Copper and angiogenesis: unravelling a relationship key to cancer progression.

1. Angiogenesis, the formation of new capillaries from existing vasculature, is a critical process in normal physiology as well as several physiopathologies. A desire to curb the supportive role angiogenesis plays in the development and metastasis of cancers has driven exploration into anti-angiogenic strategies as cancer therapeutics.

X-ray fluorescence microscopy reveals large-scale relocalization and extracellular translocation of cellular copper during angiogenesis.

Although copper has been reported to influence numerous proteins known to be important for angiogenesis, the enhanced sensitivity of this developmental process to copper bioavailability has remained an enigma, because copper metalloproteins are prevalent and essential throughout all cells. Recent developments in x-ray optics at third-generation synchrotron sources have provided a resource for highly sensitive visualization and quantitation of metalloproteins in biological samples. Here, we report the application of x-ray fluorescence microscopy (XFM) toin vitro models of angiogenesis and neurogenesis, revealing a surprisingly dramatic spatial relocalization specific to capillary formation of 80-90% of endogenous cellular copper stores from intracellular compartments to the tips of nascent endothelial cell filopodia and across the cell membrane.

Subtractive transcriptomics: establishing polarity drives in vitro human endothelial morphogenesis.

Although investigations of mature normal and tumor-derived capillaries have resulted in characterization of these structures at the phenotypic level, less is known regarding the initial molecular cues for cellular assembly of endothelial cells into human capillaries. Here, we employ a novel combination of microenvironmental manipulation and microarray data filtration over narrowly delineated temporal data series to identify the morphogenesis component apart from the proliferation component, as pooled human microvascular-derived endothelial cells are induced to form capillary-like structures in vitro in a murine tumor-derived matrix. The 217 morphogenesis-specific genes identified using this subtractive transcriptomics approach are mostly independent of the angiogenic proteins currently used as therapeutic targets for aberrant angiogenesis.

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells.

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction.

Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

To gain insight into the function of human protein kinase X (PrKX), a signal-transduction protein required for macrophage differentiation, we identified regulatory subunit I alpha of protein kinase A, T54 and Smad6 as partners for this protein using a yeast two-hybrid interaction screen. Interactions between PrKX and these proteins were substantiated by co-immunoprecipitation. Interaction between Smad6 and PrKX was also confirmed in human myeloid HL-60 cells following their phorbol 12-myristate 13-acetate (PMA)-induced differentiation into macrophages.

Analysis of protein interaction and function with a 3-dimensional MALDI-MS protein array.

Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions.

Spermine acts as a negative regulator of macrophage differentiation in human myeloid leukemia cells.

The role of putrescine, spermidine and spermine in phorbol 12-myristate-13-acetate (PMA)-induced macrophage differentiation was examined in human HL-60 and U-937 myeloid leukemia cells. Unlike other polyamines, spermine affected this differentiation by acting as a negative regulator. This negative regulation was established by showing that the PMA-induced macrophage phenotype, but not PMA-associated replication arrest, was abrogated (a) by replenishing the PMA-evoked decrease in cellular spermine levels with this polyamine from an exogenous source and (b) by blocking PMA-induced expression of the polyamine catabolic enzyme N(1)-spermidine/spermine acetyltransferase (SSAT) with antisense oligonucleotides in the presence of low substrate level.

The crystal structure of Escherichia coli MoaB suggests a probable role in molybdenum cofactor synthesis.

The crystal structure of Escherichia coli MoaB was determined by multiwavelength anomalous diffraction phasing and refined at 1.6-A resolution. The molecule displayed a modified Rossman fold.

Microarray analysis of changes in bone cell gene expression early after cadmium gavage in mice.

We developed an in vivo model for cadmium-induced bone loss in which mice excrete bone mineral in feces beginning 8 h after cadmium gavage. Female mice of three strains [CF1, MTN (metallothionein-wild-type), and MT1,2KO (MT1,2-deficient)] were placed on a low-calcium diet for 2 weeks. Each mouse was gavaged with 200 microg Cd or vehicle only.

Bridging the electrochemical and biological worlds with hybrid nanocomposites.

Recent discoveries arising from a combination of the biological, physical, chemical and materials sciences have resulted in the invention of numerous hybrid molecules that possess strengths inherent to each individual discipline. Nanocomposites that link biological molecules to inorganic moieties have led to a family of new reagents with unique capabilities for cellular imaging and macromolecule detection. A recent report has extended the applications of these hybrid molecules from their use as detection and scaffolding reagents into the realm of a biologically functional molecule.

A human peripheral blood monocyte-derived subset acts as pluripotent stem cells.

We have identified, cultured, characterized, and propagated adult pluripotent stem cells (PSC) from a subset of human peripheral blood monocytes. These cells, which in appearance resemble fibroblasts, expand in the presence of macrophage colony-stimulating factor and display monocytic and hematopoietic stem cell markers including CD14, CD34, and CD45. We have induced these cells to differentiate into mature macrophages by lipopolysaccharide, T lymphocytes by IL-2, epithelial cells by epidermal growth factor, endothelial cells by vascular endothelial cell growth factor, neuronal cells by nerve growth factor, and liver cells by hepatocyte growth factor.

Protein kinase C-beta, fibronectin, alpha(5)beta(1)-integrin, and tumor necrosis factor-alpha are required for phorbol diester-induced apoptosis in human myeloid leukemia cells.

The human myeloid HL-60 cell line and its cell variant HL-525 were used to study signaling events leading to apoptosis induction by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC) enzymes. Unlike parental cells, HL-525 cells are PKC-beta deficient and resistant to PMA-induced apoptosis. These cells regain susceptibility to apoptosis induction after transfection with a PKC-beta expression vector.

Involvement of protein kinase C-beta and ceramide in tumor necrosis factor-alpha-induced but not Fas-induced apoptosis of human myeloid leukemia cells.

The role of protein kinase C-beta (PKC-beta) in apoptosis induced by tumor necrosis factor (TNF)-alpha and anti-Fas monoclonal antibody (mAb) in the human myeloid HL-60 leukemia cell line was studied by using its variant HL-525, which is deficient in PKC-beta. In contrast to the parental HL-60 cells, HL-525 is resistant to TNF-alpha-induced apoptosis but sensitive to anti-Fas mAb-induced apoptosis. Both cell types expressed similar levels of the TNF-receptor I, whereas the Fas receptor was detected only in HL-525 cells.

A lineage-specific protein kinase crucial for myeloid maturation.

To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells.

Cloning and sequence of the human type II IMP dehydrogenase gene.

Human type II inosine 5'-monophosphate dehydrogenase (EC 1.1.1.

Differential induction of apoptosis in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A.

To investigate a possible relationship between apoptosis induction and protein phosphorylation in human breast carcinoma cells, we treated three such cell types, MB-231, MCF-7, and AU-565, with okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, or phorbol 12 myristate 13-acetate, an activator of protein kinase C. We then examined these cells for the appearance of apoptosis markers. While OA caused multiplication arrest and cytotoxicity in all three cell lines, apoptosis was induced in MB-231 and MCF-7 cells but not in AU-565 cells.

Chromosomal localization and structure of the human type II IMP dehydrogenase gene (IMPDH2).

We determined the chromosomal localization and structure of the gene encoding human type II inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.

Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner.