| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
Walther Cancer Center, W. M. Keck Center for Transgene Research, and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In addition to their roles in clot dissolution, components of the fibrinolytic system, including Pg, uPA, uPAR, PAI-1, and tPA have been shown to be important for other processes, such as ECM degradation, in some cases via activation of MMPs (3) , wound healing (4) , atherosclerosis (5) , angiogenesis (6) , and cancer (7) . In this latter case, observations that neoplastic cells express elevated levels of uPA, tPA, PAI-1, and uPAR suggest that these proteins may be involved in tumor growth, metastasis, and angiogenesis (8, 9, 10, 11) . Angiogenesis-dependent tumor progression (12) has also been associated with interaction of integrins with uPA and uPAR, thus linking cellular proteolysis and ECM interactions (13) . Confirming the importance of fibrinolytic components and the cancer phenotype, numerous clinical studies have demonstrated that high levels of uPA (14, 15, 16, 17) and PAI-1 (18 , 19) are associated with a poorer prognostic outcome in certain human cancers, and recent data show a similar correlate with the receptor for uPA, uPAR (20, 21, 22) .
On a molecular level, uPA/uPAR complex formation on the surface of tumor cells can focus Pm-catalyzed proteolysis, which in turn can facilitate tumor cell invasion and metastasis by either directly degrading ECM proteins or indirectly performing this same function via activation of MMPs. Immunocytochemical studies of primary tumor and metastasis of Lewis lung carcinoma cells using uPA antibodies have demonstrated intense immunoreactivity at the sites of invasive growth of the tumor and degradation of normal tissue (23) .
Thus, although there appears to be a correlation between enhanced expression of components of the fibrinolytic system and tumor growth and metastasis, the specific mechanisms that underlie the contributions of this system to neoplasia are unclear. In an attempt to illuminate some of these issues, the current study used a fibrosarcoma model in WT, UPA-/-, and PAI-1-/- C57BL/6J mice to directly assess the role of host expression of these proteins in tumorigenesis. The choice of this tumor model was governed by its syngenicity with, and rapid growth characteristics in, the host C57BL/6J mice; its high degree of vascularization; its expression of high levels of the angiogenic factor, VEGF; and its predictable route of metastasis. A summary of the results of this investigation is presented herein.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture.
The C57BL/6J-derived murine fibrosarcoma tumor cell line, T241, was
grown in McCoy’s modified with L-glutamine (RPMI 16290)
containing essential vitamins, 10% fetal bovine serum (Life
Technologies, Rockville, MD), and antibiotics. These cells were
maintained in a humidified incubator at 37°C, 5%
CO2.
Implantation of Tumor Cells in Mice.
At confluency, adherent cells were detached by incubation with 0.25%
trypsin (Hyclone, Rockville, MD). The cells were centrifuged and then
resuspended in a sterile solution of PBS at a final concentration of
about 1.0 x 107 cells/ml. A
100-µl aliquot of resuspended cells (about 1.0 x 106 cells) was injected s.c. between the shoulder
blades 
3 cm from the tail. Visible and small palpable tumors were
measured using a digital caliper (MyCal; Thomas Scientific, Swedesboro,
NJ). Tumor volumes were calculated using the formula:
(width2)(length)(0.52), as described
(24)
.
RT-PCR.
Total RNA was isolated from tumors implanted in WT,
UPA-/-, and
PAI-1-/- mice and from control
WT livers, kidneys, intestines, spleen, and testes.
Additionally, total RNA was obtained from T241 fibrosarcoma cells grown
in culture. The Ultraspec RNA isolation system (Biotech, Houston, TX)
was used in each case for the first strand cDNA synthesis, which was
carried out using the Ready-To-Go kit (Amersham Pharmacia Biotech,
Piscataway, NJ). The entire volume of 33 µl of cDNA was amplified by
PCR using primers specific for the gene of interest (Table 1)
. PCR conditions for PG, TPA, UPAR,
and PAI-1 were: 94°C for 2 min (1 cycle) and 35 cycles at
94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and then a final
incubation at 72°C for 8 min. PCR conditions for the UPA
cDNA were similar, except the annealing temperature was 60°C.
|
For immunohistochemical analysis, 4-µm sections were placed on silane-coated slides, deparaffinized, and rehydrated. Following this, the slides were blocked for endogenous peroxidase activity with Peroxoblock (Zymed, San Francisco, CA). Sections were then preincubated with 10% normal horse serum in PBS-5% BSA for 30 min to block nonspecific binding of the antibodies.
