Alexander Shevelev

Institute of Experimental Cardiology

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Jerzy Trojan

Inserm

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Т. Н. Власик

National Medical Research Center of Cardiology

Donald D. Anthony

Case Western Reserve University

L. Trojan

Inserm

ЗИ

З. И. Цоколаева

V.A. Negovsky Scientific Research Institute of General Reanimatology

A. Ly

Uppsala University

Natalia M. Kashirina

National Medical Research Center of Cardiology

Ming Wei

Wuhan University

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И. Н. Рыбалкин

National Medical Research Center of Cardiology

Maria Claudia Noguera

Universidad de La Sabana

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Institute of Experimental Cardiology

National Medical Research Center of Cardiology

Ministry of Health of the Russian Federation

Russian Academy of Sciences

Lomonosov Moscow State University

Moscow State University of Medicine and Dentistry

Inserm

Case Western Reserve University

Moscow State University

Jagiellonian University

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Capability of cultivated human hair-follicle cells to integrate with skin structure in vivo

Cell and Tissue Biology (2010)

E. S. Chermnykh N. V. Radyukhina P. N. Rutkevich Alexander Shevelev Т. Н. Власик

Dermal papillae

Epidermis (zoology)

Transduction (biophysics)

Human skin

10.1134/s1990519x10030053

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Citations (1)

Developmentally Imprinted Genes as Markers for Bladder Tumor Progression

The Journal of Urology (1996)

