ABSTRACT
Geophagy among orangutans is the most poorly documented in contrast to the knowledge of soil-eating practices of other great ape species. Observations of soil consumption by orangutans in the Sungai Wain Forest Preserve (Wanariset) of Borneo are presented, along with physico-mineral-chemical analyses of the ingested soil in an effort to understand what might stimulate the activity. The consumed soils are: light colored, not excessively weathered by normal standards, higher in the clay size fraction relative to controls, and are comprised of a mix of clay minerals without any specificity of 1:1, 2:1 and/or 2:1:1 (Si:Al) species. The geophagic soils contain chlorides below detection limits, effectively eliminating salt as a stimulus. Soil chemical and geochemical analyses confirm that orangutans prefer soils with pH levels near or above 4.0, while controls are consistently lower (pH = 3.5-4.0), a considerable difference in acidity for at least four out of six soils consumed. Geochemical analysis shows Al, Fe and K are high in the consumed vs control samples; higher Al follows from higher clay percentages in the consumed earth. Iron and K may play physiological roles, but Fe is mostly in the ferrous form (Fe(+2)) and may not be readily taken up by the animals. The preferential choice of consumed samples, with pH above 4.0 and higher clay contents, may promote a more beneficial intestinal environment.
Subject(s)
Pica , Pongo pygmaeus/physiology , Soil/chemistry , Animals , Borneo , IndonesiaABSTRACT
Within cells, the nucleus is surrounded by the cytoplasm which contains diffusible macromolecules at a high concentration (>100 mg/ml). When cells are broken to isolate nuclei by current methods these macromolecules are dispersed, and to reproduce the environment of nuclei in vivo more closely we have developed a method to isolate them in a medium where cytoplasmic macromolecules are replaced by an inert, volume-occupying polymer and which is essentially cation-free. Nuclei isolated by this method resemble closely those prepared by conventional procedures as seen by optical and electron microscopy, and their internal compartments (nucleoli, PML and Cajal bodies, transcription centers, and splicing speckles) and transcriptional activity are conserved. This procedure is efficient for mammalian cells that normally grow in suspension and do not have an extensive cytoskeleton, and requires ~30 min.
Subject(s)
Biomimetic Materials , Cell Fractionation/methods , Cell Nucleus , Culture Media/chemistry , Cytoplasm/metabolism , Polymers , Animals , Cell Line , Humans , MiceABSTRACT
The principles that determine the organization of the nucleus have become clearer in recent years, largely because of new insights into polymer, colloid, and soft-matter science. Macromolecules, together with the giant linear polymers that form the chromosomes, are confined at high concentrations within the nuclear envelope and their interactions are influenced strongly by short-range depletion or entropic forces which are negligible in dilute systems, in addition to the more familiar van der Waals, electrostatic, steric, hydrogen bonding, and hydrophobic forces. The studies described in this volume are consistent with the model that this complex and concentrated mixture of macromolecules is maintained in a delicate equilibrium by quite simple although unsuspected physicochemical principles. The sensitivity of this equilibrium to perturbation may underlie the controversies about the existence of a nuclear matrix or scaffold. In this volume, we underline the importance for cell biologists of being familiar with current work in colloid, polymer, soft matter, and nanoscience. This chapter presents a brief background to the aspects of the nucleus that are considered in detail in subsequent chapters.
Subject(s)
Biopolymers/metabolism , Nuclear Envelope/metabolism , Nuclear Matrix/metabolism , Animals , Biopolymers/chemistry , Entropy , Humans , Nuclear Envelope/chemistry , Nuclear Matrix/chemistryABSTRACT
To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ~170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20-30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair.
Subject(s)
Chromosomes, Artificial , DNA Breaks, Double-Stranded/radiation effects , DNA Breaks, Single-Stranded/radiation effects , DNA Repair/radiation effects , Cell Line , Chromosomes, Artificial/genetics , Chromosomes, Artificial/radiation effects , DNA Ligases/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gamma Rays , Humans , Kinetics , Poly(ADP-ribose) Polymerases/metabolism , RNA, Small Interfering , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolismABSTRACT
The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a â¼170 kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes.
