BACKGROUND: Tumor targeting of radiotherapy represents a great challenge. The addition of multimodal nanoparticles, such as 3 nm gadolinium-based nanoparticles (GdBNs), has been proposed as a promising strategy to amplify the effects of radiation in tumors and improve diagnostics using the same agents. This singular property named theranostic is a unique advantage of GdBNs. It has been established that the amplification of radiation effects by GdBNs appears due to fast electronic processes. However, the influence of these nanoparticles on cells is not yet understood. In particular, it remains dubious how nanoparticles activated by ionizing radiation interact with cells and their constituents. A crucial question remains open of whether damage to the nucleus is necessary for the radiosensitization exerted by GdBNs (and other nanoparticles). METHODS: We studied the effect of GdBNs on the induction and repair of DNA double-strand breaks (DSBs) in the nuclear DNA of U87 tumor cells irradiated with γ-rays. For this purpose, we used currently the most sensitive method of DSBs detection based on high-resolution confocal fluorescence microscopy coupled with immunodetection of two independent DSBs markers. RESULTS: We show that, in the conditions where GdBNs amplify radiation effects, they remain localized in the cytoplasm, i.e. do not penetrate into the nucleus. In addition, the presence of GdBNs in the cytoplasm neither increases induction of DSBs by γ-rays in the nuclear DNA nor affects their consequent repair. CONCLUSIONS: Our results suggest that the radiosensitization mediated by GdBNs is a cytoplasmic event that is independent of the nuclear DNA breakage, a phenomenon commonly accepted as the explanation of biological radiation effects. Considering our earlier recognized colocalization of GdBNs with the lysosomes and endosomes, we revolutionary hypothesize here about these organelles as potential targets for (some) nanoparticles. If confirmed, this finding of cytoplasmically determined radiosensitization opens new perspectives of using nano-radioenhancers to improve radiotherapy without escalating the risk of pathologies related to genetic damage.

• Keywords
• DNA double-strand breaks, DNA repair, Gadolinium, Nanomedicine, Nanoparticles, Radiosensitization, Radiotherapy, Theranostic,
• MeSH
• DNA Breaks, Double-Stranded drug effects   MeSH
• Gadolinium toxicity   MeSH
• Glioblastoma metabolism   MeSH
• Metal Nanoparticles toxicity   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• DNA Repair drug effects   MeSH
• DNA Damage drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Gadolinium MeSH

We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 μM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 μM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 μM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 μM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.

• MeSH
• Leukemia, Promyelocytic, Acute pathology   MeSH
• Apoptosis drug effects   MeSH
• Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors   MeSH
• HL-60 Cells MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• G2 Phase Cell Cycle Checkpoints drug effects   MeSH
• Humans MeSH
• Morpholines pharmacology   MeSH
• DNA Repair drug effects   MeSH
• Pyrazines pharmacology   MeSH
• Pyrones pharmacology   MeSH
• Sulfones pharmacology   MeSH
• Radiation Tolerance drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one MeSH Browser
• 3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR protein, human MeSH Browser
• Protein Kinase Inhibitors MeSH
• Morpholines MeSH
• Pyrazines MeSH
• Pyrones MeSH
• Sulfones MeSH

In the work presented here, changes in haematopoiesis of mice (B6129SF2/J) were studied 1 year after their whole-body exposure to a dose of 7 Gy (72% of mice survived). The irradiated mice were compared with non-irradiated younger (4 months of age) and older (16 months of age) mice. There was a significant increase in the relative abundance of primitive stem cells with long-term capability of the haematopoiesis recovery lin(-)/Sca-1(+)/CD117(+)/CD34(-) in the bone marrow of mice aged 16 months (irradiated and non-irradiated) compared with those aged 4 months. In terms of the ability to respond to further whole-body irradiation at a dose of 1 Gy, the presence of γH2A.X foci was studied in lin(-) bone marrow cells. There was a considerable number of persisting foci in lin(-) stem cells isolated from the bone marrow of the older irradiated mice. In the blood count from the peripheral blood of the older mice (both non-irradiated and irradiated at 7 Gy), there was a significant increase in granulocytes. In the group exposed to 7 Gy, the numbers of thrombocytes significantly increased, and on the contrary, the numbers of erythrocytes, the amount of haemoglobin, and haematocrit significantly decreased.

