of the fractal arrangement-its range and correlation/interaction between nuclear acids and proteins are specific for different cells and is related to their functionality.

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 Qc approximate to 4x10-2 nm-1 in the momentum transfer dependence Q-D. The use of contrast variation (D2O-H2O) 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 approximate to 2.5 at scales smaller than 150 nm down to 20 nm. Both NA and proteins show a logarithmic fractal behavior with D approximate to 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 103 nm resembling cycle cell activity, such as an appearance of nucleoli and a DNA replication.

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.

In this study, we present a model for visualization of the exosome transfer of p53-GFP protein between cultured mammalian cells. The temporal parameters of the accumulation of fluorescent labeled protein in recipient cells have been analyzed. We employed HEK293 cells transfected with p53∆Y126-GFP plasmid (a GFP clone) as donor of the exosomes, while the original HEK293 cell line was used as a recipient. Our results provide evidence that the transfer of the GFP protein from cell to cell is carried out via exosomes. It has been shown that the accumulation of this protein by the recipient cells in cocultures of these cells takes a prolonged time. The temporal parameters of this transfer differ between the cells within the same population.

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-toback principle. Apparently, such organization of this hub-protein allows it to better coordinate the processes taking place in the cytoplasm as well.

Exosomes are nanosized vesicles that are secreted by many types of cells. We have found that exosomes secreted by HEK293 and HT-1080 can suppress growth and proliferation of p53-deficient cells. Upon overexpression of exogenous p53-GFP in HEK293 cells, we observed p53 protein in exosomes that were secreted by these cells. We also found endogenous p53 in exosomes that were secreted by HT-1080 cells with a higher level of p53 expression. We were able to detect endogenous p53 protein in exosomes that originated from human plasma and were transferred to p53-deficient cells. Our findings indicate that p53 protein can be transferred between cells and may play an important physiological role.

Using the sectional analysis of two-dimensional electrophoretic gels with liquid chromatography-mass spectrometry, proteoform profiles for individual genes expressed in cancer (glioblastoma) and normal (FLEH) cells were obtained. Profiles of more than 5000 genes were analyzed. It turned out that many genes encoding potential biomarkers of glioblastoma are characterized by sets of proteoforms that are different in normal and cancer cells. These proteoforms could be sources of highly specific markers and targets for therapy. Using a section analysis of two-dimensional electrophoretic gels with liquid chromatography by mass spectrometry, proteoform profiles were obtained for individual genes expressed in cancer (glioblastoma) and normal (FLEH) cells. Profiles of more than 5000 genes were analyzed. It turned out that many genes encoding potential biomarkers of glioblastoma are characterized by sets of proteoforms, which are different in normal and cancer cells. These proteoforms could be sources of highly specific markers and targets for therapy.

Abstract. Were researched IgG on the surface cells of different histological types tumors of cerebrum, using fluorescing staphylococcus A-protein. The study of target IgG shows divers intensive of microscopic fluorescent illumination. This results associate related with level amount IgG. The maximum concentrate of surface’s IgG was on the cells of malignant tumors and there was direct correlate with aggressive manner and quickly recurrence of tumor’s growth, and shot survival. The fraction of IgG with specific antitumor’s antibody covers tumor’s antigens has been block this antigens for receptors of T-lymphocytes. Linked with immunological anticell’s deficit phenomenon may be one from famous reasons of malignant clinical type tumor disease. (Med. Immunol., vol. 10, N 6, pp 593-596) .

Abstract. Were researched IgG on the surface cells of different histological types tumors of cerebrum, using fluorescing staphylococcus A-protein. The study of target IgG shows divers intensive of microscopic fluorescent illumination. This results associate related with level amount IgG. The maximum concentrate of surface’s IgG was on the cells of malignant tumors and there was direct correlate with aggressive manner and quickly recurrence of tumor’s growth, and shot survival. The fraction of IgG with specific antitumor’s antibody covers tumor’s antigens has been block this antigens for receptors of T-lymphocytes. Linked with immunological anticell’s deficit phenomenon may be one from famous reasons of malignant clinical type tumor disease. (Med. Immunol., vol. 10, N 6, pp 593-596).

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 to obtain proteomics information specific for this disease. 2DE separation with following imaging, immunochemistry, spot picking, and mass-spectrometry allowed us to detecting more than 600 protein spots and identifying more than 130 of them. Proteome profiles in normal and glioblastoma cell lines are very similar but levels of several proteins have prominent differences between norm and cancer. Among these proteins are alpha-enolase (ENOA_HUMAN), pyruvate kinase 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 about protein p53. Its level was dramatically up-regulated and enriched by multiple additional isoforms in all glioblastoma cell lines. An immunological analysis (Western blot) of three hub-proteins (p53, 14-3-3, PCNA) allowed us to creating the minimal barcode of glioblastoma cell lines. These preliminary data point to this barcode as a promising diagnostic tool for testing of the biological fluids from patients.