Subject(s)
Chromatin/chemistry , DNA Cleavage , DNA, Circular/chemistry , Cell Line , Chromosomes/chemistry , DNA/chemistry , DNA Breaks, Double-Stranded , DNA Breaks, Single-Stranded , DNA Restriction Enzymes , DNA-Binding Proteins/analysis , Humans , Indicators and ReagentsABSTRACT
In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ~30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100-200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ~30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes.
Subject(s)
Chromosomes/metabolism , Adenosine Triphosphatases/metabolism , Animals , Antigens, Neoplasm/metabolism , CHO Cells , Chromosomes/chemistry , Cricetinae , Cricetulus , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Dextrans/pharmacology , Ficoll/pharmacology , Metaphase/drug effects , Microscopy, Electron , Multiprotein Complexes/metabolism , Nucleosomes/chemistry , Nucleosomes/metabolism , Polyethylene Glycols/pharmacologyABSTRACT
PURPOSE: Cells exposed to ionizing radiation release factors that induce deoxyribonucleic acid damage, chromosomal instability, apoptosis, and changes in the proliferation rate of neighboring unexposed cells, phenomena known as bystander effects. This work analyzes and compares changes in global transcript levels induced by direct irradiation and by bystander effects in K562 (human erythroleukemia) cells. METHODS AND MATERIALS: Cells were X-irradiated with 4 Gy or transferred into culture medium collected from cells 1 h after irradiation (irradiation-conditioned medium). Global transcript profiles were assessed after 36 h of growth by use of Affymetrix microarrays (Affymetrix, Santa Clara, CA) and the kinetics of change of selected transcripts by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The level of the majority (72%) of transcripts changed similarly (increase, decrease, or no change) in cells grown in irradiation-conditioned medium or irradiated, whereas only 0.6% showed an opposite response. Transcript level changes in bystander and irradiated cells were significantly different from those in untreated cells grown for the same amount of time and were confirmed by quantitative reverse transcriptase-polymerase chain reaction for selected genes. Signaling pathways in which the highest number of transcripts changed in both conditions were found in the following groups: neuroactive ligand-receptor, cytokine-cytokine receptor interaction, Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) and Mitogen-Activated Protein Kinase (MAPK) In control cells more transcripts were downregulated than in irradiated and bystander cells with transcription factors YBX1 and STAT5B, heat shock protein HSPA1A, and ribonucleic acid helicase DDX3X as examples. CONCLUSIONS: The transcriptomes of cells grown in medium from X-irradiated cells or directly irradiated show very similar changes. Signals released by irradiated cells may cause changes in the transcriptome of neighboring cells that sustain their survival.