Mark J. Cooper Martin Fischer D. Komitowski Alexander Shevelev Ekkehard Schulze

No AccessJournal of UrologyInvestigative Urology1 Jun 1996Developmentally Imprinted Genes as Markers for Bladder Tumor Progression Mark J. Cooper, Martin Fischer, Dymitr Komitowski, Alexander Shevelev, Ekkehard Schulze, Ilana Ariel, Mark L. Tykocinski, Stela Miron, Joseph Ilan, Nathan De Groot, and Abraham Hochberg Mark J. CooperMark J. Cooper , Martin FischerMartin Fischer , Dymitr KomitowskiDymitr Komitowski , Alexander ShevelevAlexander Shevelev , Ekkehard SchulzeEkkehard Schulze , Ilana ArielIlana Ariel , Mark L. TykocinskiMark L. Tykocinski , Stela MironStela Miron , Joseph IlanJoseph Ilan , Nathan De GrootNathan De Groot , and Abraham HochbergAbraham Hochberg View All Author Informationhttps://doi.org/10.1016/S0022-5347(01)66120-2AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: Developmentally imprinted genes, such as H19 and insulin-like growth factor-II (IGF-II), play an important role during human embryogenesis and also have been implicated in the pathogenesis of embryonal tumors of childhood. Since H19 is expressed in human fetal bladder, we evaluated 35 bladder carcinomas for H19 expression by in situ hybridization analysis and correlated expression with tumor grade. As a prelude to gene transfer studies to determine if H19 is a bladder tumor oncogene, we also evaluated bladder cell lines for expression of H19, IGF-II, IGF-I and the type I IGF receptor. Materials and Methods: H19 expression was evaluated by in situ hybridization analysis in bladder tumor specimens. Northern analysis was used to evaluate the expression of H19, IGF-II, IGF-I and the type I IGF receptor in bladder cell lines. Results: H19 was expressed preferentially in advanced stage tumors: 2 of 12 grade I tumors were H19 positive, whereas 9 of 11 grade II and 7 of 10 grade III tumors expressed H19 (p = 0.004). Additionally, 6 of 6 carcinoma in situ tumors were H19 positive, whereas normal bladder mucosa cells were H19 negative. We found that 3 of 11 cell lines (HT-1376, HT-1197 and 5637) express high levels of H19 mRNA, and each of these cell lines and J82 also express IGF-II. All cell lines examined expressed the type I IGF receptor, whereas there was no detectable IGF-I mRNA. Conclusions: These data demonstrate that H19 is an oncodevelopmental marker of bladder tumor progression and raise the possibility that H19 may have oncogenic properties in bladder cancer. References 1 : Genetic alterations in bladder cancer. Lancet1993; 342: 469. Google Scholar 2 : Frequency of H-ras mutations in human bladder cancer detected by direct sequencing. Br. J. Urol.1994; 73: 516. Google Scholar 3 : Overexpression of normal and mutated forms of HRAS induces orthotopic bladder invasion in a human transitional cell carcinoma. Proc. Natl. Acad. Sci. U.S.A.1990; 87: 9047. Google Scholar 4 : Neoplastic progression by EJ/ras at different steps of transformation in vitro of human uroepithelial cells. Cancer Res.1992; 52: 688. Google Scholar 5 : Expression of epidermal growth factor receptor in bladder cancer as related to established prognostic factors, oncoprotein (c-erbB-2, p53) expression and long-term prognosis. Br. J. Cancer1994; 69: 1120. Google Scholar 6 : Ha-ras induction of the invasive phenotype results in up-regulation of epidermal growth factor receptors and altered responsiveness to epidermal growth factor in human papillary transitional cell carcinoma cells. Cancer Res.1991; 51: 4486. Google Scholar 7 : Identification of p53 gene mutations in bladder cancers and urine samples. Science1991; 252: 706. Crossref, Medline, Google Scholar 8 : p53 mutations in human bladder cancer: genotypic versus phenotypic patterns. Int. J. Cancer1994; 56: 347. Crossref, Medline, Google Scholar 9 : Altered expression of the retinoblastoma gene product: prognostic indicator in bladder cancer. J. Natl. Cancer Inst.1992; 84: 1251. Crossref, Medline, Google Scholar 10 : Expression of the retinoblastoma gene product in bladder carcinoma cells associates with a low frequency of tumor formation. Cancer Res.1992; 52: 1968. Google Scholar 11 : Tumor behavior in transitional cell carcinoma of the bladder in relation to chromosomal markers and histopathology. Cancer Res.1987; 47: 6800. Google Scholar 12 : Allelotype of human bladder cancer. Cancer Res.1994; 54: 531. Medline, Google Scholar 13 : Genome imprinting and carcinogenesis. Biochim. Biophys. Acta1991; 1072: 51. Google Scholar 14 : Relaxation of imprinted genes in human cancer. Nature1993; 362: 747. Google Scholar 15 : Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms' tumour. Nature1993; 362: 749. Google Scholar 16 : Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms' tumour. Nature Genet.1994; 7: 433. Google Scholar 17 : Epigenetic lesions at the H19 locus in Wilms' tumour patients. Nature Genet.1994; 7: 440. Google Scholar 18 : Parental imprinting of the mouse insulin-like growth factor II gene. Cell1991; 64: 849. Google Scholar 19 : Parental imprinting of the mouse H19 gene. Nature1991; 351: 153. Google Scholar 20 : Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature1993; 362: 751. Google Scholar 21 : Monoallelic expression of the human H19 gene. Nature Genet.1992; 1: 40. Google Scholar 22 : Genomic imprinting: control of gene expression by epigenetic inheritance. Curr. Opin. Cell. Biol.1994; 6: 390. Google Scholar 23 : Preferential mutation of paternally derived RB gene as the initial event in sporadic osteosarcoma. Nature1989; 338: 156. Google Scholar 24 : Activation of an imprinted allele of the insulin-like growth factor II gene implicated in rhabdomyosarcoma. J. Clin. Invest.1994; 94: 445. Google Scholar 25 : A fine-structure deletion map of human chromosome 11p; analysis of J1 series hybrids. Somat. Cell Mol. Genet.1989; 15: 477. Google Scholar 26 : Nonrandom loss of maternal chromosome 11 alleles in Wilms tumors. Am. J. Hum. Genet.1987; 40: 413. Google Scholar 27 : Loss of heterozygosity for genes on 11p and the clinical course of patients with lung carcinoma. Cancer Res.1990; 50: 2302. Google Scholar 28 : Correlation of loss of alleles on the short arms of chromosomes 11 and 17 with metastasis of primary breast cancer to lymph nodes. Cancer Res.1992; 52: 3914. Google Scholar 29 : Tumour-suppressor activity of H19 RNA. Nature1993; 365: 764. Google Scholar 30 : Parental origin of 11p15 deletions in human lung cancer. Oncogene1994; 9: 3063. Google Scholar 31 : Autonomous growth of a human neuroblastoma cell line is mediated by insulin-like growth factor II. J. Clin. Invest.1989; 84: 829. Google Scholar 32 : Insulin-like growth factor II acts as an autocrine growth and motility factor in human rhabdomyosarcoma. Cell Growth Differ.1990; 1: 325. Google Scholar 33 : The product of the H19 gene may function as an RNA. Mol. Cell. Biol.1990; 10: 28. Google Scholar 34 : Locus unlinked to alpha-fetoprotein under the control of the murine raf and Rif genes. Proc. Natl. Acad. Sci. U.S.A.1984; 81: 5523. Google Scholar 35 : The structure and expression of a novel gene activated in early mouse embryogenesis. E.M.B.O.J.1988; 7: 673. Google Scholar 36 : The expression of the H-19 and IGF-2 genes during human embryogenesis and placental development. Mol. Reprod. Dev.1993; 34: 374. Google Scholar 37 : Expression of the imprinted gene H19 in the human fetus. Mol. Reprod. Dev.1994; 38: 239. Google Scholar 38 : Human imprinted genes as oncodevelopmental markers. Tumor Biol.1994; 15: 123. Google Scholar 39 : The imprinted H19 gene as a tumor marker in bladder carcinoma. Urology1995; 45: 335. Google Scholar 40 : Tumorigenicity of T24 urinary bladder carcinoma cell sublines. Int. J. Cancer1987; 40: 240. Google Scholar 41 : Quantitative description of chromatin structure during neoplasia by the method of image processing. Anal. Quant. Cytol. Histol.1985; 47: 178. Google Scholar 42 : Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry1979; 18: 5294. Google Scholar 43 : Transcription of the H19 gene in differentiating cytotrophoblasts from human placenta. Mol. Reprod. Dev.1992; 32: 196. Google Scholar 44 : Sequence of cDNA encoding human insulin-like growth factor I precursor. Nature1983; 306: 609. Google Scholar 45 : Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. E.M.B.O.J.1986; 5: 2503. Google Scholar 46 : Human neuroblastoma tumor cell lines correspond to the arrested differentiation of chromaffin adrenal medullary neuroblasts. Cell Growth Differ.1990; 1: 149. Google Scholar 47 : Histologic typing of urinary bladder tumours. Geneva: The World Health Organization1973. Google Scholar 48 : A human tissue culture cell line from a transitional cell tumor of a urinary bladder: growth, chromosome pattern and ultrastructure. Br. J. Cancer1970; 24: 746. Google Scholar 49 : Human bladder carcinoma: characterization of two new tumor cell lines and search for tumor viruses. J. Natl. Cancer Inst.1977; 58: 881. Google Scholar 50 : Ultrastructure, karyology and immunology of a cell line originated from a human transitional-cell carcinoma. Br. J. Cancer1978; 38: 64. Google Scholar 51 : Cellular and humoral immune responses to human urinary bladder carcinomas. Int. J. Cancer1970; 5: 310. Google Scholar 52 : A cell line from an anaplastic transitional cell carcinoma of human urinary bladder. Br. J. Cancer1977; 35: 142. Google Scholar 53 : Improved growth of human urothelial carcinoma cell cultures. J. Urol.1986; 136: 953. Link, Google Scholar 54 : Establishment and characterization of human bladder tumor cell lines and sublines with different degrees of malignancy. Cancer Res.1985; 45: 5070. Google Scholar 55 : The expression of the imprinted H19 and IGF-2 genes in human bladder carcinoma. F.E.B.S. Lett.1995; 374: 57. Google Scholar 56 : Physical linkage of two mammalian imprinted genes, H19 and insulin-like growth factor 2. Nature Genet.1992; 2: 61. Google Scholar 57 : Insulin-like growth factor II binding and action in human fetal fibroblasts. J. Cell Physiol.1987; 133: 560. Google Scholar 58 : Two molecular pathways to transitional cell carcinoma of the bladder. Cancer Res.1994; 54: 784. Medline, Google Scholar 59 : Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev.1993; 7: 1663. Google Scholar 60 : Tumor suppression by RNA from the 3' untranslated region of alpha-tropomyosin. Cell1993; 75: 1107. Google Scholar 61 : Evolutionary conservation of possible functional domains of the human and murine XIST genes. Hum. Mol. Genet.1993; 2: 663. Google Scholar 62 : Genetic complementation reveals a novel regulatory role for 3' untranslated regions in growth and differentiation. Cell1993; 72: 903. Google Scholar 63 : Identification of a novel paternally expressed gene in the Prader-Willi syndrome region. Hum. Mol. Genet.1994; 3: 1877. Google Scholar 64 : enod40, a gene expressed during nodule organogenesis, codes for a nontranslatable RNA involved in plant growth. E.M.B.O.J.1994; 13: 5099. Google Scholar From the Departments of Medicine and Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio; the German Cancer Research Center, Heidelberg, Germany; the Department of Pathology, Hadassah University Hospital, Mount Scopus, and the Department of Biological Chemistry, The Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.© 1996 by American Urological Association, Inc.FiguresReferencesRelatedDetails Volume 155Issue 6June 1996Page: 2120-2127 Advertisement Copyright & Permissions© 1996 by American Urological Association, Inc.MetricsAuthor Information Mark J. Cooper More articles by this author Martin Fischer More articles by this author Dymitr Komitowski More articles by this author Alexander Shevelev More articles by this author Ekkehard Schulze More articles by this author Ilana Ariel More articles by this author Mark L. Tykocinski More articles by this author Stela Miron More articles by this author Joseph Ilan More articles by this author Nathan De Groot More articles by this author Abraham Hochberg More articles by this author Expand All Advertisement PDF downloadLoading ...

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10.1016/s0022-5347(01)66120-2

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Новые плазмидные конструкции, предназначенные для терапевтического ангиогенеза и несущие гены ангиогенных факторов роста – VEGF, HGF и ангиопоэтина-1

П. И. Макаревич Alexander Shevelev И. Н. Рыбалкин Natalia M. Kashirina Л. Н. Липатова

Efficacy of critical limb ischemia gene therapy can be improved by application of novel plasmid vectors with higher transgene expression. The goal of this study is to evaluate in vitro and in vivo expression of angiogenic growth factors after gene transfer using a novel plasmid vector PC4W. Plasmid constructs with genes of human VEGF185 CpC4W-hHGFopt), HGF CpC4W-hHGFopt) and angiopoietin-1 (pC4W-hAng-1optJ were tested in vitro in a HEK293T cell culture. Cells were subjected to calcium-phosphate transfection and conditioning medium samples were assayed for transgene levels using Western blot and ELISA. Results were compared with commercially available pcDNA3 based vectors encoding the same growth factors. Reverse transcription PCR was used to assay transgene expression in BALB-c mice ischemic muscle. ELISA and Western blotting data suggest that PC4W based constructs give a higher protein output of about 2-2,5 fold compared with pcDNA3 based plasmids. Optimization of nucleotide sequence in growth factors cDNA results in additional increase in transgene expression. RT-PCR data shows that expression of human HGF persists in murine ischemic skeletal muscle up to 14 days after gene transfer. Our results indicate that novel plasmid constructs for angiogenic growth factors expression have a good efficacy in vitro and in vivo and can be used for VEGF1B5, HGF and angiopoietin-1 expression in human cell culture and in experimental animals' tissue. At the moment all developed constructs pass through a series of experiments in animal ischemia models and will be used for combined gene therapy development.

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IGF-I triple helix gene therapy of rat and human gliomas.

PubMed (2003)

L. Trojan Piotr Kopiñski Andrzej Mazurek L Chyczewski A. Ly

IGF-I anti-gene technology was applied in treatment of rat and human gliomas using IGF-I triple helix approach.CNS-1 rat glioma cell and primary human glioblastoma cell lines established from surgically removed glioblastomas multiforme were transfected in vitro with IGF-I antisense (pMT-Anti-IGF-I) or IGF-I triple helix (pMT-AG-TH) expression vectors. The transfected cells were examined for immunogenicity (immunocytochemistry and flow cytometry analysis) and apoptosis phenomena (electron microscopy). 3 x 10(6) transfected cells were inoculated subcutaneously either into transgenic Lewis rats or in patients with glioblastoma. The peripheral blood lymphocytes (PBL) derived from "vaccinated" patients were immunophenotyped for the set of CD antigens (CD4, CD8 etc).Using immunocytochemistry and Northern blot techniques, the transfected "antisense" and "triple-helix" cells showed total inhibition of IGF. Transfected cultures were positively stained either for both MHC-I and B7 antigens--60% of cloned lines, or for MHC-I only--40% of cloned lines. Moreover "triple helix" cells as compared to "antisense" cells showed slightly higher expression of MHC-I or B7. Transfected cells also showed the feature of apoptosis in 60%-70% of cells. In in vivo experiments with rats bearing tumors, the injection of "triple helix" cells expressing both MHC-I and B7 interrupted tumor growth in 80% of cases. In contrast, transfected cells expressing only MHC-I stopped development in 30% of tumors. In five patients with surgically resected glioblastoma who were inoculated with "triple helix" cells, PBL showed an increased percentage of CD4 + CD25+ and CD8 + CD11b-cells, following two vaccinations.The anti-tumor effectiveness of IGF-I anti-gene technology may be related to both MHC-I and B7 expression in cells used for therapy. The IGF-I antigene therapy of human glioblastoma multiforme increases immune response of treated patients.

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Methodology for Anti-Gene Anti-IGF-I Therapy of Malignant Tumours

Chemotherapy Research and Practice (2012)

Jerzy Trojan Yuexin Pan Ming Wei Adama Ly Alexander Shevelev

The aim of this study was to establish the criteria for methodology of cellular "anti-IGF-I" therapy of malignant tumours and particularly for glioblastoma multiforme. The treatment of primary glioblastoma patients using surgery, radiotherapy, and chemotherapy was followed by subcutaneous injection of autologous cancer cells transfected by IGF-I antisense/triple helix expression vectors. The prepared cell "vaccines" should it be in the case of glioblastomas or other tumours, have shown a change of phenotype, the absence of IGF-I protein, and expression of MHC-I and B7. The peripheral blood lymphocytes, PBL cells, removed after each of two successive vaccinations, have demonstrated for all the types of tumour tested an increasing level of CD8(+) and CD8(+)28(+) molecules and a switch from CD8(+)11b(+) to CD8(+)11. All cancer patients were supervised for up to 19 months, the period corresponding to minimum survival of glioblastoma patients. The obtained results have permitted to specify the common criteria for "anti-IGF-I" strategy: characteristics sine qua non of injected "vaccines" (cloned cells IGF-I(-) and MHC-I(+)) and of PBL cells (CD8(+) increased level).

