Synthesis, regulation and production of urokinase using mammalian cell culture: a comprehensive review.

Urokinase, a serine protease, catalyzes the conversion of plasminogen to plasmin, which is responsible for dissolution of clots in blood vessels. It is an important drug for treatment of thromboembolic disease. Production of urokinase by mammalian cell culture has the following important steps: synthesis, regulation and secretion. Production and accumulation of this product in a bioreactor is a real challenge for biochemical engineers. Considerable information at molecular level needs to be understood for production of urokinase in order to correlate different parameters, which in turn can maximize the productivity. This information will be highlighted in this review. Moreover, urokinase production is a product-inhibited process. Therefore, in situ urokinase separation strategy is required to operate a bioreactor at its maximum urokinase formation rate. Integrated urokinase production and isolation processes developed recently will also be discussed briefly in this review.

[1]  Xian-wen Hu,et al.  Pilot production of u-PA with porous microcarrier cell culture , 2000, Cytotechnology.

[2]  H. Kwaan,et al.  Plasminogen activator activity in cultures from human tissues. An immunological and histochemical study. , 1969, The Journal of clinical investigation.

[3]  F. Hucho,et al.  Downstream targets of urokinase-type plasminogen-activator-mediated signal transduction. , 1998, European journal of biochemistry.

[4]  Yusuf A. Hannun,et al.  Regulation of protein kinase C and role in cancer biology , 1994, Cancer and Metastasis Reviews.

[5]  J. Santibañez,et al.  Epidermal growth factor stimulates urokinase-type plasminogen activator expression in human gingival fibroblasts. Possible modulation by genistein and curcumin. , 2004, Journal of periodontal research.

[6]  Xingmao Liu,et al.  Temperature shift as a process optimization step for the production of pro-urokinase by a recombinant Chinese hamster ovary cell line in high-density perfusion culture. , 2004, Journal of bioscience and bioengineering.

[7]  A. Lesuk,et al.  Crystalline Human Urokinase: Some Properties , 1965, Science.

[8]  P. Ellaiah,et al.  Effect of low frequency AC electric fields on urokinase production by MPGN kidney cells , 2003 .

[9]  A. Vaheri,et al.  Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells , 1987, The Journal of cell biology.

[10]  G. Hartnell,et al.  The case of Abbokinase and the FDA: the events leading to the suspension of Abbokinase supplies in the United States. , 2000, Journal of vascular and interventional radiology : JVIR.

[11]  V. Stepanova,et al.  Urokinase as a Multidomain Protein and Polyfunctional Cell Regulator , 2004, Biochemistry (Moscow).

[12]  P. Roychoudhury,et al.  Production and purification of urokinase: a comprehensive review. , 2006, Protein expression and purification.

[13]  M. Cubellis,et al.  Receptor‐mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI‐1. , 1990, The EMBO journal.

[14]  P. Verde,et al.  An upstream enhancer and a negative element in the 5' flanking region of the human urokinase plasminogen activator gene. , 1988, Nucleic acids research.

[15]  S. Moestrup,et al.  Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes. , 1992, The Journal of biological chemistry.

[16]  M. Scully,et al.  Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes. , 1989, The Journal of biological chemistry.

[17]  D. Collen,et al.  Purification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures. , 1986, The Journal of biological chemistry.

[18]  K. Danø,et al.  The pro-urokinase plasminogen-activation system in the presence of serpin-type inhibitors and the urokinase receptor: rescue of activity through reciprocal pro-enzyme activation. , 2003, The Biochemical journal.

[19]  Bo Mattiasson,et al.  Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p‐aminobenzamidine affinity chromatography , 2006, Journal of molecular recognition : JMR.

[20]  Xian-wen Hu,et al.  High density and scale-up cultivation of recombinant CHO cell line and hybridomas with porous microcarrier Cytopore , 1999, Cytotechnology.

[21]  Bo Mattiasson,et al.  Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line. , 2006, Journal of chromatography. A.

[22]  J M Zahm,et al.  Cell migration and proliferation during the in vitro wound repair of the respiratory epithelium. , 1997, Cell motility and the cytoskeleton.

[23]  R. Medcalf,et al.  Human synovial fibroblasts produce urokinase-type plasminogen activator. , 1986, Arthritis and rheumatism.

[24]  J. Bos,et al.  Regulation of Ras-mediated signalling: more than one way to skin a cat. , 1995, Trends in biochemical sciences.

[25]  P. Sonderegger,et al.  The Axonally Secreted Serine Proteinase Inhibitor, Neuroserpin, Inhibits Plasminogen Activators and Plasmin but Not Thrombin* , 1998, The Journal of Biological Chemistry.

[26]  A. Corti,et al.  Production and characterisation of human recombinant single chain urokinase-type plasminogen activator from mouse cells , 1989 .

[27]  L. Lund,et al.  One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity. , 1988, The Journal of biological chemistry.

[28]  D. Moir,et al.  Kidney Plasminogen Activator: A Precursor Form of Human Urokinase With High Fibrin Affinity , 1984, Bio/Technology.

