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ABSTRACT: The molecular changes induced by alemtuzumab following binding of CD52 on B tumor cells were investigated. Alemtuzumab alone had no detectable impact on cell signaling but cross-linking of alemtuzumab on the surface of B tumor lines with anti-human Fc antibodies induced a transient Ca(2+) flux followed by phosphorylation of several kinases involved in stress and survival pathways, and expression of associated proteins including TNF-α. Cross-linking of alemtuzumab also induced capping and caspase-dependent apoptosis of the tumor lines. When using primary cells from B-CLL patients, alemtuzumab alone was capable of inducing protein phosphorylation and apoptosis through the cross-linking of alemtuzumab by FcγRIIb receptors on B-CLL cells. Apoptosis was prevented by blocking of FcγRIIb receptors with anti-CD32 antibody. Overall, our results indicate that cross-linking of alemtuzumab on B tumor cells can occur naturally through Fc receptor interaction and leads to the activation of specific cellular pathways and induction of apoptosis.
Leukemia & lymphoma 09/2011; 53(4):699-709. · 2.40 Impact Factor
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ABSTRACT: Netrin-4 is a 628 amino acid basement membrane component that promotes neurite elongation at low concentrations but inhibits neurite extension at high concentrations. There is a growing body of literature suggesting that several molecules, including netrins, are regulators of both neuronal and vascular growth. It is believed that molecules that guide neural growth and development are also involved in regulating morphogenesis of the vascular tree. Further, netrins have recently been implicated in controlling epithelial cell branching morphogenesis in the breast, lung and pancreas. Characterization of purified netrin-4 in in vitro angiogenesis assays demonstrated that netrin-4 markedly inhibits HMVEC migration and tube formation. Moreover, netrin-4 inhibits proliferation of a variety of human tumor cells in vitro. Netrin-4 has only modest effects on proliferation of endothelial and other non-transformed cells. Netrin-4 treatment results in phosphorylation changes of proteins that are known to control cell growth. Specifically, Phospho-Akt-1, Phospho-Jnk-2, and Phospho-c-Jun are reduced in tumor cells that have been treated with netrin-4. Together, these data suggest a potential role for netrin-4 in regulating tumor growth.
Experimental Cell Research 01/2009; 315(5):784-94. · 3.58 Impact Factor
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ABSTRACT: Lytic development of bacteriophage Mu is controlled by a regulatory cascade and involves three phases of transcription: early, middle and late. Late transcription requires the host RNA polymerase holoenzyme and a 16.5-kDa Mu-encoded activator protein C. Consistent with these requirements, the four late promoters P(lys), P(I), P(P) and P(mom) have recognizable -10 hexamers but lack typical -35 hexamers. The C protein binds to a 16-bp imperfect dyad-symmetrical sequence element centered at -43.5 and overlapping the -35 region. Based on the crystal structure of the closely related Mor protein, the activator of Mu middle transcription, we predict that two regions of C are involved in DNA binding: a helix-turn-helix region and a beta-strand region linking the dimerization and helix-turn-helix domains. To test this hypothesis, we carried out mutagenesis of the corresponding regions of the C gene by degenerate oligonucleotide-directed PCR and screened the resulting mutants for their ability to activate a P(lys)-galK fusion. Analysis of the mutant proteins by gel mobility shift, beta-galactosidase and polyacrylamide gel electrophoresis assays identified a number of amino acid residues important for C DNA binding in both regions.
