James P. Quigley

The Scripps Research Institute, لا هویا, California, United States

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Publications (164)983.62 Total impact

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    ABSTRACT: Urokinase-type plasminogen activator (uPA) is a potential therapeutic target in cancer. Yet, classical development of small molecule inhibitors of uPA has proved a daunting task, with only limited success in engineering high affinity and specificity among related proteases having conserved active site architecture In nature, a key mechanism of protease regulation is the point of zymogen activation. Thus far, controlling protease activity by targeting the zymogen activation step is an underexploited strategy. We have now designed a specific monoclonal antibody inhibitor (Mab-112) with sub-nanomolar affinity to pro-uPA. A detailed mechanistic evaluation with several biophysical methods and alanine-scanning mutagenesis elucidated a novel multifunctional mechanism whereby Mab-112 by binding to the activation domain retards the proteolytic conversion of single-chain pro-uPA into the two-chain form and subsequently averts the conformational transition to a mature protease by sequestering the two-chain form in a zymogen-like state. Furthermore, Mab-112 is a non-competitive inhibitor of two-chain uPA, stabilising the protease in a non-catalytic conformation. Functional studies employing high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the human HT-1080 cell line demonstrate that Mab-112 is an effective inhibitor of intravasation in the chorioallantoic membrane assay. These findings show the utility of pharmacological interference of zymogen activation as a plausible and robust means to regulate uPA activity and the downstream effects of plasminogen activation. Furthermore, a strategy that targets regions related to pro-enzyme activation likely provide a unique inhibitor-protease interaction surface and is thus expected to enhance the chances of engineering high inhibitor specificity.
    Apmis 05/2008; 116(5):414-414. DOI:10.1111/j.1600-0463.2008.00abs1165_5.x · 2.04 Impact Factor
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    ABSTRACT: In vivo tumor cell migration through integrin-dependent pathways is key to the metastatic behavior of malignant cells. Using quantitative in vivo assays and intravital imaging, we assessed the impact of cell migration, regulated by the integrin-associated tetraspanin CD151, on spontaneous human tumor cell metastasis. We demonstrate that promoting immobility through a CD151-specific metastasis blocking mAb prevents tumor cell dissemination by inhibiting intravasation without affecting primary tumor growth, tumor cell arrest, extravasation, or growth at the secondary site. In vivo, this loss of migration is the result of enhanced tumor cell-matrix interactions, promoted by CD151, which prevent dissociation by individual cells and leads to a subsequent inhibition of invasion and intravasation at the site of the primary tumor.
    Cancer cell 04/2008; 13(3):221-34. DOI:10.1016/j.ccr.2008.01.031 · 23.52 Impact Factor
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    ABSTRACT: The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.
    Biochemical Journal 04/2008; 410(3):595-604. DOI:10.1042/BJ20071137 · 4.40 Impact Factor
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    Andrew J Ramsay · Janet C Reid · Gloria Velasco · James P Quigley · John D Hooper ·
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    ABSTRACT: Matriptase-2 (also known as TMPRSS6) is a recently identified member of the type II transmembrane serine protease (TTSP) family. Structurally this enzyme contains a short cytoplasmic amino terminal tail, a transmembrane region, a stem region containing two CUB domains and three LDL receptor class A domains, and at the carboxy terminal a trypsin-like serine protease domain. The matriptase-2 gene and encoded protein are highly conserved in mammals. Biochemically matriptase-2 has substrate specificity similar to the structurally related protein matriptase (also known as MT-SP1). Although the patho-physiological functions of matriptase-2 are not known, its high mRNA expression in liver and several cancers indicate that this enzyme, similar to other TTSPs, will likely have important cell surface associated roles in normal and disease states. Here we overview the identification of matriptase-2, summarise its structural features, biochemistry, expression pattern and disease associations and discuss its potential functions.
    Frontiers in Bioscience 02/2008; 13(2):569-79. DOI:10.2741/2702 · 3.52 Impact Factor
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    Elena I Deryugina · James P Quigley ·
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    ABSTRACT: Angiogenesis plays a critical role in many normal physiological processes as well as in tumor neovascularization associated with cancer progression. Among various animal model systems designed to study the mechanisms underlying angiogenesis, chick embryo models have been useful tools in analyzing the angiogenic potential of purified factors and intact cells. The chorioallantoic membrane (CAM), a specialized, highly vascularized tissue of the avian embryo, serves as an ideal indicator of the anti- or pro-angiogenic properties of test compounds. In this chapter, we describe a number basic chick embryo CAM models of angiogenesis. A special emphasis is on the model system employing three-dimensional (3D) collagen grafts planted on the CAM, referred herein as onplants. This collagen onplant model allows for unambiguous quantification of angiogenesis and also for in-depth analysis of the cellular and biochemical mechanisms by which specific cells of different origin or purified effector molecules induce or inhibit the angiogenic process.
