A High-resolution, Fluorescence-based Method for Localization of Endogenous Alkaline Phosphatase Activity

Cox, William G.; Singer, Victoria L.

doi:10.1177/002215549904701110

Cited by 76 publications

(60 citation statements)

References 31 publications

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“…Genetically manipulated mouse Jmjd1a/G9a knockout (KO) ES cells as well as G9a KO plus transgene (Tg) ES cells have been described previously (14,17). For detection of alkaline phosphatase activity, cells were plated on 6-well plates at low density (300 cells/well) and stained as previously described (25) female mice were mated to obtain embryos. Blastocysts were flushed from oviducts at 3.5 days postcoitum and cultured in potassium modified simplex optimized medium (KSOM) (MR-020P-5D; Millipore, Watford, United Kingdom).…”

Section: Methodsmentioning

confidence: 99%

The Hypoxia-Inducible Epigenetic Regulators Jmjd1a and G9a Provide a Mechanistic Link between Angiogenesis and Tumor Growth

Ueda

Lee

et al. 2014

Molecular and Cellular Biology

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dHypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression. Growing solid tumors develop shortages of oxygen (hypoxia) and nutrient supplies. Cancer cells induce the formation of new blood vessels from surrounding host tissues to overcome these adverse conditions. Hypoxia-inducible factors (HIFs) drive the transcriptome of cells to adapt to low-oxygen conditions (1-3). It is now increasingly evident that cancer cells adapted to hypoxia have more-malignant phenotypes (4-7). These findings raise the possibility that hypoxia effectuates long-term adaptation to low-oxygen conditions by altering the epigenetic landscape of cancer cells. However, how hypoxia, HIFs, and possibly other coregulated proteins affect the epigenetic status of oxygen-deprived cancer cells to promote malignancy remains largely unknown.To examine the impact of hypoxia on cancers, we previously examined global gene expression profiles of seven human cell lines of diverse cancer origins and found that JMJD1A (Jumonji domain-containing 1A, also known as JHDM2A and KDM3A) is one of the epigenetic factors most commonly induced by hypoxia (3). Consistent with this result, other studies have shown that JMJD1A and other Jumonji domain-containing proteins are upregulated in response to hypoxia and that HIF-1␣ is directly involved in this activation (8, 9). Jumonji domain-containing proteins have been identified as histone lysine demethylases and require Fe(II) and ␣-ketoglutarate for their catalytic activity (10). Among them, JMJD1A demethylates mono-and dimethylated histone H3 lysine 9 (H3K9) residues. JMJD1A also acts as a coactivator in cooperation with the androgen receptor (11), and it has been proposed to operate downstream of HIF-1␣ to activate target genes under hypoxic conditions (9). While Jmjd1a Ϫ/Ϫ mice are viable, males show defects in spermatogenesis leading to infertility, and both genders display an obese phenotype (12)(13)(...

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Section: Methodsmentioning

confidence: 99%

The Hypoxia-Inducible Epigenetic Regulators Jmjd1a and G9a Provide a Mechanistic Link between Angiogenesis and Tumor Growth

Ueda

Lee

et al. 2014

Molecular and Cellular Biology

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“…For immunofluorescent detection of ERK1/2, Alexa Fluor 594-conjugated secondary antibody (Invitrogen) was used. Fluorescence-based tartrate-resistant acid phosphatase (TRAP) staining and alkaline phosphatase staining were performed using ELF97 (Invitrogen) as a phosphatase substrate (6,10). To reduce background, TRAP staining using ELF97 was performed in the presence of 50 mM tartrate.…”

Section: Methodsmentioning

confidence: 99%

Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Play Essential Roles in Osteoblast Differentiation and in Supporting Osteoclastogenesis