• MeSH
• Cell Line MeSH
• Whole-Body Irradiation methods   MeSH
• Erythrocytes metabolism   radiation effects   MeSH
• Granulocytes metabolism   radiation effects   MeSH
• Hematopoietic Stem Cells cytology   metabolism   radiation effects   MeSH
• Hematopoiesis radiation effects   MeSH
• Hemoglobins metabolism   radiation effects   MeSH
• Histones metabolism   MeSH
• Bone Marrow radiation effects   MeSH
• Mice MeSH
• Blood Platelets metabolism   radiation effects   MeSH
• Age Factors MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• gamma-H2AX protein, mouse MeSH Browser
• Hemoglobins MeSH
• Histones MeSH

During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF) and endonuclease G (endoG). Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID). We studied the structure, cellular localization, and interactions of several proteins in silico and also in cells using fluorescent microscopy. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the lipid membranes. Bioinformatic predictions were conducted to analyze the interactions of the studied proteins with each other and with other possible partners. We conducted molecular modeling of proteins with unknown 3D structures. These models were then refined by MolProbity server and employed in molecular docking simulations of interactions. Our results show data acquired using a combination of modern in silico methods and image analysis to understand the localization, interactions and functions of proteins AMID, AIF, endonuclease G, and other apoptosis-related proteins.

• MeSH
• Apoptosis * MeSH
• Models, Biological MeSH
• Endonucleases metabolism   MeSH
• Microscopy, Fluorescence methods   MeSH
• Caspases metabolism   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Models, Molecular MeSH
• Cell Line, Tumor MeSH
• Computer Simulation MeSH
• Proteomics methods   MeSH
• Software MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Computational Biology methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Endonucleases MeSH
• Caspases MeSH

Nuclear locations of the c-myc gene and its transcripts (c-myc (T)) have been investigated in relation to nuclear domains involved in RNA synthesis and processing. Transcription of the c-myc gene appears to be linked to the late G(1)- and preferentially to S-phases of the cell cycle. The c-myc gene and its transcripts were positioned non-randomly within the interphase nucleus; additionally, c-myc RNA signals accumulated at nucleoli. Using oligo-probes, designed to exon II and exon III of the c-myc gene, single c-myc (T) was preferentially observed in human carcinoma HT29 and A549 cells. Conversely, human embryonal teratocarcinoma NTERA cells were characterized by the presence of multiple c-myc RNA signals located in both the nucleoli and nucleoplasm. When accumulated at nucleoli, c-myc (T) occupied the periphery of this organelle, though not those associated with the cultivation surface. In HT29 cells, approximately 80% of c-myc (T) co-localized with the RNAP II positive regions, so-called transcription factories. However, in approximately 20% of the cells with c-myc transcripts, the c-myc (T) was released from the site of synthesis, and was not associated with either transcription factories or SC35 domains. In approximately 60% of nuclei with c-myc (T), these signals were located in close proximity to the SC35 regions, but promyelocytic leukaemia bodies were associated with c-myc (T) only in approximately 20% of the nuclei. Taken together, c-myc RNA signals were positioned in the most internal parts of the cell nuclei preferentially associated with the nucleoli. Specific nuclear and nucleolar positioning probably reflects the kinetics of c-myc RNA metabolism.

• MeSH
• Cell Nucleus genetics   metabolism   ultrastructure   MeSH
• HT29 Cells MeSH
• Gene Expression MeSH
• Transcription, Genetic MeSH
• Genes, myc * MeSH
• Humans MeSH
• Chromosomes, Human, Pair 8 MeSH
• RNA, Messenger metabolism   MeSH
• Tumor Cells, Cultured MeSH
• Proto-Oncogene Proteins c-myc metabolism   MeSH
• RNA Polymerase II metabolism   MeSH
• Tissue Distribution MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Messenger MeSH
• MYC protein, human MeSH Browser
• Proto-Oncogene Proteins c-myc MeSH
• RNA Polymerase II MeSH

Mammalian heterochromatin protein 1 (HP1alpha, HP1beta, HP1gamma subtypes) and transcriptional intermediary factor TIF1beta 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, HP1alpha and HP1beta co-localized and associated with chromocentres (clusters of centromeres), which correlated with clustering of TIF1beta at these heterochromatic regions. The cell types that we studied were also characterized by a pronounced focal distribution of HP1gamma. The above-mentioned nuclear patterns of HP1 and TIF1beta proteins were completely different from the nuclear patterns observed in the remaining cell types investigated, in which HP1alpha was associated with chromocentres while HP1beta and HP1gamma were largely localized in distinct nuclear regions. Moreover, a dispersed nuclear distribution of TIF1beta was observed. Our findings showed that the nuclear arrangement of HP1 subtypes and TIF1beta 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.

