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Polyoma middle T-induced vascular tumor formation: the role of the plasminogen activator/plasmin system.

Sabapathy KT, Pepper MS, Kiefer F, Möhle-Steinlein U, Tacchini-Cottier F, Fetka I, Breier G, Risau W, Carmeliet P, Montesano R, Wagner EF - J. Cell Biol. (1997)

Bottom Line: In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates.Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA.Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, University of Vienna, A-1030 Vienna, Austria.

ABSTRACT
The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells, leading to the formation of vascular tumors in newborn mice. Transformed endothelial (End.) cell lines established from such tumors exhibit altered proteolytic activity as a result of increased expression of urokinase-type plasminogen activator (uPA) and are capable of inducing vascular tumors efficiently when injected into adult mice. In this study we have used mice lacking components of the PA/plasmin system to analyze the role of this system in the transformation process and in tumor growth. We found that the proteolytic status of the host is not a critical determinant for PymT-induced vascular tumor formation. In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates. Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA. However, in contrast with tumors induced by PymT itself, the tumorigenic potential of mutant and wild-type End. cell lines was found to be highly dependent on the proteolytic status of both the tumor cells and the host. Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.

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Expression of mRNA (A) and zymographic analysis  (B) of End. cells lacking uPA, tPA, utPA, and PAI-1. (A) Poly  A+ RNA from the following cells were analyzed by Northern  analysis: b4, passage number 41 (p41); u23 (p12); u24 (p18); t37  (p22); t38 (p16); ut1 (p9); ut2 (p11); p1 (p28); and p3 (p20). (B)  The following End. cell extracts and culture supernatants were  analyzed by zymography (a and b) and reverse zymography (c) as  described in Materials and Methods: s2 (p29); u23 (p30); t37  (p31); ut1 (p19); ut2 (p17); and p1 (p27). (b) The same gel as  shown in a, incubated at 37°C for a longer time period.
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Figure 3: Expression of mRNA (A) and zymographic analysis (B) of End. cells lacking uPA, tPA, utPA, and PAI-1. (A) Poly A+ RNA from the following cells were analyzed by Northern analysis: b4, passage number 41 (p41); u23 (p12); u24 (p18); t37 (p22); t38 (p16); ut1 (p9); ut2 (p11); p1 (p28); and p3 (p20). (B) The following End. cell extracts and culture supernatants were analyzed by zymography (a and b) and reverse zymography (c) as described in Materials and Methods: s2 (p29); u23 (p30); t37 (p31); ut1 (p19); ut2 (p17); and p1 (p27). (b) The same gel as shown in a, incubated at 37°C for a longer time period.

Mentions: We have previously established transformed End. cell lines from tumor-bearing wild-type and various Src-kinase mutant mice, as well as by infection of primary endothelial cells by PymT in vitro (for review see Wagner and Risau, 1994; Pepper et al., 1997). To test whether the absence of components of the PA/plasmin system affects the capacity to establish permanent End. cell lines in vitro, we derived a number of independent cell lines from tumors induced in tPA-, uPA-, utPA-, and PAI-1–deficient mice (Table I). Each of these cell lines displayed the characteristic spindle-shaped, highly refractile morphology that is typical of endothelial cells (data not shown). The time required to establish the different mutant cell lines as homogeneously growing, characteristic End. cells was not significantly different using single knockout mice. However, the double mutant utPA−/− End. cells took three times as long to establish when compared with controls (data not shown). With the exception of ut1 and ut2, all End. cell lines exhibited roughly equivalent growth parameters in vitro as measured by cell doubling times after ∼10 passages (Fig. 2). The proliferation rate of ut1 and ut2 cell lines was <50% of controls. All End. cell lines obtained expressed the endothelial-specific tyrosine kinase receptor Flk-1 (Fig. 3 A) and CD31/PECAM-1 (data not shown), confirming their endothelial origin. Most of these cell lines were clonal, since only one integration site for the provirus was detected by Southern blotting (data not shown).