Matsushita

Chan

Kawanami

et al. 2009

Molecular and Cellular Biology

205

181

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Osteoblasts and chondrocytes arise from common osteo-chondroprogenitor cells. We show here that inactivation of ERK1 and ERK2 in osteo-chondroprogenitor cells causes a block in osteoblast differentiation and leads to ectopic chondrogenic differentiation in the bone-forming region in the perichondrium. Furthermore, increased mitogen-activated protein kinase signaling in mesenchymal cells enhances osteoblast differentiation and inhibits chondrocyte differentiation. These observations indicate that extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in the lineage specification of mesenchymal cells. The inactivation of ERK1 and ERK2 resulted in reduced beta-catenin expression, suggesting a role for canonical Wnt signaling in ERK1 and ERK2 regulation of skeletal lineage specification. Furthermore, inactivation of ERK1 and ERK2 significantly reduced RANKL expression, accounting for a delay in osteoclast formation. Thus, our results indicate that ERK1 and ERK2 not only play essential roles in the lineage specification of osteo-chondroprogenitor cells but also support osteoclast formation in vivo.The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK MAPK) pathway is activated by various stimuli, including a number of growth factors and cytokines. The activation of the Raf members of MAPK kinase kinase leads to the activation of the MAPK kinase, MEK1, and MEK2. MEK1 and MEK2 then phosphorylate and activate MAPK, ERK1, and ERK2. ERK1 and ERK2 phosphorylate various cytoplasmic and nuclear target proteins, ranging from cytoplasmic adaptor proteins and transcription factors to kinases, including ribosomal S6 kinase (RSK) (4,17,18,23,36,46). In this pathway, multiple mutations that cause syndromes with various skeletal manifestations have recently been identified. Missense-activating mutations in KRAS, BRAF, MEK1, and MEK2 have been identified in Costello, Noonan, LEOPARD, and Cardio-facio-cutaneous syndromes, while loss-of-function mutations in RSK2, a kinase downstream of ERK1 and ERK2, cause Coffin-Lowry syndrome (2, 42). These observations highlight the importance of the ERK MAPK pathway in human skeletal development.Both chondrocytes and osteoblasts arise from common osteo-chondro progenitor cells. Bone growth is achieved through two major ossification processes, endochondral ossification and intramembranous ossification, in which chondrocytes and osteoblasts are involved (5,13,30,31,38). In normal endochondral ossification, the skeletal element is formed as a cartilaginous template that is subsequently replaced by bone. Condensed mesenchymal cells differentiate into chondrocytes. Chondrocytes first proliferate in columnar stacks to form the growth plate and then exit the cell cycle and differentiate into hypertrophic chondrocytes. Hypertrophic chondrocytes are removed by apoptotic cell death, and the cartilaginous matrix is resorbed by chondroclasts/osteoclasts and replaced by trabecular bone. Chondroclast/osteoclast formation is supported by receptor activator of nucl...

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“…III D). [59][60][61] The procedure mentioned above significantly enhanced the transportation of SPBs compared to the case where the procedure was not performed prior to experiments.…”

Section: B Optimization Of Chip Surface and Channel Wallsmentioning

confidence: 99%

Isolation of cells for selective treatment and analysis using a magnetic microfluidic chip

et al. 2014

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This study describes the development and testing of a magnetic microfluidic chip (MMC) for trapping and isolating cells tagged with superparamagnetic beads (SPBs) in a microfluidic environment for selective treatment and analysis. The trapping and isolation are done in two separate steps; first, the trapping of the tagged cells in a main channel is achieved by soft ferromagnetic disks and second, the transportation of the cells into side chambers for isolation is executed by tapered conductive paths made of Gold (Au). Numerical simulations were performed to analyze the magnetic flux and force distributions of the disks and conducting paths, for trapping and transporting SPBs. The MMC was fabricated using standard microfabrication processes. Experiments were performed with E. coli (K12 strand) tagged with 2.8 μm SPBs. The results showed that E. coli can be separated from a sample solution by trapping them at the disk sites, and then isolated into chambers by transporting them along the tapered conducting paths. Once the E. coli was trapped inside the side chambers, two selective treatments were performed. In one chamber, a solution with minimal nutrition content was added and, in another chamber, a solution with essential nutrition was added. The results showed that the growth of bacteria cultured in the second chamber containing nutrient was significantly higher, demonstrating that the E. coli was not affected by the magnetically driven transportation and the feasibility of performing different treatments on selectively isolated cells on a single microfluidic platform.

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A High-resolution, Fluorescence-based Method for Localization of Endogenous Alkaline Phosphatase Activity

Cited by 76 publications

References 31 publications

The Hypoxia-Inducible Epigenetic Regulators Jmjd1a and G9a Provide a Mechanistic Link between Angiogenesis and Tumor Growth

The Hypoxia-Inducible Epigenetic Regulators Jmjd1a and G9a Provide a Mechanistic Link between Angiogenesis and Tumor Growth

Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Play Essential Roles in Osteoblast Differentiation and in Supporting Osteoclastogenesis

Isolation of cells for selective treatment and analysis using a magnetic microfluidic chip

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