Subject(s)
Bystander Effect/radiation effects , Culture Media, Conditioned/pharmacology , Gene Expression Profiling/methods , K562 Cells/radiation effects , Signal Transduction/radiation effects , Bystander Effect/drug effects , DNA Damage , Down-Regulation , Humans , K562 Cells/metabolism , Radiation Dosage , Reverse Transcriptase Polymerase Chain Reaction/methods , Signal Transduction/drug effects , Time Factors , Up-RegulationABSTRACT
Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small-scale motion and irregular conformation of chromatin seen in vivo are not reproduced in nuclei isolated in conventional ionic media.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Cations/metabolism , Cell Fractionation , Cell Line, Tumor , Chromatin/metabolism , Green Fluorescent Proteins/metabolism , Humans , Ions , K562 Cells , Macromolecular Substances , Microscopy, Fluorescence/methods , Nuclear Envelope/metabolism , Polymers/chemistry , Transcription, GeneticABSTRACT
Unirradiated cells which neighbor cells exposed to ionizing radiation (IR) show responses termed bystander effects, including DNA damage, chromosomal instability, mutation, and apoptosis. We used genome-wide microarrays to compare the change in transcript profiles in Me45 (human melanoma) cells grown in culture medium from irradiated cells (irradiation conditioned medium, ICM) with those which occurred after IR, sampling after more than one division cycle to detect long-term changes which could be relevant in radiotherapy. Transcripts of >10,000 genes showed an increased or decreased level in both conditions using the criterion of a >+/-10% change, and >85% of these were common to growth in ICM and after IR. When these genes were grouped into metabolic pathways according to the Kyoto Encyclopedia of Genes and Genomes (KEGG), significant differences (p<0.01) were seen between the numbers of up- and down-regulated transcripts in certain groups after both ICM and IR, particularly in the groups neuroactive ligand-receptor interactions, oxidative phosphorylation, cytokine-cytokine receptor interactions, proteasomes, and ribosomes. Quantitative RT-PCR assays of transcripts of selected genes in these pathways confirmed the similar effects of growth in ICM and IR. We conclude that factors transmitted from irradiated cells can influence transcript levels in bystander cells, and that these changes persist for more than one cell cycle consistent with the long-term transmissible effects seen in progeny cells, revealing new facets of the IR-induced bystander effect.
Subject(s)
Biomarkers, Tumor/genetics , Bystander Effect , Culture Media, Conditioned/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/radiation effects , Melanoma/genetics , X-Rays , Biomarkers, Tumor/metabolism , Humans , Melanoma/radiotherapy , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
Many of the chapters in this volume are concerned with processes or structures inside the nucleus, and it is relevant to consider the properties of their environment, or rather of the multiple different and specific environments that must exist in local regions of the highly heterogeneous intranuclear space. Relatively little is known about the fundamental physical properties of these environments, and theoretical treatments of phenomena in such concentrated mixtures of charged macromolecules are complex and as yet poorly developed. Some of the phenomena that occur at the molecular level are unexpected and counterintuitive for biologists, although well known to colloid and polymer scientists; for example, the existence of short-range attractive forces between macromolecules or structures with like charges. As a background for the chapters that follow, we consider here some of the particular features of intranuclear environments, how they may influence processes and structures in the nucleus, and their implications for working with nuclei.
Subject(s)
Cell Nucleus/metabolism , Animals , Colloids/chemistry , Diffusion , Dose-Response Relationship, Drug , Humans , Ions , Kinetics , Magnesium/chemistry , Models, Biological , Monte Carlo Method , Oxidation-Reduction , Polymers/chemistry , Stochastic ProcessesABSTRACT
The crowding of macromolecules in the cell nucleus, where their concentration is in the range of 100 mg/ml, is predicted to result in strong entropic forces between them. Here the effects of crowding on polynucleosome chains in vitro were studied to evaluate if these forces could contribute to the packing of chromatin in the nucleus in vivo. Soluble polynucleosomes approximately 20 nucleosomes in length formed fast-sedimenting complexes in the presence of inert, volume-occupying agents poly(ethylene glycol) (PEG) or dextran. This self-association was reversible and consistent with the effect of macromolecular crowding. In the presence of these crowding agents, polynucleosomes formed large assemblies as seen by fluorescence microscopy after labelling DNA with the fluorescent stain DAPI, and formed rods and sheets at a higher concentration of crowding agent. Self-association caused by crowding does not require exogenous cations. Single, approximately 800 nucleosome-long chains prepared in 100 microM Hepes buffer with no added cations, labelled with the fluorescent DNA stain YOYO-1, and spread on a polylysine-coated surface formed compact 3-D clusters in the presence of PEG or dextran. This reversible packing of polynucleosome chains by crowding may help to understand their compact conformations in the nucleus. These results, together with the known collapse of linear polymers in crowded milieux, suggest that entropic forces due to crowding, which have not been considered previously, may be an important factor in the packing of nucleosome chains in the nucleus.
Subject(s)
Nucleosomes/chemistry , Nucleosomes/ultrastructure , Macromolecular Substances/chemistry , Nucleic Acid ConformationABSTRACT
ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD(+) as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD(+) or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 microM H(2)O(2) and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a approximately 100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H(2)O(2) treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 microM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM-10 microM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r > or = 0.92, p<0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.
Subject(s)
Adenosine Diphosphate Ribose/metabolism , DNA Breaks , DNA Repair , Adenosine Diphosphate Ribose/biosynthesis , Cell Line , DNA Damage , Humans , Hydrogen Peroxide/pharmacology , Kinetics , Time FactorsABSTRACT
In the crowded intranuclear environment, entropic depletion forces between macromolecules are expected to be strong. A review of simulations of linear polymers leads to several predictions about probable conformations of a polynucleosome chain in these conditions. These include a globular conformation, variable compaction due to different local rigidity or curvature of the mosaic of isochores, satellite sequences, and nucleosomes containing different histone variants, and the possibility that chromosomes represent separate phases like those seen in heterogeneous particle mixtures by experiment and simulation. Experimental results which show that macromolecular crowding alone, in the absence of exogenous cations, can stabilise interphase chromosomes and cause self-association of polynucleosome chains are presented. Together, these considerations suggest that macromolecular crowding and entropic forces are major factors in packing polynucleosome chains in vivo.
Subject(s)
Chromatin/chemistry , Chromosomes/chemistry , Entropy , Nucleosomes/chemistry , Animals , Chromosomes/ultrastructure , Computer Simulation , Humans , Models, Biological , Molecular Conformation , Nucleosomes/ultrastructureABSTRACT
DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.
Subject(s)
DNA Damage , DNA Helicases/genetics , DNA Repair , DNA-Binding Proteins/genetics , Lymphocytes/radiation effects , O(6)-Methylguanine-DNA Methyltransferase/genetics , Polymorphism, Genetic , Transcription Factors/genetics , Adult , Aged , Apoptosis , Gamma Rays , Humans , Lymphocytes/metabolism , Micronuclei, Chromosome-Defective , Middle Aged , X-ray Repair Cross Complementing Protein 1 , Xeroderma Pigmentosum Group D ProteinABSTRACT
Many and possibly all macromolecules in the nucleus are segregated into discrete compartments, but the current model that this is achieved by a fibrillar nuclear matrix which structures the nuclear interior and compartments is not consistent with all experimental observations, as reviewed here. New results are presented which suggest that macromolecular crowding forces play a crucial role in the assembly of at least two compartments, nucleoli and PML bodies, and an in vitro system in which crowding assembles macromolecular complexes into structures which resemble nuclear compartments is described. Crowding forces, which are strong in the nucleus due to the high macromolecule concentration (in the range of 100 mg/ml), vastly increase the association constants of intermolecular interactions and can segregate different macromolecules into discrete phases. The model that they play a role in compartmentalisation of the nucleus is generally consistent with the properties of compartments, including their spherical or quasispherical form and their dynamic and mobile nature.
Subject(s)
Cell Nucleus/physiology , Macromolecular Substances/metabolism , Models, Biological , Nuclear Matrix/physiology , Animals , Cell Nucleolus/metabolism , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , DNA/metabolism , Humans , Hypotonic Solutions/pharmacology , Intranuclear Space/metabolism , Macromolecular Substances/chemistry , Matrix Attachment Regions/physiology , Neoplasm Proteins/metabolism , Nuclear Matrix/chemistry , Nuclear Matrix-Associated Proteins/metabolism , Nuclear Matrix-Associated Proteins/physiology , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , Thermodynamics , Transcription Factors/metabolism , Tumor Suppressor ProteinsABSTRACT
The mechanisms which cause macromolecules to form discrete compartments within the nucleus are not understood. Here, two ubiquitous compartments, nucleoli, and PML bodies, are shown to disassemble when K562 cell nuclei expand in medium of low monovalent cation concentration; their major proteins dispersed as seen by immunofluorescence and immunoelectron microscopy, and nucleolar transcript elongation fell by approximately 85%. These compartments reassembled and nucleolar transcription recovered in the same medium after adding inert, penetrating macromolecules (8 kDa polyethylene glycol (PEG), or 10.5 kDa dextran) to 12% w/v, showing that disassembly was not caused by the low cation concentration. These responses satisfy the criteria for crowding or volume exclusion effects which occur in concentrated mixtures of macromolecules; upon expansion the macromolecular concentration within the nucleus falls, and can be restored by PEG or dextran. These observations, together with evidence of a high concentration of macromolecules in the nucleus (in the range of 100mg/ml) which must cause strong crowding forces, suggest strongly that these forces play an essential role in driving the formation, and maintaining the function of nuclear compartments. This view is consistent with their dynamic and mobile nature and can provide interpretations of several unexplained observations in nuclear biology.