Polyoma middle T-induced vascular tumor formation: the role of the plasminogen activator/plasmin system.

Sabapathy KT, Pepper MS, Kiefer F, Möhle-Steinlein U, Tacchini-Cottier F, Fetka I, Breier G, Risau W, Carmeliet P, Montesano R, Wagner EF - J. Cell Biol. (1997)

Expression of mRNA (A) and zymographic analysis  (B) of End. cells lacking uPA, tPA, utPA, and PAI-1. (A) Poly  A+ RNA from the following cells were analyzed by Northern  analysis: b4, passage number 41 (p41); u23 (p12); u24 (p18); t37  (p22); t38 (p16); ut1 (p9); ut2 (p11); p1 (p28); and p3 (p20). (B)  The following End. cell extracts and culture supernatants were  analyzed by zymography (a and b) and reverse zymography (c) as  described in Materials and Methods: s2 (p29); u23 (p30); t37  (p31); ut1 (p19); ut2 (p17); and p1 (p27). (b) The same gel as  shown in a, incubated at 37°C for a longer time period.
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Related In: Results  -  Collection

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Figure 3: Expression of mRNA (A) and zymographic analysis (B) of End. cells lacking uPA, tPA, utPA, and PAI-1. (A) Poly A+ RNA from the following cells were analyzed by Northern analysis: b4, passage number 41 (p41); u23 (p12); u24 (p18); t37 (p22); t38 (p16); ut1 (p9); ut2 (p11); p1 (p28); and p3 (p20). (B) The following End. cell extracts and culture supernatants were analyzed by zymography (a and b) and reverse zymography (c) as described in Materials and Methods: s2 (p29); u23 (p30); t37 (p31); ut1 (p19); ut2 (p17); and p1 (p27). (b) The same gel as shown in a, incubated at 37°C for a longer time period.
Mentions: We have previously established transformed End. cell lines from tumor-bearing wild-type and various Src-kinase mutant mice, as well as by infection of primary endothelial cells by PymT in vitro (for review see Wagner and Risau, 1994; Pepper et al., 1997). To test whether the absence of components of the PA/plasmin system affects the capacity to establish permanent End. cell lines in vitro, we derived a number of independent cell lines from tumors induced in tPA-, uPA-, utPA-, and PAI-1–deficient mice (Table I). Each of these cell lines displayed the characteristic spindle-shaped, highly refractile morphology that is typical of endothelial cells (data not shown). The time required to establish the different mutant cell lines as homogeneously growing, characteristic End. cells was not significantly different using single knockout mice. However, the double mutant utPA−/− End. cells took three times as long to establish when compared with controls (data not shown). With the exception of ut1 and ut2, all End. cell lines exhibited roughly equivalent growth parameters in vitro as measured by cell doubling times after ∼10 passages (Fig. 2). The proliferation rate of ut1 and ut2 cell lines was <50% of controls. All End. cell lines obtained expressed the endothelial-specific tyrosine kinase receptor Flk-1 (Fig. 3 A) and CD31/PECAM-1 (data not shown), confirming their endothelial origin. Most of these cell lines were clonal, since only one integration site for the provirus was detected by Southern blotting (data not shown).

Bottom Line: In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates.Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA.Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, University of Vienna, A-1030 Vienna, Austria.

ABSTRACT
The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells, leading to the formation of vascular tumors in newborn mice. Transformed endothelial (End.) cell lines established from such tumors exhibit altered proteolytic activity as a result of increased expression of urokinase-type plasminogen activator (uPA) and are capable of inducing vascular tumors efficiently when injected into adult mice. In this study we have used mice lacking components of the PA/plasmin system to analyze the role of this system in the transformation process and in tumor growth. We found that the proteolytic status of the host is not a critical determinant for PymT-induced vascular tumor formation. In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates. Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA. However, in contrast with tumors induced by PymT itself, the tumorigenic potential of mutant and wild-type End. cell lines was found to be highly dependent on the proteolytic status of both the tumor cells and the host. Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.

Show MeSH
Related in: MedlinePlus