• MeSH
• Azacitidine analogs & derivatives   pharmacology   MeSH
• Cell Differentiation drug effects   physiology   MeSH
• Cell Nucleus metabolism   MeSH
• Centromere metabolism   MeSH
• Chromosomal Proteins, Non-Histone genetics   metabolism   MeSH
• Decitabine MeSH
• Chromobox Protein Homolog 5 MeSH
• Immunohistochemistry MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Histone Deacetylase Inhibitors MeSH
• Nuclear Proteins metabolism   MeSH
• Microscopy, Confocal MeSH
• Hydroxamic Acids pharmacology   MeSH
• DNA Methylation drug effects   MeSH
• Cell Line, Tumor MeSH
• Protein Subunits genetics   metabolism   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Transcription Factors metabolism   MeSH
• Blotting, Western MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Azacitidine MeSH
• Chromosomal Proteins, Non-Histone MeSH
• Decitabine MeSH
• Chromobox Protein Homolog 5 MeSH
• Enzyme Inhibitors MeSH
• Histone Deacetylase Inhibitors MeSH
• Nuclear Proteins MeSH
• Hydroxamic Acids MeSH
• Protein Subunits MeSH
• Recombinant Fusion Proteins MeSH
• transcriptional intermediary factor 1 MeSH Browser
• Transcription Factors MeSH
• trichostatin A MeSH Browser
• Green Fluorescent Proteins MeSH

To determine the influence of increased gene expression and amplification in colorectal carcinoma on chromatin structure, the nuclear distances between pairs of bacterial artificial chromosome (BAC) clones with genomic separation from 800 to 29,000 kb were measured and compared between the tumor and parallel epithelial cells of six patients. The nuclear distances were measured between the loci in chromosomal bands 7p22.3-7p21.3; 7q35-7q36.3; 11p15.5-11p15.4; 20p13; 20p12.2; 20q11.21 and 20q12 where increased expression had been found in all types of colorectal carcinoma. The loci were visualized by three-dimensional fluorescence in situ hybridization using 22 BAC clones. Our results show that for short genomic separations, mean nuclear distance increases linearly with increased genomic separation. The results for some pairs of loci fell outside this linear slope, indicating the existence of different levels of chromatin folding. For the same genomic separations the nuclear distances were frequently shorter for tumor as compared with epithelial cells. Above the initial growing phase of the nuclear distances, a plateau phase was observed in both cell types where the increase in genomic separation was not accompanied by an increase in nuclear distance. The ratio of the mean nuclear distances between the corresponding loci in tumor and epithelium cells decreases with increasing amplification of loci. Our results further show that the large-scale chromatin folding might differ for specific regions of chromosomes and that it is basically preserved in tumor cells in spite of the amplification of many loci.

• MeSH
• Gene Amplification genetics   MeSH
• Cell Nucleus genetics   ultrastructure   MeSH
• Chromatin genetics   ultrastructure   MeSH
• DNA, Neoplasm genetics   MeSH
• DNA Probes MeSH
• Adult MeSH
• Epithelial Cells pathology   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Colorectal Neoplasms genetics   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Chromosomes, Human genetics   ultrastructure   MeSH
• Chromosome Banding MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA, Neoplasm MeSH
• DNA Probes MeSH

PURPOSE: Chromosomal aberrations and the nuclear topography of retinoblastoma tumour cells as well as lymphocytes of patients suffering from the familiar or sporadic form of retinoblastoma were studied. METHODS: Fluorescence in situ hybridisation (FISH) on fresh, paraffin-embedded tumour tissues and on peripheral blood leukocytes was used for cytogenetic analysis. The cell cycle profile and induction of apoptosis was studied by flow cytometry and gene expression changes were detected by RT-PCR. RESULTS: Using the repeated FISH technique, the average distances between the nuclear membrane and the fluorescence gravity centre (FGC) of seven selected chromosomes were determined in the same tumour population and three other cell types. Chromosome order in positioning from the nuclear membrane was similar in all cell populations investigated. Our experimental studies were focused on specific genetic loci relevant for retinoblastoma tumour pathogenesis. We revealed a certain heterogeneity in the copy number of the Rb1, N-myc, and TP53 gene loci in tumour cells. In addition, in lymphocytes isolated from peripheral blood of the patients, a high degree of copy number heterogeneity was also detected. In 60% of analysed retinoblastomas we observed numerical aberration involving the centromeric region of chromosome 6. In these tumours, apoptotic bodies were found irrespective of clinical therapy. Chromosome instability seems to be a typical feature of primary retinoblastomas as well as of the human pseudodiploid cell line Y79. These cells, of a hereditary form of retinoblastoma (Y79), were irradiated by gamma rays and exposed to anti-tumour drugs such as etoposide, vincristine, and cisplatin. These treatments induced apoptosis, changes in the cell cycle profile, and specific modifications in the nuclear topography of selected loci. Treatment with a non-lethal concentration of hydroxyurea was shown to induce the loss of the amplified N-myc gene involved in the homogenously staining region (HSR) that was found to be associated with the nuclear membrane of retinoblastoma Y79 cells. CONCLUSIONS: We assume that not only cytological and cytogenetic parameters but also aberrant chromatin structures and their nuclear topography can be useful tools for optimal tumour marker specification.