[29]  J. Varani,et al.  Proteolytic enzymes and arachidonic acid metabolites produced by MRC-5 cells on various microcarrier substrates , 1986, In Vitro Cellular & Developmental Biology.

[30]  N. E. Hansen,et al.  The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils. , 1994, Blood.

[31]  K. Danø,et al.  Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor. , 1991, The Journal of biological chemistry.

[32]  J. Williams The fibrinolytic activity of urine. , 1951, British journal of experimental pathology.

[33]  E. Plow,et al.  Plasminogen receptors: Ubiquitous sites for cellular regulation of fibrinolysis , 1988 .

[34]  P. R. Gibson,et al.  Cell associated urokinase activity and colonic epithelial cells in health and disease. , 1991, Gut.

[35]  L. Holmberg,et al.  Purification of urokinase by affinity chromatography. , 1976, Biochimica et biophysica acta.

[36]  Bo Mattiasson,et al.  Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices , 2006, Biotechnology and bioengineering.

[37]  Jian-ning Liu,et al.  An efficient system for production of recombinant urokinase-type plasminogen activator. , 1997, Protein expression and purification.

[38]  D. Alonso,et al.  Overproduction of urokinase-type plasminogen activator is regulated by phospholipase D- and protein kinase C-dependent pathways in murine mammary adenocarcinoma cells. , 1997, Biochimica et Biophysica Acta.

[39]  P. Verde,et al.  Increase in urokinase plasminogen activator mRNA synthesis in human carcinoma cells is a primary effect of the potent tumor promoter, phorbol myristate acetate , 1986, The Journal of cell biology.

[40]  O. Mayboroda,et al.  The Jak/Stat Pathway and Urokinase Receptor Signaling in Human Aortic Vascular Smooth Muscle Cells* , 1998, The Journal of Biological Chemistry.

[41]  A. Oikarinen,et al.  Dexamethasone-induced plasminogen activator inhibitor: characterization, purification, and preparation of monoclonal antibodies , 2004, Archives of Dermatological Research.

[42]  L. Mallucci,et al.  A cell cycle study of the effects of Con A on synchronized mouse embryo fibroblasts: arrest at two specific stages of the cycle and dissociation between uptake of thymidine and DNA synthesis. , 1980, Journal of cell science.

[43]  L. Ossowski,et al.  A proenzyme form of human urokinase. , 1982, The Journal of biological chemistry.

[44]  Y. Nagamine,et al.  Cytoskeleton Reorganization Induces the Urokinase-type Plasminogen Activator Gene via the Ras/Extracellular Signal-regulated Kinase (ERK) Signaling Pathway* , 1997, The Journal of Biological Chemistry.

[45]  F. Blasi,et al.  The urokinase plasminogen activator system in cancer: Recent advances and implication for prognosis and therapy , 2003, Cancer and Metastasis Reviews.

[46]  F. Booyse,et al.  Purification and properties of a single-chain urokinase-type plasminogen activator form produced by subcultured human umbilical vein endothelial cells. , 1988, The Journal of biological chemistry.

[47]  J. Varani,et al.  Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates , 2006, Cytotechnology.

[48]  K. Danø,et al.  Purification and characterization of a plasminogen activator from mouse cells transformed by an oncogenic virus. , 1980, Biochimica et biophysica acta.

[49]  Lewis Lj Plasminogen activator (urokinase) from cultured cells. , 1979 .

[50]  Y. Nagamine,et al.  The plasminogen activator system: biology and regulation , 1999, Cellular and Molecular Life Sciences CMLS.

[51]  H. Fuse,et al.  Induction of urokinase-type plasminogen activator by lipopolysaccharide in PC-3 human prostatic cancer cells. , 2000, Thrombosis research.

[52]  W. Schleuning,et al.  Isolation and characterization of urokinase from human plasma. , 1982, The Journal of biological chemistry.

[53]  F. Blasi,et al.  Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes , 1997, The EMBO journal.

[54]  V. Dolo,et al.  Urokinase Plasminogen Activator and Gelatinases Are Associated with Membrane Vesicles Shed by Human HT1080 Fibrosarcoma Cells* , 1997, The Journal of Biological Chemistry.

[55]  J. Engasser,et al.  Continuous production of prourokinase in feed harvest and perfusion cultures , 1990, Biotechnology and bioengineering.

[56]  Alan J Dickson,et al.  Stability of protein production from recombinant mammalian cells , 2003, Biotechnology and bioengineering.

[57]  Denis Drapeau,et al.  Spin Filter Perfusion System for High Density Cell Culture: Production of Recombinant Urinary Type Plasminogen Activator in CHO Cells , 1990, Bio/Technology.

[58]  M. A. Bohn,et al.  Kinetics of prourokinase production by human kidney cells in culture , 1987 .

[59]  Dong-Myung Kim,et al.  Efficient production of a bioactive, multiple disulfide‐bonded protein using modified extracts of Escherichia coli , 2004, Biotechnology and bioengineering.

