Higher order genomic organization and epigenetic control maintain cellular identity and prevent breast cancer.

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial-to-mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer-compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

Core filaments of the nuclear matrix.

The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.

Association of nuclear matrix antigens with exon-containing splicing complexes.

mAbs raised against the human nuclear matrix (anti-NM)1 mAbs have been used to investigate the role of nuclear matrix antigens in pre-mRNA processing. The three anti-NM mAbs used in this study recognize antigens that are highly localized to nuclear matrix speckles. Surprisingly, all three of these mAbs preferentially immunoprecipitate splicing complexes containing exon sequences. The anti-NM mAbs efficiently immunoprecipitate the exon product complex but not complexes containing the lariat product after the second step of splicing. Two of the anti-NM mAbs completely inhibit pre-mRNA splicing in vitro. However, none of the anti-NM mAbs appear to recognize factors stably associated with splicing snRNPs. The three anti-NM mAbs predominantly react with distinct high molecular weight antigens, which belong to a class of nuclear proteins that selectively precipitate with Ser-Arg protein-splicing factors in the presence of high Mg2+ concentrations. Immunological, biochemical, and cell biological data indicate that two of the NM antigens are related to the defined set of Ser-Arg proteins. The results suggest the existence of an extended Ser-Arg family as a component of the nuclear matrix.

A normally masked nuclear matrix antigen that appears at mitosis on cytoskeleton filaments adjoining chromosomes, centrioles, and midbodies.

mAbs were generated against HeLa nuclear matrix proteins and one, HIB2, which selectively stained mitotic cells, was selected for further study. Western blot analysis showed H1B2 antibody detected a protein of 240 kD in the nuclear matrix fractions. The H1B2 antigen was completely masked in immunofluorescently stained interphase cells. However, removing chromatin with DNase I digestion and 0.25 M ammonium sulfate extraction exposed the protein epitope. The resulting fluorescence pattern was bright, highly punctate, and entirely nuclear. Further extraction of the nuclear matrix with 2 M NaCl uncovers an underlying, anastomosing network of 9-13 nm core filaments. Most of the H1B2 antigen was retained in the fibrogranular masses enmeshed in the core filament network and not in the filaments themselves. The H1B2 antigen showed remarkable behavior at mitosis. As cells approached prophase the antigen became unmasked to immunofluorescent staining without the removal of chromatin. First appearing as a bright spot, the antibody staining spread through the nucleus finally concentrating in the region around the condensed chromosomes. The antibody also brightly stained the spindle poles and, more weakly, in a punctate pattern in the cytoskeleton around the spindle. As the chromosomes separated at anaphase, H1B2 remained with the separating daughter sets of chromosomes. The H1B2 antigen returned to the reforming nucleus at telophase, but left a bright staining region in the midbody. Immunoelectron microscopy of resinless sections showed that, in the mitotic cell, the H1B2 antibody did not stain chromosomes and centrioles themselves, but decorated a fibrogranular network surrounding and connected to the chromosomes and a fibrogranular structure surrounding the centriole.

The acute myeloid leukemia-associated protein, DEK, forms a splicing-dependent interaction with exon-product complexes.

DEK is an approximately 45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.

The architectural organization of nuclear metabolism.

Nucleic acid metabolism is structurally organized in the nucleus. DNA replication and transcription have been localized to particular nuclear domains. Additional domains have been identified by their morphology or by their composition; for example, by their high concentration of factors involved in RNA splicing. The domain organization of the nucleus is maintained by the nuclear matrix, a nonchromatin nuclear scaffolding that holds most nuclear RNA and organizes chromatin into loops. The nuclear matrix is built on a network of highly branched core filaments that have an average diameter of 10 nm. Many of the intermediates and the regulatory and catalytic factors of nucleic acid metabolism are retained in nuclear matrix preparations, suggesting that nucleic acid synthesis and processing are structure-bound processes in cells. Tissue-specific and malignancy-induced variations in nuclear structure and metabolism may result from altered matrix architecture and composition.

Pulse energy dependence of subcellular dissection by femtosecond laser pulses.

Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 micrometer away.

