Jeffrey A. Nickerson

Nuclear structure in cancer cells

Nuclear architecture — the spatial arrangement of chromosomes and other nuclear components — provides a framework for organizing and regulating the diverse functional processes within the nucleus. There are characteristic differences in the nuclear architectures of cancer cells, compared with normal cells, and some anticancer treatments restore normal nuclear structure and function. Advances in understanding nuclear structure have revealed insights into the process of malignant transformation and provide a basis for the development of new diagnostic tools and therapeutics.

The Nuclear Matrix: Past and Present

Publisher Summary This chapter describes the structure and function of the nuclear matrix. The nuclear matrix is firmly connected to the extensive network of intermediate filaments occupying the cytoplasmic space and anchoring on the outer surface of the nuclear lamina. The nuclear matrix and intermediate filaments are integrated into a single cell-wide structure that retains the overall geometry and appearance of the intact cell. Changes in nuclear matrix protein composition have also been observed during development. The fetal rat calvarial osteoblast has become an important in vitro developmental system for studying phenotype-specific gene expression. Six nuclear matrix proteins have been reported in human colon adenocarcinoma tumor samples which were absent from normal colon tissue. Such malignancy-specific nuclear matrix proteins may have considerable diagnostic value and may help to explain the changes in nuclear structure that accompany malignancy. The studies on rat osteosarcoma cells suggested that these tumor cells are expressing matrix proteins normally expressed only at earlier developmental stages in the osteoblast lineage.

Mitotic occupancy and lineage-specific transcriptional control of rRNA genes by Runx2

Regulation of ribosomal RNA genes is a fundamental process that supports the growth of cells and is tightly coupled with cell differentiation. Although rRNA transcriptional control by RNA polymerase I (Pol I) and associated factors is well studied, the lineage-specific mechanisms governing rRNA expression remain elusive. Runt-related transcription factors Runx1, Runx2 and Runx3 establish and maintain cell identity, and convey phenotypic information through successive cell divisions for regulatory events that determine cell cycle progression or exit in progeny cells. Here we establish that mammalian Runx2 not only controls lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also acts as a repressor of RNA Pol I mediated rRNA synthesis. Within the condensed mitotic chromosomes we find that Runx2 is retained in large discrete foci at nucleolar organizing regions where rRNA genes reside. These Runx2 chromosomal foci are associated with open chromatin, co-localize with the RNA Pol I transcription factor UBF1, and undergo transition into nucleoli at sites of rRNA synthesis during interphase. Ribosomal RNA transcription and protein synthesis are enhanced by Runx2 deficiency that results from gene ablation or RNA interference, whereas induction of Runx2 specifically and directly represses rDNA promoter activity. Runx2 forms complexes containing the RNA Pol I transcription factors UBF1 and SL1, co-occupies the rRNA gene promoter with these factors in vivo, and affects local chromatin histone modifications at rDNA regulatory regions. Thus Runx2 is a critical mechanistic link between cell fate, proliferation and growth control. Our results suggest that lineage-specific control of ribosomal biogenesis may be a fundamental function of transcription factors that govern cell fate.

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.

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.

Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS

Dipeptide repeat (DPR) proteins are toxic in various models of FTD/ALS with GGGGCC (G4C2) repeat expansion. However, it is unclear whether nuclear G4C2 RNA foci also induce neurotoxicity. Here, we describe a Drosophila model expressing 160 G4C2 repeats (160R) flanked by human intronic and exonic sequences. Spliced intronic 160R formed nuclear G4C2 sense RNA foci in glia and neurons about ten times more abundantly than in human neurons; however, they had little effect on global RNA processing and neuronal survival. In contrast, highly toxic 36R in the context of poly(A)(+) mRNA were exported to the cytoplasm, where DPR proteins were produced at >100-fold higher level than in 160R flies. Moreover, the modest toxicity of intronic 160R expressed at higher temperature correlated with increased DPR production, but not RNA foci. Thus, nuclear RNA foci are neutral intermediates or possibly neuroprotective through preventing G4C2 RNA export and subsequent DPR production.

PTEN enters the nucleus by diffusion

Despite much evidence for phosphatidylinositol phosphate (PIP)‐triggered signaling pathways in the nucleus, there is little understanding of how the levels and activities of these proteins are regulated. As a first step to elucidating this problem, we determined whether phosphatase and tensin homolog deleted on chromosome 10 (PTEN) enters the nucleus by passive diffusion or active transport. We expressed various PTEN fusion proteins in tsBN2, HeLa, LNCaP, and U87MG cells and determined that the largest PTEN fusion proteins showed little or no nuclear localization. Because diffusion through nuclear pores is limited to proteins of 60,000 Da or less, this suggests that nuclear translocation of PTEN occurs via diffusion. We examined PTEN mutants, seeking to identify a nuclear localization signal (NLS) for PTEN. Mutation of K13 and R14 decreased nuclear localization, but these amino acids do not appear to be part of an NLS. We used fluorescence recovery after photobleaching (FRAP) to demonstrate that GFP‐PTEN can passively pass through nuclear pores. Diffusion in the cytoplasm is retarded for the PTEN mutants that show reduced nuclear localization. We conclude that PTEN enters the nucleus by diffusion. In addition, sequestration of PTEN in the cytoplasm likely limits PTEN nuclear translocation.

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. J. Cell. Biochem. 70:172–180, 1998.

Ectopic Runx2 Expression in Mammary Epithelial Cells Disrupts Formation of Normal Acini Structure: Implications for Breast Cancer Progression

The transcription factor Runx2 is highly expressed in breast cancer cells compared with mammary epithelial cells and contributes to metastasis. Here we directly show that Runx2 expression promotes a tumor cell phenotype of mammary acini in three-dimensional culture. Human mammary epithelial cells (MCF-10A) form polarized, growth-arrested, acini-like structures with glandular architecture. The ectopic expression of Runx2 disrupts acini formation, and electron microscopic ultrastructural analysis revealed the absence of lumens. Characterization of the disrupted acini structures showed increased cell proliferation (Ki-67 positive cells), decreased apoptosis (Bcl-2 induction), and loss of basement membrane formation (absence of beta(4) integrin expression). In complementary experiments, inhibition of Runx2 function in metastatic MDA-MB-231 breast cancer cells by stable expression of either short hairpin RNA-Runx2 or a mutant Runx2 deficient in subnuclear targeting resulted in reversion of acini to more normal structures and reduced tumor growth in vivo. These novel findings provide direct mechanistic evidence for the biological activity of Runx2, dependent on its subnuclear localization, in promoting early events of breast cancer progression and suggest a molecular therapeutic target.

The Ultrastructure of MCF-10A Acini

MCF‐10A human mammary epithelial cells cultured inside reconstituted basement membrane form acini that resemble the acinar structures of mammary lobules. This three‐dimensional culture system has been used for identifying and characterizing the signal transduction pathways controlling cell proliferation and death, and for studying their disregulation in malignant progression. We have compared the ultrastructure of MCF‐10A acini, MCF‐10A cells grown in monolayer, and the acinar structures of human breast lobules. The tissue architecture of MCF‐10A acini was formed by hemidesmosomes connected to a basement membrane and by abundant desmosomes between acinar cells. Intermediate filaments that joined into large and abundant filament bundles connected hemidesmosomes and desmosomes to sites at the nuclear surface. Fewer and thinner bundles of filaments were observed in monolayer MCF‐10A cells and even fewer in breast tissue. Tight junctions were observed between cells in breast tissue but missing in MCF‐10A acini. The cytoplasm of MCF‐10A acinar cells had a polar organization similar to that observed in breast tissue, with centrosomes and the Golgi apparatus on the apical side of the nucleus. MCF‐10A acinar nuclei had an irregular, frequently invaginated surface and had a single nucleolus. The distribution of heterochromatin was similar to that in the epithelial cells of breast tissue. The nuclei of monolayer MCF‐10A cells had multiple nucleoli, a more regular profile, and less heterochromatin. Electron microscopy has the resolution required to survey features of MCF‐10A cell and acinus architecture that may change with manipulations designed to induce malignant phenotypes. J. Cell. Physiol.