[60]  Y. Chisti,et al.  Tissue-type plasminogen activator: characteristics, applications and production technology. , 1996, Biotechnology advances.

[61]  A. Siegbahn,et al.  Expression of fibrinolytic and coagulation factors in cocultured human endothelial and smooth muscle cells. , 2004, Tissue engineering.

[62]  S. L. Gonias,et al.  Binding of Urokinase-type Plasminogen Activator to Its Receptor in MCF-7 Cells Activates Extracellular Signal-regulated Kinase 1 and 2 Which Is Required for Increased Cellular Motility* , 1998, The Journal of Biological Chemistry.

[63]  K. Okada,et al.  Effect of hyperthermia on the viability and the fibrinolytic potential of human cancer cell lines. , 2000, Clinica chimica acta; international journal of clinical chemistry.

[64]  D. Moir,et al.  Optimization of pro‐urokinase secretion from recombinant Saccharomyces cerevisiae , 1991, Biotechnology and bioengineering.

[65]  M. E. Garcés,et al.  Acetaldehyde increases the activity and gene expression of urokinase type plasminogen activator in a hepatic stellate cell line. , 1999, Toxicology.

[66]  R. Medcalf,et al.  Glucocorticoid-modulated gene expression of tissue- and urinary-type plasminogen activator and plasminogen activator inhibitor 1 and 2 , 1988, The Journal of cell biology.

[67]  N. Chaniotakis,et al.  Enzyme stabilization strategies based on electrolytes and polyelectrolytes for biosensor applications , 2004, Analytical and bioanalytical chemistry.

[68]  M. Weibel,et al.  Affinity chromatography of urokinase. , 1974, Methods in enzymology.

[69]  Jung Hoe Kim,et al.  Performance study of perfusion cultures for the production of single-chain urokinase-type plasminogen activator (scu-PA) in a 2.5 l spin-filter bioreactor , 1998 .

[70]  R. Roblin,et al.  Immunological quantitation and immunoadsorption of urokinase-like plasminogen activators secreted by human cells. , 1980, The Journal of biological chemistry.

[71]  J. Ando,et al.  Differential regulation of urokinase-type plasminogen activator expression by fluid shear stress in human coronary artery endothelial cells. , 2004, American journal of physiology. Heart and circulatory physiology.

[72]  M. Satoh,et al.  Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability , 2004, Cytotechnology.

[73]  D. Severson,et al.  Signal transduction in vascular smooth muscle: diacylglycerol second messengers and PKC action. , 1994, The American journal of physiology.

[74]  P. Verde,et al.  Identification and primary sequence of an unspliced human urokinase poly(A)+ RNA. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[75]  P. Roychoudhury,et al.  Production of urokinase by HT 1080 human kidney cell line. , 1999, Artificial cells, blood substitutes, and immobilization biotechnology.

[76]  T. Gelehrter,et al.  Glucocorticoid induction of plasminogen activator and plasminogen activator-inhibitor messenger RNA in rat hepatoma cells. , 1989, Molecular endocrinology.

[77]  S. Yasothornsrikul,et al.  The Local Chromaffin Cell Plasminogen/Plasmin System and the Regulation of Catecholamine Secretion , 2002, Annals of the New York Academy of Sciences.

[78]  S. Rabbani,et al.  Transcriptional regulation of urokinase (uPA) gene expression in breast cancer cells: Role of DNA methylation. , 1999, International journal of cancer.

[79]  R. Pardi,et al.  alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)- dependent internalization of the urokinase receptor , 1995, The Journal of cell biology.

[80]  D. Gomez,et al.  Deregulation of the signaling pathways controlling urokinase production. Its relationship with the invasive phenotype. , 1999, European journal of biochemistry.

[81]  J. Griffiths,et al.  Production of tissue plasminogen activators from animal cells. , 1987, Advances in biochemical engineering/biotechnology.

[82]  K. Danø,et al.  Potentiation of plasminogen activation by an anti-urokinase monoclonal antibody due to ternary complex formation. A mechanistic model for receptor-mediated plasminogen activation. , 1993, The Journal of biological chemistry.

[83]  D. Vetterlein,et al.  Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain , 1983, Thrombosis and Haemostasis.

[84]  P. Quax,et al.  Plasminogen activators are involved in keratinocyte and fibroblast migration in wounded cultures in vitro , 1994 .

[85]  D. Collen Ham-Wasserman lecture: role of the plasminogen system in fibrin-homeostasis and tissue remodeling. , 2001, Hematology. American Society of Hematology. Education Program.

[86]  Kinetics of producing pro-urokinase in perfusion cultivations , 1990, Biotechnology Letters.

[87]  T. Orfeo,et al.  α-Thrombin Stimulates Urokinase Production and DNA Synthesis in Cultured Human Cerebral Microvascular Endothelial Cells , 1995 .

[88]  H. Granger,et al.  Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF. , 1997, Circulation research.