    Methods in enzymology 02/2008; 444:21-41. DOI:10.1016/S0076-6879(08)02802-4 · 2.09 Impact Factor
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    ABSTRACT: Several lines of evidence have implicated matrix metalloproteinase 9 (MMP-9) as a protease inducing an angiogenic switch critical for tumor progression. Among MMP-9-expressing cell types, including cancer cells and tumor-associated leukocytes, inflammatory neutrophils appear to provide an important source of MMP-9 for tumor angiogenesis. However, delivery of MMP-9 by neutrophils has not been mechanistically linked to its catalytic activity at the angiogenic site. By using a modified angiogenic model, allowing for a direct analysis of exogenously added cells and their products in collagen onplants grafted on the chorioallantoic membrane of the chicken embryo, we demonstrate that intact human neutrophils and their granule contents are highly angiogenic. Furthermore, purified neutrophil MMP-9, isolated from the released granules as a zymogen (proMMP-9), constitutes a distinctly potent proangiogenic moiety inducing angiogenesis at subnanogram levels. The angiogenic response induced by neutrophil proMMP-9 required activation of the tissue inhibitor of metalloproteinases (TIMP)-free zymogen and the catalytic activity of the activated enzyme. That the high angiogenic potency of neutrophil proMMP-9 is associated with its unique TIMP-free status was confirmed when a generated and purified stoichiometric complex of neutrophil proMMP-9 with TIMP-1 failed to induce angiogenesis. Recombinant human proMMP-9, operationally free of TIMP-1, also induced angiogenesis at subnanomolar levels, but lost its proangiogenic potential when stoichiometrically complexed with TIMP-1. Similar proMMP-9/TIMP-1 complexes, but naturally produced by human monocytic U937 cells and HT-1080 fibrosarcoma cells, did not stimulate angiogenesis. These findings provide biochemical evidence that infiltrating neutrophils, in contrast to other cell types, deliver a potent proangiogenic moiety, i.e., the unencumbered TIMP-free MMP-9.
    Proceedings of the National Academy of Sciences 01/2008; 104(51):20262-7. DOI:10.1073/pnas.0706438104 · 9.67 Impact Factor
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    ABSTRACT: The role of tumor-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in cancer cell dissemination was analyzed by employing two variants of human HT-1080 fibrosarcoma, HT-hi/diss and HT-lo/diss, which differ by 50-100-fold in their ability to intravasate and metastasize in the chick embryo. HT-hi/diss and HT-lo/diss were compared by quantitative reverse transcription-PCR and Western blot analyses for mRNA and protein expression of nine MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13, and -14) and three TIMPs (TIMP-1, -2, and -3) in cultured cells in vitro and in primary tumors in vivo. MMP-1 and MMP-9 were more abundant in the HT-hi/diss variant, both in cultures and in tumors, whereas the HT-lo/diss variant consistently expressed higher levels of MMP-2, TIMP-1, and TIMP-2. Small interfering RNA-mediated down-regulation of MMP-2 and TIMP-2 increased intravasation of HT-lo/diss cells. Coordinately, treatment of the developing HT-hi/diss tumors with recombinant TIMP-1 and TIMP-2 significantly reduced HT-hi/diss cell intravasation. However, a substantial increase of HT-hi/diss dissemination was observed upon small interfering RNA-mediated down-regulation of three secreted MMPs, including the interstitial collagenase MMP-1 and the two gelatinases, MMP-2 and MMP-9, but not the membrane-tethered MMP-14. The addition of recombinant pro-MMP-9 protein to the HT-hi/diss tumors reversed the increased intravasation of HT-hi/diss cells, in which MMP-9 was stably down-regulated by short hairpin RNA interference. This rescue did not occur if the pro-MMP-9 was stoichiometrically complexed with TIMP-1, pointing to a direct role of the MMP-9 enzyme in regulation of HT-hi/diss intravasation. Collectively, these findings demonstrate that tumor-derived MMPs may have protective functions in cancer cell intravasation, i.e. not promoting but rather catalytically interfering with the early stages of cancer dissemination.