• MeSH
• Apoptosis MeSH
• In Situ Hybridization, Fluorescence MeSH
• Humans MeSH
• Retinal Neoplasms genetics   pathology   MeSH
• Ploidies MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Flow Cytometry MeSH
• Retinoblastoma genetics   pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The spatial arrangement of some genetic elements relative to chromosome territories and in parallel with the cell nucleus was investigated in human lymphocytes. The structure of the chromosome territories was studied in chromosomes containing regions (clusters) of highly expressed genes (HSA 9, 17) and those without such clusters (HSA 8, 13). In chromosomes containing highly expressed regions, the elements pertaining to these regions were found close to the centre of the nucleus on the inner sides of chromosome territories; those pertaining to regions with low expression were localized close to the nuclear membrane on the opposite sides of the territories. In chromosomes with generally low expression (HSA 8, 13), the elements investigated were found symmetrically distributed over the territories. Based on the investigations of the chromosome structure, the following conclusions are suggested: (1) Chromosome territories have a non-random internal 3D structure with defined average mutual positions between elements. For example, RARalpha, TP53 and Iso-q of HSA 17 are nearer to each other than they are to the HSA 17 centromere. (2) The structure of a chromosome territory reflects the number and chromosome location of clusters of highly expressed genes. (3) Chromosome territories behave to some extent as solid bodies: if the territory is found closer to the nuclear centre, the individual genetic elements of this chromosome are also found, on average, closer the centre of the nucleus. (4) The positions of centromeres are, on average, nearer to the fluorescence weight centre of the territory (FWCT) than to genes. (5) Active genes are not found near the centromeres of their own territory. A simple model of the structure of chromosome territory is proposed.

• MeSH
• Cell Nucleus genetics   MeSH
• Centromere genetics   MeSH
• Euchromatin genetics   MeSH
• Genes MeSH
• Heterochromatin genetics   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Nuclear Envelope genetics   MeSH
• Cell Compartmentation MeSH
• Humans MeSH
• Chromosomes, Human, Pair 17 ultrastructure   MeSH
• Chromosomes, Human ultrastructure   MeSH
• Lymphocytes diagnostic imaging   MeSH
• Monte Carlo Method MeSH
• Models, Genetic MeSH
• Computer Simulation MeSH
• Image Processing, Computer-Assisted MeSH
• Ultrasonography MeSH
• Imaging, Three-Dimensional * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Euchromatin MeSH
• Heterochromatin MeSH

Higher-order compartments of nuclear chromatin have been defined according to the replication timing, transcriptional activity, and information content (Ferreira et al. 1997, Sadoni et al. 1999). The results presented in this work contribute to this model of nuclear organization. Using different human blood cells, nuclear positioning of genes, centromeres, and whole chromosomes was investigated. Genes are located mostly in the interior of cell nuclei; centromeres are located near the nuclear periphery in agreement with the definition of the higher-order compartments. Genetic loci are found in specific subregions of cell nuclei which form distinct layers at defined centre-of-nucleus to locus distances. Inside these layers, the genetic loci are distributed randomly. Some chromosomes are polarized with genes located in the inner parts of the nucleus and centromere located on the nuclear periphery; polar organization was not found for some other chromosomes. The internal structure of the higher-order compartments as well as the polar and non-polar organization of chromosomes are basically conserved in different cell types and at various stages of the cell cycle. Some features of the nuclear structure are conserved even in differentiated cells and during cellular repair after irradiation, although shifted positioning of genetic loci was systematically observed during these processes.

• MeSH
• Cell Nucleus genetics   radiation effects   ultrastructure   MeSH
• Cell Cycle MeSH
• Bone Marrow Cells radiation effects   ultrastructure   MeSH
• Centromere radiation effects   MeSH
• Genes radiation effects   MeSH
• HL-60 Cells MeSH
• In Situ Hybridization, Fluorescence MeSH
• Interphase MeSH
• Cell Compartmentation MeSH
• Leukopoiesis MeSH
• Humans MeSH
• Chromosomes, Human radiation effects   MeSH
• Lymphocytes cytology   radiation effects   ultrastructure   MeSH
• U937 Cells MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH