Surgical and Anesthetic Management of a Mediastinal Fatty Tumor: Lipoblastoma.

Lipoblastoma is a rare fatty tumor that is diagnosed almost exclusively in children. Presentation often consists of respiratory symptoms; chest computed tomography shows a hypodense, low, attenuated mediastinal mass. Surgical approach and anesthetic management are dependent on the location of the tumor and the degree of airway compression; in most cases, a thoracotomy is performed, although a sternotomy is used in selected cases. Final diagnosis can be confirmed using molecular genetic analysis; a genetic hallmark of lipoblastoma is the rearrangement of chromosomal region 8q12 and the PLAG1 gene. Tumor recurrence is rare when a complete resection is performed.

Genomic promoter occupancy of runt-related transcription factor RUNX2 in Osteosarcoma cells identifies genes involved in cell adhesion and motility.

Runt-related transcription factors (RUNX1, RUNX2, and RUNX3) are key lineage-specific regulators of progenitor cell growth and differentiation but also function pathologically as cancer genes that contribute to tumorigenesis. RUNX2 attenuates growth and stimulates maturation of osteoblasts during bone formation but is also robustly expressed in a subset of osteosarcomas, as well as in metastatic breast and prostate tumors. To assess the biological function of RUNX2 in osteosarcoma cells, we examined human genomic promoter interactions for RUNX2 using chromatin immunoprecipitation (ChIP)-microarray analysis in SAOS-2 cells. Promoter binding of both RUNX2 and RNA polymerase II was compared with gene expression profiles of cells in which RUNX2 was depleted by RNA interference. Many RUNX2-bound loci (1550 of 2339 total) exhibit promoter occupancy by RNA polymerase II and contain the RUNX consensus motif 5'-((T/A/C)G(T/A/C)GG(T/G). Gene ontology analysis indicates that RUNX2 controls components of multiple signaling pathways (e.g. WNT, TGFß, TNFα, and interleukins), as well as genes linked to cell motility and adhesion (e.g. the focal adhesion-related genes FAK/PTK2 and TLN1). Our results reveal that siRNA depletion of RUNX2, PTK2, or TLN1 diminishes motility of U2OS osteosarcoma cells. Thus, RUNX2 binding to diverse gene loci may support the biological properties of osteosarcoma cells.

Brain cavernomas associated with en coup de sabre linear scleroderma: Two case reports.

Linear scleroderma is a form of localized scleroderma that primarily affects the pediatric population. When it occurs on the scalp or forehead, it is termed "en coup de sabre". In the en coup de sabre subtype, many extracutaneous associations, mostly neurological, have been described. A patient with linear scleroderma en coup de sabre was noted to have ipsilateral brain cavernomas by magnetic resonance imaging. Using a worldwide pediatric rheumatology electronic list-serve, another patient with the same 2 conditions was identified. These two patients are reported in this study. Consideration of neuroimaging studies to disclose abnormal findings in patients with linear scleroderma en coup de sabre is important for potentially preventing and treating neurological manifestations associated with this condition.

Corticosteroid-induced spinal epidural lipomatosis in the pediatric age group: report of a new case and updated analysis of the literature.

Spinal epidural lipomatosis is a rare complication of chronic corticosteroid treatment. We report a new pediatric case and an analysis of this and 19 pediatric cases identified in the international literature. The youngest of these combined 20 patients was 5 years old when lipomatosis was diagnosed. Lipomatosis manifested after a mean of 1.3 (+/- 1.5) years (SD) (median, 0.8 years; range, 3 weeks - 6.5 years) of corticosteroid treatment. The corticosteroid dose at the time of presentation of the lipomatosis ranged widely, between 5 and 80 mg of prednisone/day. Back pain was the most common presenting symptom. Imaging revealed that lipomatosis almost always involved the thoracic spine, extending into the lumbosacral region in a subset of patients. Predominantly lumbosacral involvement was documented in only two cases. Although a neurological deficit at presentation was documented in about half of the cases, surgical decompression was not performed in the cases reported after 1996. Instead, reducing the corticosteroid dose (sometimes combined with dietary restriction to mobilize fat) sufficed to induce remission. In summary, pediatric spinal epidural lipomatosis remains a potentially serious untoward effect of corticosteroid treatment, which, if recognized in a timely manner, can have a good outcome with conservative treatment.

An architectural genetic and epigenetic perspective.

The organization and intranuclear localization of nucleic acids and regulatory proteins contribute to both genetic and epigenetic parameters of biological control. Regulatory machinery in the cell nucleus is functionally compartmentalized in microenvironments (focally organized sites where regulatory factors reside) that provide threshold levels of factors required for transcription, replication, repair and cell survival. The common denominator for nuclear organization of regulatory machinery is that each component of control is architecturally configured and every component of control is embedded in architecturally organized networks that provide an infrastructure for integration and transduction of regulatory signals. It is realistic to anticipate emerging mechanisms that account for the organization and assembly of regulatory complexes within the cell nucleus can provide novel options for cancer diagnosis and therapy with maximal specificity, reduced toxicity and minimal off-target complications.

Architectural epigenetics: mitotic retention of mammalian transcriptional regulatory information.

Epigenetic regulatory information must be retained during mammalian cell division to sustain phenotype-specific and physiologically responsive gene expression in the progeny cells. Histone modifications, DNA methylation, and RNA-mediated silencing are well-defined epigenetic mechanisms that control the cellular phenotype by regulating gene expression. Recent results suggest that the mitotic retention of nuclease hypersensitivity, selective histone marks, as well as the lineage-specific transcription factor occupancy of promoter elements contribute to the epigenetic control of sustained cellular identity in progeny cells. We propose that these mitotic epigenetic signatures collectively constitute architectural epigenetics, a novel and essential mechanism that conveys regulatory information to sustain the control of phenotype and proliferation in progeny cells by bookmarking genes for activation or suppression.

Mitotic bookmarking of genes: a novel dimension to epigenetic control.

Regulatory machinery is focally organized in the interphase nucleus. The information contained in these focal nuclear microenvironments must be inherited during cell division to sustain physiologically responsive gene expression in progeny cells. Recent results suggest that focal mitotic retention of phenotypic transcription factors at promoters together with histone modifications and DNA methylation--a mechanism collectively known as gene bookmarking--is a novel parameter of inherited epigenetic control that sustains cellular identity after mitosis. The epigenetic signatures imposed by bookmarking poise genes for activation or suppression following mitosis. We discuss the implications of phenotypic transcription factor retention on mitotic chromosomes in biological control and disease.

Organization, integration, and assembly of genetic and epigenetic regulatory machinery in nuclear microenvironments: implications for biological control in cancer.

There is growing awareness that the fidelity of gene expression necessitates coordination of transcription factor metabolism and organization of genes and regulatory proteins within the three-dimensional context of nuclear architecture. The regulatory machinery that governs genetic and epigenetic control of gene expression is compartmentalized in nuclear microenvironments. Temporal and spatial parameters of regulatory complex organization and assembly are functionally linked to biological control and are compromised with the onset and progression of tumorigenesis. High throughput imaging of cells, tissues, and tumors, including live cell analysis, is expanding research's capabilities toward translating components of nuclear organization into novel strategies for cancer diagnosis and therapy.

The leukemogenic t(8;21) fusion protein AML1-ETO controls rRNA genes and associates with nucleolar-organizing regions at mitotic chromosomes.

RUNX1/AML1 is required for definitive hematopoiesis and is frequently targeted by chromosomal translocations in acute myeloid leukemia (AML). The t(8;21)-related AML1-ETO fusion protein blocks differentiation of myeloid progenitors. Here, we show by immunofluorescence microscopy that during interphase, endogenous AML1-ETO localizes to nuclear microenvironments distinct from those containing native RUNX1/AML1 protein. At mitosis, we clearly detect binding of AML1-ETO to nucleolar-organizing regions in AML-derived Kasumi-1 cells and binding of RUNX1/AML1 to the same regions in Jurkat cells. Both RUNX1/AML1 and AML1-ETO occupy ribosomal DNA repeats during interphase, as well as interact with the endogenous RNA Pol I transcription factor UBF1. Promoter cytosine methylation analysis indicates that RUNX1/AML1 binds to rDNA repeats that are more highly CpG methylated than those bound by AML1-ETO. Downregulation by RNA interference reveals that RUNX1/AML1 negatively regulates rDNA transcription, whereas AML1-ETO is a positive regulator in Kasumi-1 cells. Taken together, our findings identify a novel role for the leukemia-related AML1-ETO protein in epigenetic control of cell growth through upregulation of ribosomal gene transcription mediated by RNA Pol I, consistent with the hyper-proliferative phenotype of myeloid cells in AML patients.

Genetic and epigenetic regulation in nuclear microenvironments for biological control in cancer.

The regulatory machinery that governs genetic and epigenetic control of gene expression is compartmentalized in nuclear microenvironments. Temporal and spatial parameters of regulatory complex organization and assembly are functionally linked to biological control and are compromised with the onset and progression of tumorigenesis providing a novel platform for cancer diagnosis and treatment.

Runx2 regulates G protein-coupled signaling pathways to control growth of osteoblast progenitors.

Runt-related transcription factor 2 (Runx2) controls lineage commitment, proliferation, and anabolic functions of osteoblasts as the subnuclear effector of multiple signaling axes (e.g. transforming growth factor-beta/BMP-SMAD, SRC/YES-YAP, and GROUCHO/TLE). Runx2 levels oscillate during the osteoblast cell cycle with maximal levels in G(1). Here we examined what functions and target genes of Runx2 control osteoblast growth. Forced expression of wild type Runx2 suppresses growth of Runx2(-/-) osteoprogenitors. Point mutants defective for binding to WW domain or SMAD proteins or the nuclear matrix retain this growth regulatory ability. Hence, key signaling pathways are dispensable for growth control by Runx2. However, mutants defective for DNA binding or C-terminal gene repression/activation functions do not block proliferation. Target gene analysis by Affymetrix expression profiling shows that the C terminus of Runx2 regulates genes involved in G protein-coupled receptor signaling (e.g. Rgs2, Rgs4, Rgs5, Rgs16, Gpr23, Gpr30, Gpr54, Gpr64, and Gna13). We further examined the function of two genes linked to cAMP signaling as follows: Gpr30 that is stimulated and Rgs2 that is down-regulated by Runx2. RNA interference of Gpr30 and forced expression of Rgs2 in each case inhibit osteoblast proliferation. Notwithstanding its growth-suppressive potential, our results surprisingly indicate that Runx2 may sensitize cAMP-related G protein-coupled receptor signaling by activating Gpr30 and repressing Rgs2 gene expression in osteoblasts to increase responsiveness to mitogenic signals.

Chromatin immunoprecipitation assays: application of ChIP-on-chip for defining dynamic transcriptional mechanisms in bone cells.

Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activation and repression of distinct sets of genes support the anabolic functions of osteoblasts and the catabolic properties of osteoclasts. Furthermore, metastasis of tumors to the bone environment is controlled by transcriptional mechanisms. Insights into the transcriptional regulation of genes in bone cells may provide a conceptual basis for improved therapeutic approaches to treat bone fractures, genetic osteopathologies, and/or cancer metastases to bone. Chromatin immunoprecipitation (ChIP) is a powerful technique to establish in vivo binding of transcription factors to the promoters of genes that are either activated or repressed in bone cells. Combining ChIP with genomic microarray analysis, colloquially referred to as "ChIP-on-chip," has become a valuable method for analysis of endogenous protein/DNA interactions. This technique permits assessment of chromosomal binding sites for transcription factors or the location of histone modifications at a genomic scale. This chapter discusses protocols for performing chromatin immunoprecipitation experiments, with a focus on ChIP-on-chip analysis. The information presented is based on the authors' experience with defining interactions of Runt-related (RUNX) transcription factors with bone-related genes within the context of the native nucleosomal organization of intact osteoblastic cells.

Integrating high-content screening and ligand-target prediction to identify mechanism of action.

High-content screening is transforming drug discovery by enabling simultaneous measurement of multiple features of cellular phenotype that are relevant to therapeutic and toxic activities of compounds. High-content screening studies typically generate immense datasets of image-based phenotypic information, and how best to mine relevant phenotypic data is an unsolved challenge. Here, we introduce factor analysis as a data-driven tool for defining cell phenotypes and profiling compound activities. This method allows a large data reduction while retaining relevant information, and the data-derived factors used to quantify phenotype have discernable biological meaning. We used factor analysis of cells stained with fluorescent markers of cell cycle state to profile a compound library and cluster the hits into seven phenotypic categories. We then compared phenotypic profiles, chemical similarity and predicted protein binding activities of active compounds. By integrating these different descriptors of measured and potential biological activity, we can effectively draw mechanism-of-action inferences.

Chemogenomic data analysis: prediction of small-molecule targets and the advent of biological fingerprint.

Chemogenomics comprises a systematic relationship between targets and ligands that are used as target modulators in living systems such as cells or organisms. In recent years, data on small molecule-bioactivity relationships have become increasingly available, and consequently so have the number of approaches used to translate bioactivity data into knowledge. This review will focus on two aspects of chemogenomics. Firstly, in cases such as cell-based screens, the question of which target(s) a compound is modulating in order to cause the observed phenotype is crucial. In silico target prediction tools can suggest likely biological targets of small molecules via data mining in target-annotated chemical databases. This review presents some of the current tools available for this task and shows some sample applications relevant to a pharmaceutical industry setting. These applications are the prediction of false-positives in cell-based reporter gene assays, the prediction of targets by linking bioassay data with protein domain annotations, and the direct prediction of adverse reactions. Secondly, in recent years a shift from structure-derived chemical descriptors to biological descriptors has occurred. Here, the effect of a compound on a number of biological endpoints is used to make predictions about other properties, such as putative targets, associated adverse reactions, and pathways modulated by the compound. This review further summarizes these "performance" descriptors and their applications, focusing on gene expression profiles and high-content screening data. The advent of such biological fingerprints suggests that the field of drug discovery is currently at a crossroads, where single target bioassay results are supplanted by multidimensional biological fingerprints that reflect a new awareness of biological networks and polypharmacology.

Nuclear microenvironments in biological control and cancer.

Nucleic acids and regulatory proteins are compartmentalized in microenvironments within the nucleus. This subnuclear organization may support convergence and the integration of physiological signals for the combinatorial control of gene expression, DNA replication and repair. Nuclear organization is modified in many cancers. There are cancer-related changes in the composition, organization and assembly of regulatory complexes at intranuclear sites. Mechanistic insights into the temporal and spatial organization of machinery for gene expression within the nucleus, which is compromised in tumours, provide a novel platform for diagnosis and therapy.