    Journal of Biological Chemistry 01/2008; 282(49):35964-77. DOI:10.1074/jbc.M705993200 · 4.57 Impact Factor
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    Annals of the New York Academy of Sciences 12/2006; 712(1):131 - 145. DOI:10.1111/j.1749-6632.1994.tb33568.x · 4.38 Impact Factor
  • A. Zijlstra · J. D. Lewis · H. Stuhlmann · J. P. Quigley ·

    Matrix Biology 11/2006; 25. DOI:10.1016/j.matbio.2006.08.141 · 5.07 Impact Factor
  • M. Madsen · E. Deryugina · S. Niessen · B. Cravatt · J. Quigley ·
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    ABSTRACT: Entry of malignant cells into the vasculature, i.e. intravasation, requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they establish metastatic lesions. We utilized activity based protein profiling (ABPP) to compare the active serine hydrolase repertoire of high-intravasating (HT-hi/diss) and low-intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line, recently isolated in our laboratory, to determine which enzyme(s) play a role in intravasation. ABPP is a chemical proteomic technique that uses active site directed probes linked to a reporter group to measure the activity levels of individual enzymes of a given class in a complex mixture of proteins. ABPP combined with mass spectroscopy can identify enzymes that have altered activity levels between experimental groups, such as metastatic and non-metastatic tumor cells. Since proteins are detected based on activity rather than abundance, ABPP can distinguish between active enzymes and their inactive zymogen or their inhibitor bound forms, thereby providing an advantage compared to other methods of profiling. ABPP analysis of HT-hi/diss and HT-lo/diss cells revealed multiple serine hydrolases with altered activity between the two variants, with the largest difference being the activity of urokinase plasminogen activator (uPA). While the level of uPA expression has been implicated in the metastatic process, we show that activation of uPA is a key step due to the fact that uPA protein levels in the two variants are the same. The failure to activate uPA may play a large role in the inability of HT-lo/diss cells to intravasate and metastasize, while the active uPA in HT-hi/diss cells likely contributes to the intravasation ability of these cells. To determine the biological significance of this difference in uPA activity, we used the HT-lo/diss and HT-hi/diss variants in a chick-embryo chorioallantoic membrane (CAM) assay to quantitatively measure intravasation and metastasis in vivo. We also took advantage of the activity based nature of the ABPP system to screen small molecule inhibitors of uPA for their specificity and efficacy, to determine the optimal uPA inhibitor for use in the CAM assay. Inhibition of uPA activity with natural (PAI-1) or synthetic (amiloride) inhibitors significantly diminished HT-hi/diss intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo 2-3 fold. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression. These experiments highlight the efficiency of ABPP analysis in the identification of specific proteases that regulate tumor progression. The combination of ABPP analysis with the chick embryo CAM assay has proven to be a powerful and rapid technique for identifying potential regulators of tumor dissemination and for modulating their activities in vivo to confirm their effect on the metastatic process.
    Journal of Thrombosis and Haemostasis 10/2006; 4(s1):194-194. DOI:10.1111/j.1538-7836.2006.00303.x · 5.72 Impact Factor
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    Andries Zijlstra · James P Quigley ·
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    ABSTRACT: Tumor cell metastasis to distant organs is an inefficient process that is limited in part by recently identified metastasis suppressors. Interactions between tumor cells and the surrounding stroma are thought to control much of cancer progression. In the August issue of Nature Medicine, demonstrate that specific cell surface interactions between the metastasis suppressor KAI1 on tumor cells and the decoy cytokine receptor DARC on adjacent vascular cells triggers senescence in the tumor cells and suppresses metastasis. These new observations demonstrate how metastasis suppressors can relay the restraint imposed by the stroma onto disseminating tumor cells.
    Cancer Cell 10/2006; 10(3):177-8. DOI:10.1016/j.ccr.2006.08.012 · 23.52 Impact Factor
  • J. Hooper · A. Ramsay · J. Quigley · J. Reid · M. Hunt ·
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    ABSTRACT: It has long been recognized that serine proteases initiate cellular changes. However it was not until the identification of the “protease activated receptor” (PAR) family in the 1990s that many of these changes could be explained at the molecular level. Using calcium flux and ERK activation assays and microscopy approaches we have shown that a recently identified serine protease signals via a member of the protease activated receptor family. Interestingly it also appears that cellular changes induced by this serine protease occur via a non-PAR mediated pathway. This result and our work, and that of others, on the cancer associated transmembrane protein SIMA135, suggest that there are non-PAR cell surface proteins which function to transduce extracellular serine protease initiated signals across the plasma membrane. This proposal is consistent with the plethora of known cell surface serine protease targets.
    Journal of Thrombosis and Haemostasis 10/2006; 4(s1):189-189. DOI:10.1111/j.1538-7836.2006.00368.x · 5.72 Impact Factor
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    ABSTRACT: Proteolytic modifications of the extracellular matrix proteins and cell surface receptors by matrix metalloproteinases (MMPs) are critically important for tumor cell dissemination and metastasis. Functional activity of MMPs is tightly regulated through gene and protein expression as well as the degree of proenzyme activation and inhibition of the activated enzyme by natural tissue inhibitors of metalloproteinases (TIMPs). A great deal of information has accumulated regarding the role of MMPs and TIMPs in tumor cell invasion and tumor-induced angiogenesis. However, only limited data are available on the role of the MMP/TIMP system in tumor cell intravasation, although it is one of the rate-limiting steps in the metastatic cascade. To analyze intravasation of tumor cells, we have generated two variants of human HT-1080 fibrosarcoma, which differ 100-fold in their ability to enter the vasculature of the chorioallantoic membrane (CAM) and metastasize to the secondary organs of the chick embryo. The availability of these high (HT-hi/diss) and low (HT-lo/diss) disseminating cell variants allowed us to investigate the role of MMPs and TIMPs specifically in tumor cell intravasation. By quantitative PCR and Western blot analyses, HT-hi/diss and HT-lo/diss cells were initially profiled for in vitro mRNA and protein expression levels of nine MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13, and -14) and three TIMPs (TIMP-1, -2, and -3), known to be expressed in the parental HT-1080 cells. To delineate tumor and host MMPs and TIMPs, which are actually involved in tumor cell intravasation, in vivo mRNA and protein profiling analyses were performed on CAM primary tumors derived from the two HT-1080 cell variants. Human MMP-1 and MMP-9 were more abundant in HT-hi/diss variant than in HT-lo/diss variant both in cell cultures in vitro and in primary tumors in vivo. In contrast, human MMP-2 and TIMP-2 were consistently expressed at higher levels in the HT-lo/diss cells and tumors. To further assess the role of MMP/TIMP system in tumor cell intravasation, activity based protein profiling (ABPP) of the two HT-1080 cell variants was performed with a metalloprotease-specific probe. Surprisingly, ABPP indicated only minor differences in MMP activity between the two HT-1080 cell variants. This discrepancy between results of the different profiling methods could indicate that many of the tumor MMPs are functionally inactive due to complex formation with TIMPs. Functional roles of human TIMP-1, -2 and -3 in intravasation of HT-1080 cell variants were evaluated by downregulation of protein expression with the respective siRNA. Our findings on downregulation of individual TIMPs and their combinations indicate that a fine balance between tumor MMPs and their natural inhibitors TIMPs might determine success of an intravasation event during tumor cell dissemination.
    Journal of Thrombosis and Haemostasis 10/2006; 4(s1). DOI:10.1111/j.1538-7836.2006.00305.x · 5.72 Impact Factor
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    ABSTRACT: Entry of malignant cells into the vasculature (i.e. intravasation) requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they extravasate and establish metastatic lesions. This study utilizes activity-based protein profiling to compare the active serine hydrolase repertoire in high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line to determine which enzyme(s) play a role in intravasation. Activity-based protein profiling revealed multiple serine hydrolases with altered activity between HT-hi/diss and HT-lo/diss cells, with the largest difference being the activity of urokinase-type plasminogen activator (uPA). Levels of inactive uPA zymogen were similar between the two cell variants, but only HT-hi/diss conditioned medium contained active uPA, suggesting that uPA activation may contribute to the enhanced intravasation of HT-hi/diss cells. To analyze the role of uPA activity specifically in the process of intravasation, we grafted cells from the two HT-1080 variants onto the chorioallantoic membrane of chick embryos and measured levels of tumor cell intravasation in the distal chorioallantoic membrane using quantitative human-specific Alu PCR. Inhibition of uPA activity with natural (plasminogen activator inhibitor-1) or synthetic (amiloride) inhibitors diminished HT-hi/diss Matrigel invasion in vitro and intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression.
    Journal of Biological Chemistry 07/2006; 281(23):15997-6005. DOI:10.1074/jbc.M601223200 · 4.57 Impact Factor
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    Elena I Deryugina · James P Quigley ·
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    ABSTRACT: Functions of individual matrix metalloproteinases (MMPs) differentially expressed by tumor cells and stromal cells, are finely regulated by their spatial as well as temporal interactions with distinct cellular and extracellular components of the tumor microenvironment and also distant pre-metastatic sites. Certain aspects of MMP involvement in tumor metastasis such as tumor-induced angiogenesis, tumor invasion, and establishment of metastatic foci at the secondary site, have received extensive attention that resulted in an overwhelming amount of experimental and observational data in favor of critical roles of MMPs in these processes. In particular, dependency of tumor angiogenesis on the activity of MMPs, especially that of MMP-9, renders this step possibly the most effective target of synthetic MMP inhibitors. MMP functioning in other stages of metastasis, including the escape of individual tumor cells from the primary tumor, their intravasation, survival in circulation, and extravasation at the secondary site, have not yet received enough consideration, resulting in insufficient or controversial data. The major pieces of evidence that are most compelling and clearly determine the role and involvement of MMPs in the metastatic cascade are provided by molecular genetic studies employing knock-out or transgenic animals and tumor cell lines, modified to overexpress or downregulate a specific MMP. Findings from all of these studies implicate different functional mechanisms for both tumor and stromal MMPs during distinct steps of the metastatic cascade and indicate that MMPs can exhibit pro-metastatic as well as anti-metastatic roles depending on their nature and the experimental setting. This dual function of individual MMPs in metastasis has become a major focus of this review.
    Cancer and metastasis reviews 04/2006; 25(1):9-34. DOI:10.1007/s10555-006-7886-9 · 7.23 Impact Factor
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    ABSTRACT: A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.
    Nature Medicine 04/2006; 12(3):354-60. DOI:10.1038/nm1368 · 27.36 Impact Factor
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    ABSTRACT: A quantitative in vivo angiogenesis model employing collagen onplants placed on the chick embryo chorioallantoic membrane (CAM) has been used in this study to assess the spatial and temporal associations between neutrophil-like inflammatory cells, namely chicken heterophils, and the development of new blood vessels. Previously we have demonstrated that monocytes/macrophages infiltrating the onplants were associated with extracellular matrix remodeling and angiogenesis, in particular by delivering MMP-13 collagenase. By introducing chicken gelatinase B (chMMP-9) as a specific marker for heterophils, we now show that the onset and extent of angiogenesis induced by purified growth factors or by human HT-1080 fibrosarcoma cells correlated with the initial influx of chMMP-9-positive heterophils. This early heterophil arrival was followed by the infiltration of monocytes/macrophages and appeared to sustain further blood vessel formation. The disruption of inflammatory cell influx by 2 mechanistically distinct anti-inflammatory drugs, cortisone and ibuprofen, significantly inhibited angiogenesis, indicating a functional involvement of these inflammatory cells in new blood vessel development. A direct addition of isolated heterophils or purified chMMP-9 into the HT-1080 onplants engrafted into cortisone- or ibuprofen-treated embryos reversed the antiangiogenic effects of the drugs. The exogenously added heterophils induced in vivo a further infiltration of endogenous heterophils and monocytes and dramatically rescued the impaired angiogenesis, highlighting the importance of early inflammatory leukocytes in tumor-induced angiogenesis. Moreover, purified heterophils incorporated into onplants lacking growth factors or tumor cells induced angiogenesis in nontreated embryos, further indicating a direct proangiogenic role for neutrophil-like leukocytes.
    Blood 02/2006; 107(1):317-27. DOI:10.1182/blood-2005-04-1458 · 10.45 Impact Factor
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    ABSTRACT: The human tumor/chick embryo model involving grafting of human HT-1080 fibrosarcoma cells on the chorioallantoic membrane was used in conjunction with quantitative real-time Alu PCR to select in vivo a pair of isogenic cell lines (HT-hi/diss and HT-lo/diss), dramatically differing in their ability to disseminate from the primary tumor (i.e., intravasate into the chorioallantoic membrane vasculature and metastasize to the lungs). During an immunohistochemical time course study, HT-hi/diss cells were sequentially visualized having escaped from the primary tumors, engaged with the blood vessels, and eventually observed inside the chorioallantoic membrane capillaries, thus reflecting early intravasating events. In contrast, HT-lo/diss cells seemed restricted to their primary tumor. Importantly, after i.v. inoculation, both variants arrested, extravasated, and proliferated in host tissues with similar efficiencies, highlighting that the observed earlier events at the periphery of the primary tumor could account for their differential dissemination. In a mechanistic probing of these events, we determined that HT-hi/diss intravasation was sensitive to a broad-range matrix metalloproteinase (MMP) inhibitor. To analyze the possible role of individual MMPs, membrane-bound MMP-14 and secreted MMP-9 were individually down-regulated in HT-hi/diss cells with their corresponding small interfering RNAs. Despite efficient down-regulation of MMP-14, neither intravasation nor metastasis of HT-hi/diss cells was affected significantly. However, a substantial down-regulation of MMP-9 was accompanied by a surprising 3-fold increase in intravasation and metastasis. The results emphasize a rising awareness that targeting certain MMPs might result in an enhanced malignancy, exemplified herein at the intravasation level as this step of the metastatic cascade is dissected and quantified.
    Cancer Research 01/2006; 65(23):10959-69. DOI:10.1158/0008-5472.CAN-05-2228 · 9.33 Impact Factor
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    ABSTRACT: Cancer arises by the accumulation of genetic alterations in DNA leading to aberrant gene transcription. Expression-profiling studies have correlated genomewide expression signatures with malignancy. However, functional analysis elucidating the contribution and synergy of genes in specific cancer cell phenotypes remains a formidable obstacle. Herein, we describe an alternative genetic approach for identification of genes involved in tumor progression by using a library of zinc finger artificial transcription factors (ATFs) and functional screening of tumor cells as a source of genetic plasticity and clonal selection. We isolated a six-zinc finger transcriptional activator (TF 20-VP, TF 20 containing the VP64 activator domain) that acts to reprogram a drug-sensitive, poorly invasive, and nonmetastatic cell line into a cell line with a drug-resistant, highly invasive, and metastatic phenotype. Differential expression profiles of cells expressing TF 20-VP followed by functional studies, both in vitro and in animal models, revealed that invasion and metastasis requires co-regulation of multiple target genes. Significantly, the E48 antigen, associated with poor metastasis-free survival in head and neck cancer, was identified as one specific target of TF 20-VP. We have shown phenotypic modulation of tumor cell behavior by E48 expression, including enhanced cell migration in vitro and tumor cell dissemination in vivo. This study demonstrates the use of ATFs to identify the group of genes that cooperate during tumor progression. By co-regulating multiple targets, ATFs can be used as master genetic switches to reprogram and modulate complex neoplastic phenotypes.
    Proceedings of the National Academy of Sciences 09/2005; 102(33):11716-21. DOI:10.1073/pnas.0501162102 · 9.67 Impact Factor
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    ABSTRACT: TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1-4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.
    Biochemical Journal 06/2005; 388(Pt 3):967-72. DOI:10.1042/BJ20041066 · 4.40 Impact Factor

Publication Stats

9k Citations
983.62 Total Impact Points


  • 1999-2015
    • The Scripps Research Institute
      • Department of Cell and Molecular Biology
      لا هویا, California, United States
  • 2013
    • University of California, San Diego
      San Diego, California, United States
  • 1990-2011
    • Aarhus University
      • Danish-Chinese Centre for Proteases and Cancer
      Aarhus, Central Jutland, Denmark
  • 1978-2006
    • Marine Biological Laboratory
      Falmouth, Massachusetts, United States
  • 2001
    • Queensland University of Technology
      • Institute of Health and Biomedical Innovation
      Brisbane, Queensland, Australia
  • 1991-2000
    • State University of New York
      New York City, New York, United States
  • 1994-1999
    • Stony Brook University
      • Department of Pathology
      Stony Brook, NY, United States
  • 1988-1997
    • University of California, Davis
      • Department of Molecular and Cellular Biology
      Davis, California, United States
  • 1989
    • NYU Langone Medical Center
      • Department of Pathology
      New York, New York, United States
  • 1978-1986
    • State University of New York Downstate Medical Center
      • Department of Microbiology and Immunology
      Brooklyn, New York, United States
  • 1983
    • The Ohio State University
      • Department of Pathology
      Columbus, Ohio, United States