For vWF immunostaining, antihuman vWF antibody (DAKO EPOS, Carpinteria, CA), conjugated to horseradish peroxidase, was applied to the slides and allowed to incubate for 1 h. Detection of the antibody was accomplished with the chromogen, 3-amino-9-ethylcarbazole (Vector, Burlingame, CA) or DAB (Immuno-Pure metal-enhanced DAB substrate kit; Pierce, Rockford, IL). For VEGF immunostaining, murine antihuman VEGF antibody (Oncogene, Research Products, Boston, MA) was used after pretreatment with 10 mM Tris-HCl (pH 10). The slides were incubated with the antibody overnight at room temperature in a humidified chamber. After washing, the sections were incubated for 30 min with a biotinylated horse antimouse antibody (Vector) and with the ABC Vector kit (Vector) for 30 min. Color development was achieved with 3-amino-9-ethylcarbazole kit (Vector).
For cell proliferation studies, BrdUrd (Amersham Pharmacia, Buckinghamshire, United Kingdom) injections were performed 1 h prior to sacrificing the animal. BrdUrd immunohistochemistry was performed using a murine monoclonal antibody (Dako). Before antibody application (1 µg/ml), sections were immersed in a solution of 2 N HCl at 37°C for 10 min, followed by trypsin treatment as above. Antibody was developed as described for anti-VEGF, except that DAB was used as the substrate.
TUNEL.
The TUNEL assay was performed according to a published method
(25)
. Tissue sections were incubated with 5 µg/ml of
proteinase K for 15 min at room temperature to remove proteins.
Sections were then covered with a buffer containing 30 mM
Tris-HCl (pH 7.2), 140 mM sodium cacodylate, and 1
mM CoCl2. An aliquot of 0.2 µl of
terminal deoxynucleotidyl transferase (Boehringer Mannheim,
Gaithersburg, MD) and 10 µM biotinylated dUTP (Boehringer
Mannheim) were added to the sections. The slides were incubated in a
humidified chamber at 37°C for 60 min, washed with 50 mM
Tris-HCl (pH 7), and finally with PBS (0.06 M sodium
phosphate/0.15 M NaCl, pH 7.3). The sections were then
incubated for 30 min with ABC system, and DAB was used as the
chromogen.
Analysis and Scoring.
MVCs were determined by operational modifications of a published method
(26)
. MVCs were calculated by analyzing vWF-stained
sections. Sections were scanned at low magnification (x100); three
areas per section were considered to have the highest microvessel
densities within the tumor, and adjacent peripheral tissues were
defined as hot spots. Each hot spot was examined at high magnification
(x400), and a computer-digitized image was taken and used for counting
the vessels. The mean of the MVCs of three hot spots was designated as
the total MVCs/hot spot. Two investigators blinded to the genotype of
the animal analyzed the same samples. Three microscopic fields/slide
were counted.
Cell proliferation was determined by counting the number of BrdUrd-positive cells. The results were expressed as the percentage of the total number of tumor cells within a field. The apoptotic index was obtained by dividing the number of apoptotic cells by the total number of cells counted within a field and then multiplying the results by 1000.
Electron Microscopy.
Ultrastructural analyses were performed on tumors from WT,
as well as UPA-/- and
PAI-1-/- mice. The tumors were fixed with
Karnovsky solution (27)
, rinsed twice with a solution of
0.1 M sodium cacodylate (pH 7.3), postfixed in
1% osmium tetroxide, dehydrated in a graded series of ethanol
solutions, and then embedded in epoxy resins (Polysciences, Warrington,
PA). Thick sections (0.5 µm), stained with toluidine blue, were used
to choose the area of interest, after which corresponding ultrathin
sections (90 nm) were cut and stained in 2% uranyl acetate and
Reynold’s lead stain (28)
. Sections were viewed and
photographed using a transmission electron microscope (Hitachi H600,
Tokyo, Japan) at 75 KV accelerating voltage.
Corneal Micropocket Assay.
Mice (4–12 weeks of age) of each genotype were anesthetized with i.p
injections of rodent cocktail containing 0.0135 mg of xylazine, 0.0675
mg of ketamine, and 0.00225 mg of ACE prozamine/g weight of animal. A
corneal pocket was made in one eye by a transverse incision, centrally,
halfway through the cornea. The pocket was 1 mm from the limbic
vessel toward the corneal center (29)
. Hydron-coated
pellets of sucrose aluminum sulfate containing 100 ng of bFGF were
placed in the corneal pocket. On days 3 and 7, bFGF-induced corneal
angiogenesis was evaluated using a slit-lamp ophthamology scope (Nikon)
and photographed with a digital camera (Olympus DP-10). Through the use
of a 180° reticule in the slit-lamp, two measurements were made:
maximal vessel length (mm) between the limbic vessel and the bFGF
pellet and the circumference of neovascularization in clock hours
(15° of arc = 1 clock hour; Ref. 29
). The
neovascular area (mm2) was calculated as
described (29
, 30)
.
Statistical Analysis.
Statistical analyses of the tumor growth measurements and
immunohistochemistry scores were performed with nonparametric ANOVA and
Fisher’s test. The calculations were performed using the StatView
system for Macintosh (Abacus Concepts, Berkeley, CA).
P < 0.05 was considered significant. Where
appropriate, values were expressed as the mean ± SE.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Tumor Growth Suppression in uPA- and
PAI-1-deficient Mice.
To compare tumor progression in UPA-/-
and PAI-1-/- mice relative to their
WT counterparts, equivalent amounts of T241 fibrosarcoma
cells were injected, s.c., in all animals. The sizes of the primary
tumors were measured weekly. All of the WT mice grew tumors,
which appeared earlier and were larger in mass relative to those in the
UPA-/- and
PAI-1-/- mice; some of which never grew
tumors (Fig. 1A)
. These differences proved to be statistically significant
by the third week (Fig. 1B)
. After 3 weeks, the
tumor-bearing mice were sacrificed, and the primary tumors were
removed. Tumors from WT mice (Fig. 2A)
were consistently larger in mass than the tumors of the
UPA-/- and
PAI-1-/- mice and were also more poorly
delineated and hemorrhagic (Fig. 2A)
. Tumors that grew in
UPA-/- and
PAI-1-/- mice were small, well
circumscribed, and easy to dissociate from host tissue (Fig. 2B)
. Masson’s Trichrome staining of the tumors extracted
from UPA-/- and
PAI-1-/- mice (Fig. 2, D and E)
showed an increase in collagen deposition, predominantly
in the periphery of the tumor, which appeared to form a fibrous and
thick pseudocapsule. This pseudocapsule was almost undetectable in the
tumors of WT mice (Fig. 2C)
.
|
|
. Although almost all of the WT mice displayed
these metastases, only a very small number of
UPA-/- and
PAI-1-/- mice, i.e., those
that survived for longer periods (2 months) after the
injection, had evidence of metastasis to these same locations. Because
the numbers of gene-deficient animals that grew tumors were so small
and had significantly aged during this time, more meaningful
commentary on this aspect of the study is not possible.
Expression of Fibrinolytic Factors by Implanted Fibrosarcoma Tumors
in WT, UPA-/-, and
PAI-1-/- Mice.
Some of the tumors excised from the mice were immediately
frozen in liquid nitrogen, and RT-PCR was performed using the same
probes as that described for RT-PCR of cultured cells. All tumors were
found to express the same fibrinolytic proteins as observed in the
cultured cell line (Fig. 3)
.
|
and D, whereas only scattered BrdUrd-positive
cells were detected in the periphery of the tumors in
UPA-/- and
PAI-1-/- mice (4.07 ± 1.82, n = 3, and 2.20 ± 0.47,
n = 3, respectively), as is evident in Fig. 4, B–D
.
|
. Apoptotic nuclei in the tumors were scattered and
located in the surrounding tissue of the tumors in WT mice,
whereas in UPA-/- and
PAI-1-/- mice, they were located within
the tumor and less so in the surrounding tissue and pseudocapsule.
Central necrosis was observed in all of the mice, and some distinct
apoptotic nuclei were observed in these areas. The apoptotic indices
were significantly different between the
UPA-/- and
PAI-1-/- mice, relative to WT
mice (Fig. 5D)
.
|
. Vessels were scattered or not evident at all in
PAI-1-/- mice. Tumors in
UPA-/- mice demonstrated vascular
densities (26.7 ± 1.7, n = 3)
approximately the same as for tumors in WT mice
(23.5 ± 4.8, n = 4; Fig. 6G
). However, tumors in
PAI-1-/- mice showed significantly lower
vascular densities (4.0 ± 2.1, n = 3) when compared with tumors in WT or
UPA-/- mice (Fig. 6G)
.
Positive vessels were localized both in the periphery of the tumor as
well as in the center of the tumors close to necrotic areas (Fig. 6, A–C)
. Because vascularity is not necessarily related to
angiogenesis, immunohistochemical analysis of the VEGF expression in
these tumors was performed (Fig. 6, D–F)
. In general, VEGF
expression was higher in the center and periphery of the tumors. A few
cells in necrotic areas were also positive for VEGF. VEGF staining was
intense in the WT mice and moderate in
UPA-/- mice (Fig. 6, D and E)
. A decrease in intensity and number of positive cells was
observed in tumors from PAI-1-/- mice
(Fig. 6F)
and no increase in staining intensity was observed
in the periphery of these tumors.
|
for
UPA-/- mice (Fig. 7, B and E)
, and for PAI-1-/- mice
(Fig. 7, C and F)
.
|
consists of three or more endothelial
cells with large cytoplasmic processes directed toward the periphery of
the vessel wall. A collagenous interstitium is present around the
vessels (Fig. 7A)
, and pericyte precursors with moderate
differentiation are seen without foot processes near the endothelial
layer or surrounding the vessel (Fig. 7D)
. These cells have
high physiological activity and numerous organelles.
On the other hand, in the case of UPA-/-
mice (Fig. 7B)
, the vessel contains two to three endothelial
cells forming the lumen (Fig. 7B)
, which are larger than
those found in tumors from the WT mice. Endothelial cells
contained intracytoplasmic vacuoles, suggestive of sprouting and
neolumen formation (new lumens in these cells could form by fusing
these spaces and generating new branches). Closely packed perivascular
tumor cells were seen surrounding thin-walled vessels with no
identifiable basement membrane. Cellular junctions, or close cellular
contacts, are formed between endothelial cells and these tumor cells
(Fig. 7E)
. Pseudopodia-like structures from the endothelial
cells appear along the lumen, and no pericyte precursors were observed.
With regard to PAI-1-/- mice, the vessels
formed contain small lumens, formed by one to two endothelial cells
with numerous invaginations and pseudopodia-like extensions (Fig. 7, C and F)
. A collagenous interstitium is present,
and pericyte precursors next to the vessels are noted.
Corneal Micropocket Assays.
Neovascularization was further analyzed using a corneal micropocket
model. Growth of new capillary vessels from the corneal limbus toward
the bFGF pellet was observed in WT animals. Similarly,
corneal vascularization was also present in
UPA-/- mice. However, as observed in the
tumors, PAI-1-deficient mice showed a decrease in vessel
growth, and these vessels were considerably shorter than those in
UPA-/- and WT animals (Fig. 8, A–C)
. When areas of neovascularization were calculated in
each group, a significant reduction was only observed in
PAI-deficient mice (Fig. 9A)
.
|
|
. After a time of 7 days after implantation of the bFGF
pellet, vWF immunostaining of corneal sections of WT (Fig. 9B)
and UPA-/- (Fig. 9C)
mice revealed extensive neovascularization, whereas
PAI-1-/- animals (Fig. 9D)
showed far less vWF staining, suggesting, in concert with the data of
Figs. 8
and 9
A, that bFGF-induced neovascularization is
greatly attenuated in PAI-1-deficient animals. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The present study focused on the contribution of host uPA and PAI-1 in tumor growth and dissemination. To this end, WT, UPA-/-, and PAI-1-/- mice were implanted with the murine fibrosarcoma, T241, and morphological and histological studies were performed to assess alterations in proliferation, cell death, and vascularization of the tumor in these gene-deficient mice, relative to WT mice. The angiogenic capabilities of these same mice, in the absence of tumor burden, were also investigated.
Analyses of the expression of fibrinolytic proteins demonstrated expression of a number of these proteins both in vitro and in vivo. However, ELISA-based analyses indicated a lack of an immune response to these proteins in the relevant gene-deleted mouse. Therefore, the inability of the fibrosarcoma cells to effectively grow in UPA-/- and PAI-1-/- mice relative to WT mice, although these tumors express many of the components of the fibrinolytic system, indicates that host expression of these proteins plays a major role in the growth and dissemination of this tumor.