Subject(s)
Cell Nucleolus/metabolism , Cell Nucleus/ultrastructure , Intranuclear Space/metabolism , Macromolecular Substances/metabolism , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Buffers , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Dextrans/pharmacology , Humans , Intranuclear Space/chemistry , Intranuclear Space/ultrastructure , K562 Cells , Microscopy, Electron , Polyethylene Glycols/pharmacology , Promyelocytic Leukemia Protein , Tumor Suppressor ProteinsABSTRACT
The organization of the human dystrophin gene into loop domains has been studied using two different experimental approaches: excision of DNA loops mediated by nuclear matrix-bound topoisomerase II and in situ hybridization of different probes with histone-depleted nuclei (nuclear halos). Our objective was to examine if the DNA loops mapped by this biochemical approach coincide with loops visualized by microscopy. The results obtained using both approaches were in good agreement. Eight loops separated by attachment regions of different length were mapped in the upstream part (up to exon 54) of the gene by topoisomerase II-mediated excision. One of these loops was then directly visualized by in situ hybridization of the corresponding bacmid clone with nuclear halos. This is the first direct demonstration that a DNA domain mapped as a loop using a biochemical approach corresponds to a loop visible on cytological preparations. The validity of this result and of the whole map of loop domains was confirmed by in situ hybridization using probes derived from other attachment regions or loops mapped by topoisomerase II-mediated cleavage; these probes hybridized on the core or halo region, respectively, of nuclear halos. Our results demonstrate that a single transcription unit may be organized into several loops and that DNA loop attachment regions may be fairly long. Three out of four replication origins mapped in this gene co-localize with loop attachment regions, and the major deletion hot spot is harbored in an attachment region. These results strongly suggest that partitioning of genomic DNA into specific loops attached to a skeletal structure is a characteristic feature of eukaryotic chromosome organization in interphase.
Subject(s)
DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , Dystrophin/genetics , Nucleic Acid Conformation , Cell Line, Tumor , Cell Nucleus/chemistry , Cell Nucleus/metabolism , DNA Topoisomerases, Type II/metabolism , DNA, Neoplasm/metabolism , Humans , In Situ Hybridization/methods , Indoles/chemistry , Male , Microscopy, FluorescenceABSTRACT
We compared the extent to which apoptosis is induced and clonogenicity reduced in three tumour cell lines - the human melanoma Me45 and promyelocytic leukaemia HL-60, and the rat rhabdomyosarcoma R1 - after exposure to the anticancer drugs etoposide and cis-platinum or to gamma radiation; each induces different types of DNA damage. Cells which readily underwent apoptosis did not necessarily show a correlated loss of clonogenicity; for example, Me45 cells showed the highest sensitivity to all three agents in clonogenic assays but much lower levels of apoptotic cells than R1 or HL-60 cells. These results show that the efficiency of the eradication of clonogenic cells by genotoxic agents does not solely depend on the induction of apoptotic processes, and suggest that the induction of apoptosis and suppression of clonogenicity are independent processes.