Association of adenovirus DNA transcribed activity with nuclear matrix of host cells.

With gentle cell extraction techniques, various DNA components in the HeLa cells after 6 h of adenovirus infection have been obtained. Adenovirus, early transcribed regions (E2a, E1b) and a late transcribed region (L2) were used as probes in Southern hybridization, respectively. The experiment showed that only actively transcribed adenovirus DNA fragments would tightly bind to the nuclear matrix of host cells. We inferred that the nuclear matrix of host cells plays an important role in viral DNA transcription.

Oncogenic targeting of BRM drives malignancy through C/EBPß-dependent induction of α5 integrin.

Integrin expression and activity are altered in tumors, and aberrant integrin signaling promotes malignancy. However, how integrins become altered in tumors remains poorly understood. We discovered that oncogenic activation of MEK signaling induces cell growth and survival, and promotes the malignant phenotype of mammary epithelial cells (MECs) by increasing α5 integrin expression. We determined that MEK activates c-Myc to reduce the transcription of the SWI/SNF chromatin remodeling enzyme Brahma (BRM). Our studies revealed that reduced BRM expression and/or activity drives the malignant behavior of MECs by epigenetically promoting C/EBPß expression to directly induce α5 integrin transcription. Consistently, we could show that restoring BRM levels normalized the malignant behavior of transformed MECs in culture and in vivo by preventing C/EBPß-dependent α5 integrin transcription. Our findings identify a novel mechanism whereby oncogenic signaling promotes malignant transformation by regulating transcription of a key chromatin remodeling molecule that regulates integrin-dependent stromal-epithelial interactions.

Nuclear dreams: the malignant alteration of nuclear architecture.

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets.

Localization of nuclear matrix core filament proteins at interphase and mitosis.

The gentle removal of chromatin uncovers a nuclear matrix consisting of two parts: a nuclear lamina connected to the intermediate filaments of the cytoskeleton and an internal matrix of thick, polymorphic fibers connecting the lamina to masses in the nuclear interior. This internal nuclear matrix can be further fractionated to uncover a highly branched network of 9 nm and 13 nm core filaments retaining some enmeshed bodies. The core filament network retains most of the nuclear RNA, as well as the fA12RNP antigen, and may be the most basic or core element of internal nuclear structure. One high molecular weight protein component of the core filament network, the H1B2 antigen, is normally masked in the interphase nucleus and is uncovered as the chromatin condenses at mitosis. This protein is associated with a fibrogranular network surrounding and connected to the chromosomes. The core filament-associated fA12 antigen also becomes associated with this perichromosomal network. We propose that the core filament nuclear matrix structure may not completely disassemble at mitosis but, rather, that parts remain as a structural network connected to chromosomes and other mitotic structures. These mitotic networks may, in turn, serve as the core structures on which the nuclear matrices of daughter cells are built.

Bio Vision: microscopy in three dimensions.

Conventional electron microscopy is inadequate for visualizing the three-dimensional networks supporting cell architecture: the cytoskeleton and nuclear matrix. Consequently, we have not appreciated the extent to which the cell, its biochemistry, and its molecular biology are structured. A new technology combining in situ cell fractionation and resinless section electron microscopy allows the visualization of cell structure in three dimensions and permits the localization of individual components. These techniques reveal a far richer cell architecture than had been assumed and will allow important problems of biology, which have not surrendered their secrets to a purely biochemical approach, to be addressed.

The microtubule-associated nucleoside diphosphate kinase.