Nucleic Acids Research 11/2008; 36(20):6396-405. · 8.03 Impact Factor
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ABSTRACT: Saccharomyces cerevisiae forms monounsaturated fatty acids using the ER membrane-bound Delta-9 fatty acid desaturase, Ole1p, an enzyme system that forms a double bond in saturated fatty acyl CoA substrates. Ole1p is a chimeric protein consisting of an amino terminal desaturase domain fused to cytochrome b5. It catalyzes the formation of the double bond through an oxygen-dependent mechanism that requires reducing equivalents from NADH. These are transferred to the enzyme via NADH cytochrome b5 reductase to the Ole1p cytochrome b5 domain and then to the diiron-oxo catalytic center of the enzyme. The control of OLE1 gene expression appears to mediated through the ER membrane proteins Spt23p and Mga2p. N-terminal fragments of these proteins are released by an ubiquitin/proteasome mediated proteolysis system and translocated to the nucleus where they appear to act as transcription coactivators of OLE1. OLE1 is regulated through Spt23p and Mga2p by multiple systems that control its transcription and mRNA stability in response to diverse stimuli that include nutrient fatty acids, carbon source, metal ions and the availability of oxygen.
Biochimica et Biophysica Acta 04/2007; 1771(3):271-85. · 4.66 Impact Factor
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Belinda S Parker,
Pedram Argani,
Brian P Cook,
Han Liangfeng,
Scott D Chartrand,
Mindy Zhang,
Saurabh Saha,
Alberto Bardelli, Yide Jiang,
Thia B St Martin,
Mariana Nacht,
Beverly A Teicher,
Katherine W Klinger,
Saraswati Sukumar,
Stephen L Madden
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ABSTRACT: The molecular signature that defines tumor microvasculature will likely provide clues as to how vascular-dependent tumor proliferation is regulated. Using purified endothelial cells, we generated a database of gene expression changes accompanying vascular proliferation in invasive breast cancer. In contrast to normal mammary vasculature, invasive breast cancer vasculature expresses extracellular matrix and surface proteins characteristic of proliferating and migrating endothelial cells. We define and validate the up-regulated expression of VE-cadherin and osteonectin in breast tumor vasculature. In contrast to other tumor types, invasive breast cancer vasculature induced a high expression level of specific transcription factors, including SNAIL1 and HEYL, that may drive gene expression changes necessary for breast tumor neovascularization. We demonstrate the expression of HEYL in tumor endothelial cells and additionally establish the ability of HEYL to both induce proliferation and attenuate programmed cell death of primary endothelial cells in vitro. We also establish that an additional intracellular protein and previously defined metastasis-associated gene, PRL3, appears to be expressed predominately in the vasculature of invasive breast cancers and is able to enhance the migration of endothelial cells in vitro. Together, our results provide unique insights into vascular regulation in breast tumors and suggest specific roles for genes in driving tumor angiogenesis.
Cancer Research 12/2004; 64(21):7857-66. · 7.86 Impact Factor
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Stephen L Madden,
Brian P Cook,
Mariana Nacht,
William D Weber,
Michelle R Callahan, Yide Jiang,
Michael R Dufault,
Xiaoming Zhang,
Wen Zhang,
Jennifer Walter-Yohrling, [......],
Xiaohong Cao,
Thia B St Martin,
Bruce L Roberts,
Beverly A Teicher,
Katherine W Klinger,
Radu-Virgil Stan,
Brenden Lucey,
Eleanor B Carson-Walter,
John Laterra,
Kevin A Walter
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ABSTRACT: Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression.
American Journal Of Pathology 09/2004; 165(2):601-8. · 4.89 Impact Factor
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ABSTRACT: The von Hippel-Lindau tumor suppressor, pVHL, is a key player in one of the best characterized hypoxia signaling pathways, the VHL-hypoxia-inducible factor (VHL-HIF) pathway. To better understand the role of VHL in the hypoxia signaling pathways of tumor cells, we used serial analysis of gene expression (SAGE) to investigate hypoxia-regulated gene expression in renal carcinoma cells (786-0), with and without VHL. The gene expression profiles of the cancer cells were compared to SAGE profiles from normal renal proximal tubule cells grown under both normoxia and hypoxia. The data suggest that the role of VHL as a tumor suppressor may be more complex than previously thought. Further, the data reveal that renal carcinoma cells have evolved an alternative hypoxia signaling pathway(s) compared with normal renal cells. These alternative hypoxia pathways demonstrate VHL-dependent and VHL-independent regulation. The genes involved in such pathways include those with potential importance in the physiological and pathological regulation of tumor growth and angiogenesis. Some of the genes identified as showing overexpression in the cancer cells, particularly those encoding secreted or membrane-bound proteins, could be potential biomarkers for tumors or targets for rational therapeutics that are dependent on VHL status.
Molecular Cancer Research 05/2003; 1(6):453-62. · 4.29 Impact Factor
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ABSTRACT: In Saccharomyces cerevisiae, OLE1 encodes a delta9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions.
Eukaryotic Cell 07/2002; 1(3):481-90. · 3.60 Impact Factor
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ABSTRACT: The Saccharomyces OLE1gene encodes the Δ-9 fatty acid desaturase, an enzyme that converts saturated fatty acyl-CoAs into cis-Δ-9 unsaturated fatty acids. OLE1 gene expression is regulated by unsaturated fatty acids, which repress transcription and destabilize theOLE1 mRNA. Expression of OLE1 is activated by N-terminal proteolytic fragments of two homologous endoplasmic reticulum membrane proteins, Spt23p and Mga2p.
Disruption of either gene does not significantly affect cell growth or fatty acid metabolism; cells that contain null alleles
of both genes, however, are unsaturated fatty acid auxotrophs. An analysis of spt23Δand mga2Δ strains shows that Spt23p and Mga2p differentially activate and regulate OLE1 transcription. In glucose-grown cells, both genes activate transcription to similar levels of activity. Expressed alone,
Mga2p induces high levels ofOLE1 transcription in cells exposed to cobalt or grown in glycerol-containing medium. Spt23p expressed alone activatesOLE1 transcription to levels similar to those in wild type cells. OLE1 expression is strongly repressed by unsaturated fatty acids in spt23Δ or mga2Δ cells, under all growth conditions. To test if OLE1 expression is controlled by fatty acids at the level of membrane proteolysis, soluble N-terminal fragments of Spt23p and
Mga2p that lack their membrane-spanning regions (Δtm) were expressed under the control of their native promoters in spt23Δ;mga2Δcells. Under those conditions, Mga2pΔtm acts as a powerful transcription activator that is strongly repressed by unsaturated
fatty acids. By comparison, the Spt23pΔtm polypeptide weakly activates transcription and shows little regulation by unsaturated
fatty acids. Co-expression of the two soluble fragments results in activation to levels observed with the Mga2pΔtm protein
alone. The fatty acid repression of transcription under those conditions is attenuated by Spt23Δtm, however, suggesting that
the two proteins may interact to modulate OLE1 gene expression.
Journal of Biological Chemistry 11/2001; 276(47):43548-43556. · 4.77 Impact Factor
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ABSTRACT: An organism's ability to respond to changes in oxygen tension depends in large part on alterations in gene expression. The
oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood. To further define these processes, we
have studied the Δ9 fatty acid desaturase gene OLE1 inSaccharomyces cerevisiae. We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals.OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3′ to
the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs. Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia
and cobalt. Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation. A 20-base pair
oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed
upstream of a heterologous promoter in alacZ reporter construct. Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar toOLE1. A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators. Hence, the identification
of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in
eukaryotes.
Journal of Biological Chemistry 04/2001; 276(17):14374-14384. · 4.77 Impact Factor
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Ann Byrne,
Rajashree P. McLaren,
Paul Mason,
Lilly Chai,
Michael R. Dufault,
Yinyin Huang,
Beirong Liang,
Joseph D. Gans,
Mindy Zhang,
Kara Carter,
Tatiana B. Gladysheva,
Beverly A. Teicher,
Hans-Peter N. Biemann,
Michael Booker,
Mark A. Goldberg,
Katherine W. Klinger,
James Lillie,
Stephen L. Madden, Yide Jiang
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ABSTRACT: The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1–CDK1–CDC25C, down-regulation of cyclin D1 and up-regulation of p21/Cip and p27/Kip1. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.
Experimental Cell Research.
