David Glesne

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. The pluripotent nature of individual PSC was further confirmed by a clonal analysis. The ability to store, expand, and differentiate these PSC from autologous peripheral blood should make them valuable candidates for transplantation therapy.

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. Key to this, angiogenesis additionally displays an exquisite sensitivity to bioavailable copper. Depletion of copper has been shown to inhibit angiogenesis in a wide variety of cancer cell and xenograft systems. Several clinical trials using copper chelation as either an adjuvant or primary therapy have been conducted. Yet, the biological basis for the sensitivity of angiogenesis remains unclear. Numerous molecules important to angiogenesis regulation have been shown to be either directly or indirectly influenced by copper, yet a clear probative answer to the connection remains elusive. 2 Measurements of copper in biological systems have historically relied on techniques that, although demonstrably powerful, provide little or no information as to the spatial distribution of metals in a cellular context. Therefore, several new approaches have been developed to image copper in a biological context. One such approach relies on synchrotron‐derived X‐rays from third‐generation synchrotrons and the technique of high resolution X‐ray fluorescence microprobe (XFM) analysis. 3 Recent applications of XFM approaches to the role of copper in regulating angiogenesis have provided unique insight into the connection between copper and cellular behaviour. Using XFM, copper has been shown to be highly spatially regulated, as it is translocated from perinuclear areas of the cell towards the tips of extending filopodia and across the cell membrane into the extracellular space during angiogenic processes. Such findings may explain the heightened sensitivity of this cellular process to this transition metal and set a new paradigm for the kinds of regulatory roles that the spatial dynamics of cellular transition metals may play.

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. Although copper chelation had no effect on process formation, an almost complete ablation of network formation was observed. XFM of highly vascularized ductal carcinomas showed copper clustering in putative neoangiogenic areas. This use of XFM for the study of a dynamic developmental process not only sheds light on the copper requirement for endothelial tube formation but highlights the value of synchrotron-based facilities in biological research.

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. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.

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. Fecal calcium was monitored daily for 9 days, beginning 4 days before cadmium gavage, to document the bone response. For CF1 mice, bones were taken from four groups: +/- Cd, 2 h after Cd and +/- Cd, 4 h after Cd. MTN and MT1,2KO strains had two groups each: +/-Cd, 4 h after Cd. PolyA+ RNA preparations from marrow-free shafts of femura and tibiae of each +/- Cd pair were submitted to Incyte Genomics for microarray analysis. Fecal Ca results showed that bone calcium excreted after cadmium differed for the three mouse strains: CF1, 0.24 +/- 0.08 mg; MTN, 0.92 +/- 0.22 mg; and MT1,2KO, 1.7 +/- 0.4 mg. Gene array results showed that nearly all arrayed genes were unaffected by cadmium. However, MT1 and MT2 had Cd+/Cd- expression ratios >1 in all four groups, while all ratios for MT3 were essentially 1, showing specificity. Both probes for MAPK 14 (p38 MAPK) had expression ratios >1, while no other MAPK responded to cadmium. Vacuolar proton pump ATPase and integrin alpha v (osteoclast genes), transferrin receptor, and src-like adaptor protein genes were stimulated by Cd; other src-related genes were unaffected. Genes for bone formation, stress response, growth factors, and signaling molecules showed little or no response to cadmium. Results support the hypothesis that Cd stimulates bone demineralization via a p38 MAPK pathway involving osteoclast activation.

Involvement of Protein Kinase C-β and Ceramide in Tumor Necrosis Factor-α-induced but Not Fas-induced Apoptosis of Human Myeloid Leukemia Cells

The role of protein kinase C-β (PKC-β) in apoptosis induced by tumor necrosis factor (TNF)-α 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-β. In contrast to the parental HL-60 cells, HL-525 is resistant to TNF-α-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. Transfecting the HL-525 cells with an expression vector containing PKC-β reestablished their susceptibility to TNF-α-induced apoptosis. The apoptotic effect of TNF-α in HL-60 and the transfectants was abrogated by fumonisin, an inhibitor of ceramide generation, and by the peptide Ac-YVAD-BoMK, an inhibitor of caspase-1 and -4. Supplementing HL-525 cells with exogenous ceramides bypassed the PKC-β deficiency and induced apoptosis, which was also restrained by the caspase-1 and -4 inhibitor. The apoptotic effect of anti-Fas mAb in HL-525 cells was abrogated by the antioxidantsN-acetylcysteine and glutathione and by the peptide z-DEVD-FMK, an inhibitor of caspase-3 and -7. We suggest that TNF-α-induced apoptosis involves PKC-β and then ceramide and, in turn, caspase-1 and/or -4, whereas anti-Fas mAb-induced apoptosis utilizes reactive oxygen intermediates and, in turn, caspase-3 and/or -7.

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. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.

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. Quantitative real-time PCR was used to validate 20% of these transcripts. Immunofluorescent analysis of proliferating and tube-forming cells validates at the protein level the morphogenesis-specific expression pattern of 16 of the 217 gene products identified. The transcripts that are selectively up-regulated in tube-forming endothelial cells reveal a temporal expression pattern of genes primarily associated with intracellular trafficking, guided migration, cytoskeletal reorganization, cellular adhesion, and proliferation inhibition. These data show that a sequential up-regulation of genes that establish and maintain polarity occurs during migration and morphogenesis of in vitro human endothelial cells undergoing tubulogenesis; some of which may well be effective as novel antiangiogenic drug targets.

Protein kinase C-β, fibronectin, α5β1-integrin, and tumor necrosis factor-α 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‐β deficient and resistant to PMA‐induced apoptosis. These cells regain susceptibility to apoptosis induction after transfection with a PKC‐β expression vector. By using this vector and specific neutralizing monoclonal antibodies (mAbs), it was established that PMA‐induced apoptosis also called for an interaction between cell‐surface α5β1‐integrin and its deposited ligand fibronectin (FN), which is downstream of PKC‐β activation. Experiments with mAbs, the PKC‐β vector, and exogenous FN revealed that the next step entailed an interaction between secreted tumor necrosis factor‐α and its type I receptor. By using a sphingomyelinase inhibitor, it was concluded that the subsequent step involved ceramide production. Moreover, a permeable ceramide was effective in inducing apoptosis in both HL‐60 and HL‐525 cells, and this induction was caspase‐1 and/or ‐4 dependent because an inhibitor of these caspases abrogated the induced apoptosis. Based on these and related differentiation studies, we conclude that the above signaling events, the early ones in particular, are shared with PMA‐induced macrophage differentiation in the HL‐60 cells. It is likely that once these cells acquire their macrophage phenotype and perform their tasks, they become superfluous and are eliminated from the body by a self‐triggered apoptotic process that involves our proposed signaling scheme.

Regulation and Role of Inosine-5’-Monophosphate Dehydrogenase in Cell Replication, Malignant Transformation, and Differentiation

Inosine-5’-monophosphate dehydrogenase (EC 1.1.1.205, IMPDH) is a branch point enzyme in the synthesis of adenine and guanine nucleotides and is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides.1 This enzyme has an essential role in providing necessary precursors for DNA and RNA biosynthesis, a role that can be verified by the abrupt cessation of DNA synthesis when cells are treated with IMPDH inhibitors and by the circumvention of this effect with the addition of exogenous guanosine.2