Indication of a role of plasminogen activator inhibitor type 1 in protecting murine fibrosarcoma cells against apoptosis

Maria Unni Rømer; Anne Kirkebjerg Due; Jørgen Knud Larsen; Kenneth Francis Hofland; Ib Jarle Christensen; Peter Buhl-Jensen; Kasper Almholt; Ole Lerberg Nielsen; Nils Brünner; Ulrik Lademann

doi:10.1160/TH05-01-0011

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 94(04): 859-866
DOI: 10.1160/TH05-01-0011

Cellular Proteolysis and Oncology

Schattauer GmbH

Indication of a role of plasminogen activator inhibitor type 1 in protecting murine fibrosarcoma cells against apoptosis

Authors

Maria Unni Rømer

¹Institute of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark

²Department of Oncology, H:S Rigshospitalet, Copenhagen Ø., Denmark
Anne Kirkebjerg Due

¹Institute of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark
Jørgen Knud Larsen^*

¹Institute of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark
Kenneth Francis Hofland

²Department of Oncology, H:S Rigshospitalet, Copenhagen Ø., Denmark
Ib Jarle Christensen

³Department of Surgical Gastroenterology, H:S Hvidovre Hospital, Hvidovre, Denmark
Peter Buhl-Jensen

²Department of Oncology, H:S Rigshospitalet, Copenhagen Ø., Denmark
Kasper Almholt^*

¹Institute of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark
Ole Lerberg Nielsen

¹Institute of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark
Nils Brünner

¹Institute of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Frederiksberg C, Denmark
Ulrik Lademann

Abstract

Summary

In a number of cancer types high tumor tissue levels of plasminogen activator inhibitor type 1 (PAI-1) protein are strongly associated with shorter cancer patient survival. This association has been intriguing since PAI-1 is known to inhibit urokinase plasminogen activator (uPA) that converts plasminogen to plasmin, which is actively involved in tumor progression and invasion. In order to further explore the biological role of PAI-1 in cancer, we have prepared fibroblasts from PAI-1 gene deficient mice and from their wild type littermates. From these fibroblasts fibrosarcoma cell lines were established and characterized. Both types of fibroblasts underwent spontaneous transformation as indicated by aneuploidy, immortalization, clonogenicity in soft agar and tumor formationin vivo. While both PAI-1 deficient and PAI-1 expressing cell lines showed similar proliferation ratesin vitro, cells devoid of PAI-1 were significantly more sensitive to apoptotic stimuli. When inoculated subcutaneously into nude mice PAI-1 expressing cells rapidly established tumors, while PAI-1 deficient cells had a significantly longer lag-phase before they started to grow (p<0.0001). The present study suggests that PAI-1,besides its uPA inhibiting function, has a role in cancer progression by protecting tumor cells from undergoing apoptosis.

Keywords

PAI-1 - murine fibrosarcoma cell lines - apoptosis

Full Text

References

References
1 Sidenius N, Blasi F. The urokinase plasminogen activator system in cancer: Recent advances and implication for prognosis and therapy. Cancer Metastasis Reviews 2003; 22: 205-22.
2 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92: 827-39.
3 Dano K, Romer J, Nielsen BS. et al. Cancer invasion and tissue remodeling–cooperation of protease systems and cell types. Apmis 1999; 107: 120-7.
4 Andreasen PA, Egelund R, Petersen HH. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 2000; 57: 25-40.
5 Hagedorn HG, Bachmeier BE, Nerlich AG. Synthesis and degradation of basement membranes and extracellular matrix and their regulation by TGF-beta in invasive carcinomas (Review). Int J Oncol 2001; 18: 669-81.
6 Rosenthal EL, Johnson TM, Allen ED. et al. Role of the plasminogen activator and matrix metalloproteinase systems in epidermal growth factor- and scatter factor-stimulated invasion of carcinoma cells. Cancer Res 1998; 58: 5221-30.
7 Padro T, Mesters RM, Dankbar B. et al. The catalytic domain of endogenous urokinase-type plasminogen activator is required for the mitogenic activity of platelet- derived and basic fibroblast growth factors in human vascular smooth muscle cells. J Cell Sci 2002; 115 Pt 9 1961-71.
8 Bajou K, Noel A, Gerard RD. et al. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat Med 1998; 4: 923-8.
9 Bajou K, Masson V, Gerard RD. et al. The plasminogen activator inhibitor PAI-1 controlsin vivo tumor vascularization by interaction with proteases, not vitronectin: Implications for antiangiogenic strategies. J Cell Biol 2001; 152: 777-84.
10 Kjoller L. The urokinase plasminogen activator receptor in the regulation of the actin cytoskeleton and cell motility. Biol Chem 2002; 383: 5-19.
11 Behrendt N, Ronne E, Dano K. The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface. Biol Chem Hoppe Seyler 1995; 376 (05) 269-79.
12 Durand MK, Bodker JS, Christensen A. et al. Plasminogen activator inhibitor-I and tumour growth, invasion, and metastasis. Thromb Haemost 2004; 91 (03) 438-49.
13 Look MP, Foekens JA. Clinical relevance of the urokinase plasminogen activator system in breast cancer. Apmis 1999; 107: 150-9.
14 Schmitt M, Harbeck N, Thomssen C. et al. Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy. Thromb Haemost 1997; 78: 285-96.
15 Papadopoulou S, Scorilas A, Yotis J. et al. Significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 (PAI-1) expression in human colorectal carcinomas. Tumour Biol 2002; 23: 170-8.
16 Sier CF, Vloedgraven HJ, Ganesh S. et al. Inactive urokinase and increased levels of its inhibitor type 1 in colorectal cancer liver metastasis. Gastroenterology 1994; 107: 1449-56.
17 Look MP, van Putten WL, Duffy MJ. et al. Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst 2002; 94: 116-28.
18 Harbeck N, Kates RE, Gauger K. et al. Urokinasetype plasminogen activator (uPA) and ist inhibitor PAI-1: novel tumor-derived factors with a high prognostic and predictive impact in breast cancer. Thromb Haemost 2004; 91: 450-6.
19 Stefansson S, McMahon GA, Petitclerc E. et al. Plasminogen activator inhibitor-1 in tumor growth, angiogenesis and vascular remodeling. Curr Pharm Des 2003; 9: 1545-64.
20 Kwaan HC, Wang J, Svoboda K. et al. Plasminogen activator inhibitor 1 may promote tumour growth through inhibition of apoptosis. Br J Cancer 2000; 82: 1702-8.
21 Chen Y, Kelm Jr. RJ, Budd RC. et al. Inhibition of apoptosis and caspase-3 in vascular smooth muscle cells by plasminogen activator inhibitor type-1. J Cell Biochem 2004; 92: 178-88.
22 Carmeliet P, Kieckens L, Schoonjans L. et al. Plasminogen- activator inhibitor-1 gene deficient mice .1. generation by homologous recombination and characterization. J Clin Invest 1993; 9: 2746-55.
23 Brunner N, Boysen B, Romer J. et al. The nude mouse as anin vivo model for human breast cancer invasion and metastasis. Breast Cancer Res Treat 1993; 24 (03) 257-64.
24 Laird PW, Zijderveld A, Linders K. et al. Simplified mammalian DNA isolation procedure. Nucleic Acids Res 1991; 19: 4293.
25 Almholt K, Nielsen BS, Frandsen TL. et al. Metastasis of transgenic breast cancer in plasminogen activator inhibitor-1 gene-deficient mice. Oncogene 2003; 22: 4389-97.
26 Vindelov LL, Christensen IJ. A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis. Cytometry 1990; 11: 753-70.
27 Jensen PB, Christensen IJ, Sehested M. et al. Differential cytotoxicity of 19 anticancer agents in wild type and etoposide resistant small cell lung cancer cell lines. Br J Cancer 1993; 67: 311-20.
28 Bancroft JD. Theory and Practice of Histological Techniques.. Stevens A. [4th ed.] 1-766. Hong Kong, Churchill Livingstone.: Ref Type: Generic.
29 Goldenberg DM, Pavia RA. In vivo horizontal oncogenesis by a human tumor in nude mice. Proc Natl Acad Sci U S A 1982; 79: 2389-92.
30 Schrohl AS, Christensen IJ, Pedersen AN. et al. Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer. Mol Cell Proteomics 2003; 2: 164-72.
31 Pappot H. The plasminogen activation system in lung cancer–with special reference to the prognostic role in “non-small cell lung cancer”. APMIS Suppl 1999; 92: 1-29.
32 Bindal AK, Hammoud M, Shi WM. et al. Prognostic significance of proteolytic enzymes in human brain tumors. J Neurooncol 1994; 22: 101-10.
33 Nozaki S, Endo Y, Kawashiri S. et al. Immunohistochemical localization of a urokinase-type plasminogen activator system in squamous cell carcinoma of the oral cavity: association with mode of invasion and lymph node metastasis. Oral Oncol 1998; 34: 58-62.
34 Bianchi E, Cohen RL, Dai A. et al. Immunohistochemical localization of the plasminogen activator inhibitor- 1 in breast cancer. Int J Cancer 60: 597-603.
35 Gutierrez LS, Schulman A, Brito-Robinson T. et al. Tumor development is retarded in mice lacking the gene for urokinase-type plasminogen activator or its inhibitor, plasminogen activator inhibitor-1. Cancer Research 2000; 60: 5839-47.
36 Nykjaer A, Conese M, Christensen EI. et al. Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes. EMBO J 1997; 16: 2610-20.