10.1155/2012/721873

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The Chemotactic Action of Urokinase on Smooth Muscle Cells Is Dependent on Its Kringle Domain

Journal of Biological Chemistry (2000)

Svetlana Mukhina Victoria Stepanova Dmitri Traktouev Alexei Poliakov R.Sh. Beabealashvilly

Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His204 to Gln) (r-uPAH/Q), urokinase with mutation of His204to Gln together with a deletion of growth factor-like domain (r-uPAH/Q-GFD), the catalytic domain of urokinase (r-uPALMW), and its kringle domain (r-KD) were expressed inEscherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPAH/Q, and r-uPAH/Q-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC50) were apparent at approximately 2 nm with all the uPA variants. The kringle domain induced cell migration with an EC50 of about 6 nm, whereas the denaturated r-KD and r-uPALMW were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPAH/Q-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPAH/Q-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPAH/Q-GFD and uPAwt, but not r-uPALMW. HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPAH/Q-GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR. Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His204 to Gln) (r-uPAH/Q), urokinase with mutation of His204to Gln together with a deletion of growth factor-like domain (r-uPAH/Q-GFD), the catalytic domain of urokinase (r-uPALMW), and its kringle domain (r-KD) were expressed inEscherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPAH/Q, and r-uPAH/Q-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC50) were apparent at approximately 2 nm with all the uPA variants. The kringle domain induced cell migration with an EC50 of about 6 nm, whereas the denaturated r-KD and r-uPALMW were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPAH/Q-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPAH/Q-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPAH/Q-GFD and uPAwt, but not r-uPALMW. HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPAH/Q-GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR. urokinase-type plasminogen activator growth factor-like domain of uPA kringle domain of uPA proteolytic domain of uPA recombinant single-chain urokinase with wild type structure proteolytically inactive urokinase with substitution of His204 in active center for Gln proteolytically inactive urokinase with substitution of His204 in active center for Gln, lacking GFD low molecular weight form of uPA, containing mainly PD uPA deletion mutant lacking kringle domain uPA receptor tissue-type plasminogen activator human airway smooth muscle cells human embryonic kidney cells monoclonal antibody phosphate-buffered saline Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis bovine serum albumin Plasminogen activators are directly involved in inflammation, angiogenesis, tissue remodeling, and tumor growth and invasion (for review, see Refs. 1.Blasi F. Immunol. Today. 1997; 18: 415-417Abstract Full Text PDF PubMed Scopus (240) Google Scholar, 2.Carmeliet P. Collen D. Kidney Int. 1998; 53: 1519-1549Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 3.Tkachuk V. Stepanova V. Little P.J. Bobik A. Clin. Exp. Pharm. Physiol. 1996; 23: 759-765Crossref PubMed Scopus (57) Google Scholar, 4.Mohanam S. Go Y. Sawaya R. Venkaiah B. Mohan P.M. Kouraklis G.P. Gokaslan Z.L. Lagos G.K. Rao J.S. Int. J. Oncol. 1999; 14: 169-174PubMed Google Scholar). Of the two types of plasminogen activators expressed by tissues, urokinase-type plasminogen activator (uPA1 or urokinase) and tissue-type plasminogen activator (tPA), uPA rather than tPA promotes cell migration (5.Busso N. Masur S.K. Lazega D. Waxman S. Ossowski L. J. Cell Biol. 1994; 126: 259-270Crossref PubMed Scopus (260) Google Scholar, 6.Anichini E. Fibbi G. Pucci M. Caldini R. Chevanne M. Del Rosso M. Exp. Cell Res. 1994; 213: 438-448Crossref PubMed Scopus (56) Google Scholar, 7.Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 8.Nguyen D.H.D. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) and proliferation (9.Rabbani S.A. Mazar A.P. Bernier S.M. Haq M. Bolivar I. Henkin J. Goltzman D. J. Biol. Chem. 1992; 267: 14151-14156Abstract Full Text PDF PubMed Google Scholar, 10.Fischer K. Lutz V. Wilhelm O. Schmitt M. Graeff H. Heiss P. Nishiguchi T. Harbeck N. Kessler H. Luther T. Magdolen V. Reuning U. FEBS Lett. 1998; 438: 101-105Crossref PubMed Scopus (61) Google Scholar, 11.Aguirre Ghiso J.A. Kovalski K. Ossowski L. J. Cell Biol. 1999; 147: 89-103Crossref PubMed Scopus (466) Google Scholar) via interactions with high affinity cell surface receptors (uPAR/CD87). In addition to interacting with uPA, the significance of uPAR in chemotaxis also appears dependent upon interaction with integrins (12.Chapman H.A. Curr. Opin. Cell Biol. 1997; 9: 714-724Crossref PubMed Scopus (424) Google Scholar), and/or exposure of a chemotactic epitope within uPAR following ligand binding or proteolytic cleavage (13.Blasi F. APMIS. 1999; 107: 96-101Crossref PubMed Scopus (84) Google Scholar). The urokinase/uPAR system also participates in the chemotaxis initiated by growth factors which activate tyrosine kinase receptors, as well as several chemoattractant molecules such as formylmethionylleucylphenylalanine (14.Stepanova V. Bobik A. Bibilashvily R. Belogurov A. Rybalkin I. Domogatsky S. Little P.J. Goncharova E. Tkachuk V. FEBS Lett. 1997; 414: 417-474Google Scholar, 15.Herbert J.-M. Lamarche I. Carmeliet P. J. Biol. Chem. 1997; 272: 23585-23591Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16.Cavallaro U. Wu Z. Di Palo A. Montesano R. Pepper M.S. Maier J.A. Soria M.R. FASEB J. 1998; 12: 1027-1034Crossref PubMed Scopus (21) Google Scholar, 17.Gyetko M.R. Todd 3 rd, R.F. Wilkinson C.C. Sitrin R.G. J. Clin. Invest. 1994; 93: 1380-1387Crossref PubMed Scopus (289) Google Scholar). Urokinase is secreted by many cell types, as a single chain polypeptide that possesses little or no proteolytic activity (18.Petersen L.C. Lund L.R. Nielsen L.S. Dano K. Skriver L. J. Biol. Chem. 1988; 263: 11189-11195Abstract Full Text PDF PubMed Google Scholar). It is composed of three structurally independent domains, a proteolytic domain (PD), a kringle domain (KD), and a growth factor-like domain (GFD). The active serine protease is generated from a single cleavage between Lys158 and Ile159 (18.Petersen L.C. Lund L.R. Nielsen L.S. Dano K. Skriver L. J. Biol. Chem. 1988; 263: 11189-11195Abstract Full Text PDF PubMed Google Scholar), and further plasminolysis generates the two-chain low molecular weight form uPALMW, commencing at Lys136 and consisting of mainly PD (19.Barlow G.H. Francis C.W. Marder V.J. Thromb. Res. 1981; 23: 541-547Abstract Full Text PDF PubMed Scopus (35) Google Scholar), and amino-terminal fragment (ATF) (20.Mazar A.P. Buko A. Petros A. Barnathan E.S. Henkin J. Fibrinolysis. 1992; 6 Suppl. 1: 49-55Crossref Scopus (30) Google Scholar). Urokinase PA and the ATF have been shown to bind with similar high affinity to uPAR/CD87 via the GFD (9.Rabbani S.A. Mazar A.P. Bernier S.M. Haq M. Bolivar I. Henkin J. Goltzman D. J. Biol. Chem. 1992; 267: 14151-14156Abstract Full Text PDF PubMed Google Scholar, 21.Appella E. Robinson E.A. Ullrich S.J. Stopelli M.P. Corti A. Cassani G. Blasi F. J. Biol. Chem. 1987; 262: 4437-4440Abstract Full Text PDF PubMed Google Scholar). This interaction induces cell migration and the activation of a number of signal transduction pathways within the cell cytoplasm and transcriptional apparatus; events which are independent of proteolysis (6.Anichini E. Fibbi G. Pucci M. Caldini R. Chevanne M. Del Rosso M. Exp. Cell Res. 1994; 213: 438-448Crossref PubMed Scopus (56) Google Scholar, 7.Dumler I. Weis A. Mayboroda O.A. Maasch C. Jerke U. Haller H. Gulba D.C. J. Biol. Chem. 1998; 273: 315-321Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 8.Nguyen D.H.D. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 22.Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (303) Google Scholar, 23.Sillaber C. Baghestanian M. Hofbauer R. Virgolini I. Bankl H.C. Füreder W. Agis H. Willheim M. Leimer M. Scheiner O. Binder B.R. Kiener H.P. Bevec D. Fritsch G. Majdic O. Kress H.G. Gadner H. Lechner K. Valent P. J. Biol. Chem. 1997; 272: 7824-7832Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 24.Fibbi G. Pucci M. Serni U. Cerinic M.M. Del Rosso M. Proc. Assoc. Am. Physicians. 1998; 110: 340-350PubMed Google Scholar, 25.MacDonald T.J. DeClerck Y.A. Laug W.E. J. Neurooncol. 1998; 40: 215-226Crossref PubMed Scopus (38) Google Scholar, 26.Bohuslav J. Hoρejsi V. Hansmann C. Stockl J. Weidle U.H. Majdic O. Bartke I. Knapp W. Stockinger H. J. Exp. Med. 1995; 181: 1381-1390Crossref PubMed Scopus (355) Google Scholar, 27.Tang H. Kerins D.M. Hao Q. Inagami T. Vaughan D.E. J. Biol. Chem. 1998; 273: 18268-18272Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 28.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar). Several studies indicate that the ability of uPA to initiate a chemotactic response is dependent only on its proteolytic activity (29.Wang W. Chen H.J. Gieddi K.N. Schwartz A. Cannon P.J. Circ. Res. 1995; 77: 1095-1106Crossref PubMed Scopus (20) Google Scholar, 30.Jackson C.L. Reidy M.A. Ann. N. Y. Acad. Sci. 1992; 667: 141-150Crossref PubMed Scopus (81) Google Scholar), implicating a direct or plasmin-mediated activation, or release of the mitogenic growth factors-basic fibroblast growth factor, hepatocyte growth factor/scatter factor, and VEGF− from the extracellular matrix (31.Saksela O. Rifkin D.B. J. Cell Biol. 1990; 110: 767-775Crossref PubMed Scopus (433) Google Scholar, 32.Naldini L. Tamagnone L. Vigna E. Sachs M. Hartmann G. Birchmaier W. Daikuhara Y. Tsubouchi H. Blasi F. Comoglio P.M. EMBO J. 1992; 11: 4825-4833Crossref PubMed Scopus (523) Google Scholar, 33.Plouët J. Moro F. Bertagnolli S. Coldeboeuf N Mazarguil H. Clamens S. Bayard F. J. Biol. Chem. 1997; 272: 13390-13396Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Others have demonstrated that uPA-induced cell migration is dependent on its proteolytic activity, interaction with uPAR, as well as interactions with low density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP/α2-MR) or very low density lipoprotein receptor (34.Okada S.S. Grobmeyer S.R. Barnathan E.S. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1269-1276Crossref PubMed Scopus (90) Google Scholar, 35.Wijnberg M.J. Quax P.H. Nieuwenbroek N.M. Verheijen J.H. Thromb. Haemost. 1997; 78: 880-886Crossref PubMed Scopus (60) Google Scholar). Together these studies indicate that signal transduction pathways utilized by uPA in initiating chemotaxis exhibit marked diversity depending upon cell type. The importance of uPA in cell migration and tissue remodeling was recently demonstrated in knockout mice (36.Carmeliet P. Moons L. Herbert J.M. Crawley J. Lupu F. Lijnen R. Collen D. Circ. Res. 1997; 81: 829-839Crossref PubMed Scopus (191) Google Scholar), where there was marked reduction in the size of the neointima which forms after intra-vessel injury in the uPA-deficient mice, when compared with tPA-deficient and normal animals. In contrast, when mice deficient in uPAR were similarly injured neointima size was unaffected (37.Carmeliet P. Moons L. Dewerchin M. Rosenberg S. Herbert J.M. Lupu F. Collen D. J. Cell Biol. 1998; 140: 233-245Crossref PubMed Scopus (117) Google Scholar). Together these observations suggest that mechanisms, distinct or additional to uPA/uPAR interaction might mediate processes of the uPA-dependent tissue remodeling. Since uPAR is attached to the cell membrane via glycosylphosphatidylinositol anchor and lacks transmembrane and cytoplasmic regions, alone it is not capable of initiating a chemotactic signal. An obligatory partner of yet unknown nature is probably required for uPAR to act as a signaling receptor (13.Blasi F. APMIS. 1999; 107: 96-101Crossref PubMed Scopus (84) Google Scholar, 22.Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (303) Google Scholar), that can be activated either by a uPAR fragment (38.Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar) or following binding of uPA. Such an adaptor might be integrins, which can interact directly with uPAR and promote cell adhesiveness and chemotaxis (39.Gyetko M.R. Sitrin R.G. Fuller J.A. Todd 3 rd, R.F. Petty H.R. Standiford T.J. J. Leukocyte Biol. 1995; 58: 533-538Crossref PubMed Scopus (92) Google Scholar, 40.Petty H.R. Todd 3 rd, R.F. Immunol. Today. 1996; 17: 209-212Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 41.Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (697) Google Scholar, 42.Kindzelskii A.L. Eszes M.M. Todd 3 rd, R.F. Petty H.R. Biophys. J. 1997; 73: 1777-1784Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 43.Xue W. Mizukami I. Todd 3 rd, R.F. Petty H.R. Cancer Res. 1997; 57: 1682-1689PubMed Google Scholar, 44.May A.E. Kanse S.M. Lund L.R. Gisler R.H. Imhof B.A. Preissner K.T. J. Exp. Med. 1998; 188: 1029-1037Crossref PubMed Scopus (266) Google Scholar). Most investigating uPA-dependent chemotaxis has utilized full-length uPA, DFP-inactivated uPA, or its ATF. However, the ATF includes the growth factor-like and kringle domains, which behave as independently folded domains (45.Novokhatny V. Medved L. Mazar A. Marcotte P. Henkin J. Ingham K. J. Biol. Chem. 1992; 267: 3878-3885Abstract Full Text PDF PubMed Google Scholar). Urokinase PA-kringle is highly homologous to the kringle structure-containing fragments of blood plasma proteins, which affect cell motility and/or proliferation (46.Ji W.