Microtubule protein prepared by cycles of assembly-disassembly contains a nucleoside diphosphate kinase (NDP kinase) activity. We have isolated the NDP kinase responsible for this activity from twice-polymerized bovine brain microtubule protein by a five-step chromatographic procedure. The molecular weight of this enzyme was 103,000 +/- 7,000 daltons as determined by sedimentation equilibrium experiments performed with a Beckman Airfuge. A doublet of subunit bands with molecular masses of about 18,000 daltons was detected by silver staining after gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this preparation. We conclude that the enzyme is a hexamer, although we cannot identify the mix of subunits. We were able to isolate only nanogram quantities of this enzyme, too little for extensive studies, so we isolated the enzyme directly from bovine brain without a preliminary microtubule protein isolation. The whole-brain NDP kinase was isolated by the same chromatographic steps as the enzyme from microtubule protein preparations. Both enzymes had a doublet of subunits at the same molecular weights and both were the same isozyme, chromatofocusing at a pH of 8.0. Both enzymes had similar kinetic properties and similar thermal inactivation profiles. These similar properties of the two enzymes suggest that they are identical. Both subunits of NDP kinase could be reversibly phosphorylated by ATP. Phosphorylation of the native enzyme created multiple, more acidic forms that retained activity. The isolation of this NDP kinase, which can copurify with microtubule protein through cycles of assembly-disassembly, will facilitate future studies on the role of this enzyme in the mechanism and regulation of microtubule assembly.

Polyamine regulation of the microtubule-associated protein kinase.

Microtubule protein prepared by cycles of assembly-disassembly contains a cyclic AMP-dependent protein kinase that phosphorylates the high-molecular-weight microtubule-associated protein MAP-2. The polyamine spermine at 2mM affected the phosphorylation of MAP-2 in a manner that depended on the cyclic AMP concentration. At cyclic AMP concentrations below 10(-6) M, spermine increased the rate of phosphorylation, while at cyclic AMP concentrations above 10(-6) M, spermine decreased the rate of phosphorylation. Spermine also decreased the final extent of cyclic AMP-dependent phosphorylation but did not affect the protein substrate specificity of the microtubule-associated protein kinase. MAP-2 was the principal substrate both in the presence and in the absence of spermine. Because of these results, we propose that microtubule protein phosphorylation may be regulated in vivo by spermine as well as by cyclic AMP levels.

Chromatin architecture and nuclear RNA.

The maintenance of normal chromatin morphology requires ongoing RNA synthesis. We have examined the role of RNA in chromatin organization, using selective detergent extraction of cells, RNA synthesis inhibitors, and enzymatic digestion of nuclear RNA. Comparison of extracted and unextracted cells showed that the important features of chromatin architecture were largely unchanged by the extraction procedure. Normally, chromatin was distributed in small heterochromatic regions and dispersed euchromatic strands. Ribonucleoprotein granules were dispersed throughout the euchromatic regions. Exposure to actinomycin led to the redistribution of chromatin into large clumps, leaving large empty spaces and a dense clustering of the remaining ribonucleoprotein granules. When the nuclei of extracted cells were digested with RNase A, there was a rearrangement of chromatin similar to but more pronounced than that seen in cells exposed to actinomycin. The inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole also inhibits RNA synthesis but by a different mechanism that leaves no nascent RNA chains. The drug had little effect on chromatin after brief exposure but resembled actinomycin in its effect at longer times. We also examined the structure of the nuclear matrix to which most heteronuclear RNA remains associated. Pretreatment of cells with actinomycin or digestion of the nuclear matrix with RNase A caused the matrix fibers to collapse and aggregate. The experiments show a parallel decay of chromatin and of nuclear matrix organization with the depletion of nuclear RNA and suggest that RNA is a structural component of the nuclear matrix, which in turn may organize the higher order structure of chromatin.

Alterations in nuclear matrix structure after adenovirus infection.

Infection of HeLa cells with adenovirus serotype 2 causes rearrangements in nuclear matrix morphology which can best be seen by gentle cell extraction and embedment-free section electron microscopy. We used these techniques to examine the nuclear matrices and cytoskeletons of cells at 6, 13, 28, and 44 h after infection. As infection progressed, chromatin condensed onto the nucleoli and the nuclear lamina. Virus-related inclusions appeared in the nucleus, where they partitioned with the nuclear matrix. These virus centers consisted of at least three distinguishable areas: amorphously dense regions, granular regions whose granulations appeared to be viral capsids, and filaments connecting these regions to each other and to the nuclear lamina. The filaments became decorated with viral capsids of two different densities, which may be empty capsid shells and capsids with DNA-protein cores. The interaction of some capsids with the filaments persisted even after lysis of the cell. We propose that granulated virus-related structures are sites of capsid assembly and storage and that the filaments may be involved in the transport of capsids and capsid intermediates. The nuclear lamina became increasingly crenated after infection, with some extensions appearing to bud off and form blebs of nuclear material in the cytoplasm. The perinuclear cytoskeleton became rearranged after infection, forming a corona of decreased filament number around the nucleus. In summary, we propose that adenovirus rearranges the nuclear matrix and cytoskeleton to support its own replication.

Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections.

We describe two methods for staining resinless thin sections with antibodies and gold-conjugated second antibodies. Immunolocalization of specific proteins is a powerful tool for cell structure studies but current techniques do not develop its full potential. Immunofluorescence provides only low-resolution localization, whereas conventional thin-section electron microscopy images and immunostains only the section surface. Resinless sections of extracted cell structures offer a simple and effective means of immuno-electron microscopy. Without embedding plastic or soluble proteins, the cell cytostructure produces high-contrast, three-dimensional images. Resinless sections of detergent-extracted cells are prepared by embedding in diethylene glycol distearate, sectioning, and removing diethylene glycol distearate before microscopy. In the first method of immunostaining, extracted cells were fixed and stained with antibodies before embedment, sectioning, removal of the embedding resin, and critical point drying. In the postembedment method, the sample was embedded and sectioned, the diethylene glycol distearate was removed, and the sample was rehydrated before antibody staining. With these techniques, specific proteins were localized with high resolution throughout the entire section. Stereoscopic micrographs of resinless sections revealed the precise localization of specific cytoskeleton and nuclear matrix proteins in three dimensions with unprecedented clarity.

The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein.

The retinoblastoma gene product (Rb) has been established as a tumor suppressor and cell cycle regulator, although its mechanism of action remains obscure. The observations that several Rb-binding viral oncoproteins all associate with the nuclear matrix suggest that these interactions may occur on this structure. To determine whether Rb itself is a component of the matrix, we extracted synchronized cultured cells to isolate matrix proteins while preserving nuclear architecture. Immunoblot and immunolabeling data show that a significant portion of hypophosphorylated Rb associates with the matrix only during early G1. Mutant Rb in tumor cells did not associate with the matrix, whereas Rb-reconstituted cells contained abundant matrix-bound Rb. Rb is distributed widely throughout the matrix, particularly concentrated at the nuclear periphery and in nucleolar remnants. Core filaments of the matrix contained no detectable Rb. Our screening of expression libraries for potential Rb-associated proteins has identified several that are part of the matrix. Specifically, the peripheral matrix proteins lamin A and C bound Rb in vitro. We therefore suggest that Rb interactions with the nuclear matrix may be important for its ability to regulate cell cycle progression.

The B1C8 protein is in the dense assemblies of the nuclear matrix and relocates to the spindle and pericentriolar filaments at mitosis.

The B1C8 monoclonal antibody detects a 180-kDa nuclear matrix-specific protein. The protein is a component of the dense, metabolically active bodies or assemblies revealed by resinless section electron microscopy of the nuclear matrix. These assemblies are scattered through the nuclear interior, enmeshed in a complex network of 11-nm filaments. Resinless section electron microscopy of immunogold-stained nuclear matrix preparations shows B1C8 located in many but apparently not all the assemblies. In this regard, the B1C8 antigen resembles previously studied nuclear matrix proteins such as the H1B2 protein. The speckled pattern of nuclear immunofluorescence by B1C8 reflects this labeling of the dense assemblies in the nuclear matrix. Somewhat unusual is the faint staining of cytoplasmic microtubules by B1C8, which appears to be due to a weakly cross-reacting protein. During cell division, the B1C8 antigen redistributed drastically, showing the dispersion of nuclear matrix assemblies at mitosis. Speckles of B1C8 fluorescence first coalesced at prophase within the nuclear interior and then scattered into numerous cytoplasmic speckles by prometaphase. At metaphase, the B1C8 speckled cytoplasmic staining had become even more widely distributed and finely grained. Also, intense labeling appeared at the mitotic pole and on the spindle fibers themselves. The reassembly of B1C8 antigens into larger cytoplasmic speckles began at anaphase and finally, at telophase, most B1C8 labeling redistributed into speckles in the re-forming nuclei.