-R. Barrientos L.G. Llinas M. Gray H. Villarreal X. DeFord M.E. Castellino F.J. Kramer R.A. Trail P. Biochem. Biophys. Res. Commun. 1998; 247: 414-419Crossref PubMed Scopus (94) Google Scholar,47.Cao Y. Ji R.W. Davidson D. Schaller J. Marti D. Söhndel S. McCance S.G. O'Reilly M.S. Llinás M. Folkman J. J. Biol. Chem. 1996; 271: 29461-29467Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Despite these observations, the precise mechanism mediating the uPA-dependent effects on cell migration and the impact of the different domains of uPA on cell migration remain unclear. To elucidate the role of uPA structural domains in cell migration, we have constructed and produced recombinant uPA forms deficient in the GFD, as well as the individual domains of urokinase. Recently we reported that a form of uPA lacking proteolytic activity and the growth factor-like domain is capable of inducing cell migration to a similar extent as uPA. It exhibited the atypical binding of urokinase, which was unrelated to any interaction with uPAR via GFD (48.Poliakov A.A. Mukhina S.A. Traktouev D.O. Bibilashvily R. Sh Gursky Y.G. Minashkin M.M. Stepanova V.V. Tkachuk V.A. J. Recept. Signal Transduct. Res. 1999; 19: 939-951Crossref PubMed Scopus (26) Google Scholar). Here we report that besides the growth factor-like domain, which interacts with uPAR, the kringle domain of urokinase can also be involved in the induction of cell chemotaxis by uPA. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and LipofectinTM were purchased from Life Technologies, Inc.. Chromogenic substrate S-2444 was obtained from Chromogenix, Mölndal, Sweden. Glycosylated high molecular weight urokinase purified from urine (glycosylated uPA) was from Medac, Hamburg, Germany, and tPA (Actilyse) was from Roche Molecular Biochemicals. A mouse monoclonal antibody clone 3 (R-3-01) which reacts with domain I of human uPAR was from Monozyme, Denmark. The goat anti-mouse IgG conjugated with horseradish peroxidase and chemiluminescent substrate "Super Signal Substrate" were from Pierce. Plasminogen, a mouse IgG, and two different murine monoclonal antibodies of the IgG1 subtype (UIG-1 and UNG-5) raised against human urinary uPA (49.Kratasyuk G.A. Jakubov L.Z. Sinitsyn V.V. Domogatsky S.P. Rohklin O.V. Koltsova S.V. Bynyaeva N.A. Fedorova Z.D. Samsonov G.V. Biopolim. Kletka. 1989; 5: 95-101Google Scholar) were kindly provided by Dr. S. P. Domogatsky. An anti-uPA mAb UIG-1 detected high and low molecular forms of uPA by Western blots. An anti-uPA mAb UNG-5 detected urokinase forms containing the kringle domain, (specifically, glycosylated uPA, r-uPAwt, r-uPAH/Q, r-uPAH/Q-GFD, and r-KD); it does not interact with r-uPALMW, lacking the kringle domain, tPA, and plasminogen. UNG-5 did not interact with denaturated r-KD, uPAwt, and r-uPAH/Q-GFD. Recombinant uPA with wild-type structure (r-uPAwt), proteolytically inactive urokinase with substitution of His204 in active center for Gln (r-uPAH/Q), and proteolytically inactive urokinase with deletion of NH2-terminal 1–43 amino acids called "growth factor-like domains" (r-uPAH/Q-GFD) were expressed inEscherichia coli and purified as described previously (14.Stepanova V. Bobik A. Bibilashvily R. Belogurov A. Rybalkin I. Domogatsky S. Little P.J. Goncharova E. Tkachuk V. FEBS Lett. 1997; 414: 417-474Google Scholar,48.Poliakov A.A. Mukhina S.A. Traktouev D.O. Bibilashvily R. Sh Gursky Y.G. Minashkin M.M. Stepanova V.V. Tkachuk V.A. J. Recept. Signal Transduct. Res. 1999; 19: 939-951Crossref PubMed Scopus (26) Google Scholar). Two-chain low molecular weight urokinase with the catalytic domain r-uPALMW was prepared by proteolytic cleavage of r-uPAwt by plasmin, followed by purification with affinity chromatography, using monoclonal antibody against urokinase catalytic domain coupled to CNBr-Sepharose 4B (Amersham Pharmacia Biotech). The recombinant kringle domain (r-KD) was made in E. coli as follows. The region of uPA cDNA corresponding to amino acids 42–210 (50.Riccio A. Grimaldi G. Verde P. Sebastio G. Boast S. Blasi F. Nucleic Acids Res. 1985; 13: 2759-2771Crossref PubMed Scopus (172) Google Scholar, 51.Holmes W.E. Pennica D. Blaber M. Rey M.W. Guenzler W.A. Steffens G.J. Heyneker H.L. Bio/Technology. 1985; 3: 923-929Crossref Scopus (199) Google Scholar) was amplified using primers: M3, 5′-CTGTGATCTAGATAAGTCAAAAACCTGCTATGAGGG-3′, and M7, 5′-AGCACTGTGTGGCGCTGATCACCCAGCAAGGGCTG-3′. Primer M3 was designed to introduce an XbaI site (underlined) between GFD and KD of uPA without changing the amino acid sequence. The PCR product was digested with XbaI andEcoRI enzymes, generating a fragment coding urokinase amino acids 42–166. The expression vector was constructed by modifying a pTZ19 plasmid between HindIII and XbaI sites. This modification was done using a synthetic fragment containing a new translation initiation region and coding for the first 5 amino acids (MKSTL). This plasmid allowed the cloning of coding inserts between theXbaI and BamHI sites. TheXbaI-EcoRI fragment of uPA and theEcoRI and BamHI ending synthetic duplex: 5′-AATTCACCACCCTGCACCATCACCATCACCATTAATAG-3′; 3′-GTGGTGGGACGTGGTAGTGGTAGTGGTAATTATCCTAG-5′ were ligated between the XbaI and BamHI sites; the duplex sequence contains a hexahistidine tag and stop codon after theEcoRI site. The structure of the resulting construct was confirmed by sequencing. The cloning procedures outlined above resulted in a pKR-his6 plasmid coding for a polypeptide identical to r-uPAwt amino acids 43–166 as shown:MKSTLEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDA- LQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVKECMVHDCADGK- KPSSPPEELKFQCGQKTLRP RF KIIGGEFTTLHHHHHH. The non-urokinase amino acids acquired from the vector are underlined. E. coli strain JM109 was transformed by the resulting pKR-his6 plasmid. Purification of r-KD was carried out as follows: inclusion bodies were isolated and the proteins denatured in buffer containing 6 m guanidine chloride. Subsequent reconstitution was carried out by gradually removing the denaturing agents while maintaining appropriate redox conditions with a glutathione-containing buffer (52.Marston F.A.O. Biochem. J. 1986; 240: 1-12Crossref PubMed Scopus (752) Google Scholar). The primary structure of r-KD allowed us to use nickel-chelate affinity chromatography for its initial purification. Subsequent purification to homogeneity was achieved by affinity chromatography on Sepharose coupled with the monoclonal antibody UNG-5. Then the kringle polypeptide was treated with thrombin, to cleave the peptide bond (Arg156-Phe157) in urokinase (53.Ichinose A. Fujikawa K. Suyama T. J. Biol. Chem. 1986; 261: 3486-3489Abstract Full Text PDF PubMed Google Scholar) (shown in bold); affinity chromatography using anti-kringle mAb removed the hexahistidine tag, any uncleaved peptides and thrombin. The resulting purified r-KD corresponded to urokinase amino acids Glu43-Arg156. The homogeneity of recombinant urokinase forms was confirmed by SDS-PAGE and Western blotting using monoclonal antibodies directed against the urokinase catalytic and kringle domains. The amidolytic activities of the glycosylated uPA and recombinant uPA forms were determined using the chromogenic urokinase substrate S-2444, as described previously (54.Stepanova V. Mukhina S. Köhler E. Resink T.J. Erne P. Tkachuk V.A. Mol. Cell. Biochem. 1999; 195: 199-206Crossref PubMed Scopus (44) Google Scholar). The proteolytic activities of glycosylated uPA, r-uPAwt, and r-uPALMW were ∼5000 units/nmol; the other urokinase forms with substitution of His204 in active center for Gln (r-uPAH/Q and r-uPAH/Q-GFD) as well as r-KD were proteolytically inactive. R-uPAwt and r-uPAH/Q coupled to CNBr-Sepharose 4B (Amersham Pharmacia Biotech) specifically precipitated uPAR from cell lysates with similar efficiency, while r-uPAH/Q-GFD and r-uPALMW did not. This was performed as described previously (46.Ji W.-R. Barrientos L.G. Llinas M. Gray H. Villarreal X. DeFord M.E. Castellino F.J. Kramer R.A. Trail P. Biochem. Biophys. Res. Commun. 1998; 247: 414-419Crossref PubMed Scopus (94) Google Scholar, 47.Cao Y. Ji R.W. Davidson D. Schaller J. Marti D. Söhndel S. McCance S.G. O'Reilly M.S. Llinás M. Folkman J. J. Biol. Chem. 1996; 271: 29461-29467Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Briefly, r-KD (110 μg, 1 ml) was reduced by dithiothreitol (0.5m, 15 min at room temperature). The reduced r-KD was alkylated by the addition of 0.25 m iodoacetamide for 1 h at 4 °C and then dialyzed against PBS. Both the denatured r-KD and the intact r-KD were analyzed by SDS-PAGE and Western blotting using anti-kringle mAb UNG-5. Human airway smooth muscle cells (hAWSMC) were isolated from trachea and characterized as previously described (55.Panettieri R.A. Murray R.K. DePalo L.R. Yadvish P.A. Kotlikoff M.I. Am. J. Physiol. 1989; 256: C329-C355Crossref PubMed Google Scholar). Human embryonic kidney cells HEK 293 were from Cardiology Research Center Cell Collection, Moscow. The cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 10 mm HEPES, 5 mm glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin at 37 °C and 5% CO2. The XbaI-EcoRI fragment from pB SK-(uPAR) plasmid containing human uPAR-cDNA (GenBank accession number X51675), was ligated into pUC19 (New England Biolabs) producing an intermediate plasmid pUC-uPAR. Then theHindIII-EcoRI fragment from pUC-uPAR was inserted into the pcDNA3 expression vector (Invitrogen). The resultant plasmid pcDNA-uPAR transfected into HEK 293 cells using Lipofectin, according to the manufacturers protocol. Two days later the cells were plated onto 96-well plates for clone selection. The transfected cells were cultured in DMEM with 10% fetal bovine serum containing 1.5 mg/ml G418 (Life Technologies, Inc.) for 25 days and G418-resistant clones were analyzed. Recombinant-uPAR expressed by these HEK 293 cells specifically bound recombinant uPAwt, assessed using an affinity uPAR precipitation method from cell lysates and immobilized r-uPAwt. Cells HEK 293 transfected by plasmid pcDNA3-anti-βGAL bearing a 3-kilobase fragment of E. coli β-galactosydase gene inserted in the antisense orientation (HEK-control cells) were used as controls. Cells were scraped into 10 mm HEPES buffer (pH 7.2), containing 150 mmNaCl, 1 mm EDTA, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride, and centrifuged at 1500 × g for 3 min at 4 °C. The pellet was resuspended in 10 mm Tris-HCl buffer (pH 8.0), containing 150 mm NaCl, 2 mm EDTA, 1% Triton X-100, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mmphenylmethylsulfonyl fluoride. After incubation for 20 min at 4 °C, the suspension was centrifuged at 5,000 × g for 15 min at 4 °C. The proteins in the supernatant were mixed with SDS sample buffer and subjected to 10% SDS-PAGE/Western blotting onto polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was immersed overnight at 4 °C in PBS containing 1% casein, 0.05% Tween 20, and 1 μg/ml anti-uPAR mAb (Monozyme). After multiple washings the membranes were incubated with a goat anti-mouse IgG conjugated to horseradish peroxidase and visualized using the chemiluminescent substrate "Super Signal Substrate." Migration of hAWSMC was determined as described previously, using a micro-Boyden chamber (14.Stepanova V. Bobik A. Bibilashvily R. Belogurov A. Rybalkin I. Domogatsky S. Little P.J. Goncharova E. Tkachuk V. FEBS Lett. 1997; 414: 417-474Google Scholar). Briefly, to determine the role of uPAR in uPA-induced cell migration, a hAWSMC cell suspension in DMEM supplemented with 0.1% BSA was preincubated with the anti-uPAR mAb (50 μg/ml) or a mouse Ig (50 μg/ml) for 1 h prior to seeding the cells into the upper wells of the Boyden chamber. When the role of the urokinase-kringle domain in uPA-induced cell migration was to be assessed, r-uPAwt, r-uPAH/Q-GFD, or r-KD and the anti-kringle mAb UNG-5 (20 μg/ml) or with total mouse Ig (20 μg/ml) were preincubated for 1 h prior to placement into the lower wells. HEK cell suspension was prepared in DMEM supplemented with 1% BSA. The extent of cell migration was evaluated after incubation for 3 h at 37 °C in the CO2 incubator. Data are presented as peak area on scanned fields with stained cells and expressed as percentage of cells that migrate across a filter compared with control chemotaxis. Comparisons between cell migration under the different conditions were analyzed using Student's t test. A p < 0.05 value was considered statistically significant. All results are expressed as mean ± S.E. Urokinase constructs (60 μg) were iodinated with 1 mCi of Na125I and 0.1 mg of IODO-GEN (Pierce) for 8 min at room temperature and the reaction was terminated with excessl-tyrosine. Purification of iodinated peptides was carried out by chromatography on PD-10 columns (Amersham Pharmacia Biotech) equilibrated with PBS. Iodinated peptides could be precipitated (95–98%) by trichloroacetic acid and had specific activities from 3.0 to 4.0 × 105 cpm/pmol. Binding studies with the iodinated peptides were carried out on

Kringle domain

10.1074/jbc.m909080199

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КАЛЬДЕCМОН ИЗМЕНЯЕТ CТPУКТУPУ АКТИНА НА ЛИДИPУЮЩЕМ КPАЕ И ПОДАВЛЯЕТ МИГPАЦИЮ КЛЕТОК

Биофизика (2008)

П Н Pуткевич Т Н Влаcик А В Воpотников Т В Кудpяшова Alexander Shevelev

Исследован эффект подавления экспрессии актинсвязывающего белка кальдесмона на подвижность немышечных клеток. Более чем пятикратное снижение содержания этого белка в клетках с помощью РНК-интерференции приводило к нарушению образования актиновых стресс-фибрилл и ускорению миграции клеток в зону повреждения монослоя. Стимуляция стационарных клеток сывороткой вызывала более чем 1,5-кратное накопление стресс-фибрилл только в контрольных клетках, но не в клетках, дефицитных по кальдесмону. Аналогично, накопление филаментов актина наблюдалось в активно мигрирующих клетках только дикого типа, но не в клетках с пониженным содержанием кальдесмона. Эти изменения происходили в основном на лидирующем крае мигрирующей клетки, где характерная для контрольных клеток четкая структура актиновых филаментов не выявлялась в отсутствие кальдесмона. Предполагается, что кальдесмон тормозит миграцию клеток за счет стабилизации актина в филаментах и снижения динамики мономерного актина на лидирующем крае мигрирующей клетки.

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Antisense anti-IGF-I therapy of primary hepatic cancer

Journal africain du cancer / African Journal of Cancer (2009)

L. Trojan A. Ly Lia C. Upegui-Gonzalez Piotr Jarocki Alexander Shevelev

Teratocarcinoma

10.1007/s12558-008-0005-9

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The Effect of 15-Ketosterol Analogues with a 5,6-Dimethylhept-3-en-2-yl Chain at C17 on Cholesterol Metabolism in Hep G2 Hepatoma Cells

Russian Journal of Bioorganic Chemistry (2004)