Other studies have appeared using different model systems that considered the role of components of the fibrinolytic system in tumorigenesis and metastasis. It was concluded that host PAI-1 expression had minimal effects on growth and metastasis of B16 cells (34) in mice, and that elimination of the PG gene slightly influenced tumor size but did not significantly affect lung metastasis in a murine model of Lewis Lung carcinoma (35) . On the other hand, there is evidence for a role for uPA in progression of malignancy in a model of cutaneous melanocytic neoplasm (36) . Although some of the dilemmas may involve issues such as the appropriateness of i.v. injections (34) , in which early metastatic events are bypassed, cell passage numbers, or in the case of Lewis Lung carcinoma, whether the tumor was of the high or low metastatic phenotype, these results also imply that the tumor microenvironment and the effects of host deficiencies of the fibrinolytic system may vary for different tumor models. For example, activation of metalloproteases could directly facilitate tumor growth and indirectly activate other degradative proteins, such as cathepsin, which is secreted by B16 melanoma cells in proportion to their metastatic potential (37) . Thus, it is clear, and perhaps even expected, that different tumors could respond variably to any relevant host deficiency. The fibrosarcoma model may be more reflective of general effects of PAI-1 on angiogenesis, because similar defective neovascularization accompanying a PAI-1 deficiency is seen herein with non-tumor-bearing mice in a corneal angiogenesis model and in inflammatory angiogenesis in a completely separate model of remodeling of the vessel wall after oxidative arterial injury (38) .
The protease, uPA, is known to regulate the degradation of ECM proteins. Thus, it is not surprising that tumor progression is impeded in UPA-/- mice, wherein a higher apoptotic tumor index and diminished cellular proliferation occur in the fibrosarcoma model, relative to WT mice. Although the tumor itself produces uPA, studies have shown that uPA expression by stromal cells may play an equally important role in cancer cell invasion (39) . However, a uPA deficiency did not appear to have a substantial effect on angiogenesis and neovascularization of the tumor. Other studies have demonstrated a temporal relationship between endothelial cell migration and the expression of uPA activity (40) . Additionally, fetal bovine aortic endothelial GM 7373 cells overexpressing human uPA demonstrated an enhanced invasive capacity in a Matrigel chemoinvasion assay (41) . Thus, it is expected that a lack of host expression of uPA might have a negative effect on endothelial cell migration and resultant neovascularization, to the extent that Pm is involved in this process, unless tPA activation of Pg provides an amount of Pm suitable for migratory events that lead to neovascularization. On the other hand, other proteolytic pathways for supporting neovascularization may compensate for a loss of uPA activity. For example, MMP activities are tightly regulated during angiogenesis, and their activation, and resulting function in this regard, may not necessarily be dependent on Pm activity (42) . Clinically, it has been shown in patients with colorectal adenocarcinoma that MMP-2 and MMP-9 expression positively correlated with angiogenesis (43) . Additionally, the transcription factor, ETS-1, has been implicated in regulation of endothelial cell conversion to an angiogenic phenotype. Cells that overexpress ETS-1 are highly invasive and demonstrate increased expression of MMP-1, MMP-3, and MMP-9 (44) .
VEGF is a potent neovascularization agent in some cells (45, 46, 47, 48) , and its expression is up-regulated by bFGF and other growth factors (49 , 50) . VEGF, in turn, also up-regulates the expression of Pg activators and PAI-1 (51) , thus providing a possible link between an array of growth factors, angiogenesis, and extracellular protease activity. Furthermore, expression of VEGF, itself, is up-regulated by bFGF. Importantly, PAI-1-/- mice displayed greatly reduced expression of VEGF and, probably linked to this effect, diminished angiogenesis and neovascularization of the tumor. As a result, the apoptotic index was higher in the tumors of these mice than in WT animals, and cellular proliferation was diminished, thus offering an explanation as to the lack of growth of tumors cells in these gene-deficient animals. This is coupled with ultrastructural analysis, suggesting that neovessel formations in UPA-/- and PAI-/- mice have abnormal features, such as larger and less mature vessels in the UPA-/- mice and small short neovessels in PAI-1-/- mice. Without appropriate angiogenesis in the tumor cells, tumors cannot grow and spread effectively.
Previous studies have also shown that PAI-1 blocks cellular binding of uPAR to vitronectin and thus promotes detachment of uPAR-bearing cells from vitronectin, with a resultant increase in cell motility (52) . A loss of host PAI-1 expression could result in enhanced adhesion of uPAR-bearing endothelial cells to the matrix protein, vitronectin, and thus adversely affect cell motility and resultant neovascularization. Additionally, balanced cell adhesion and proteolytic-mediated detachment through regulation of uPA activity and inhibition could be necessary for protecting the neovascularizing tissue (53) . All of these mechanisms may be functioning to enhance angiogenesis in the presence of host PAI-1, and the absence of this protein would then attenuate neovascularization in tumors and other types of cells.
In summary, the studies presented in this report underscore the importance of host expression of components of the fibrinolytic system in supporting growth and dissemination of the murine fibrosarcoma tumor, T241. Lack of host expression of either uPA or its physiological inhibitor, PAI-1, attenuates growth and survival of this tumor. uPA can provide the necessary targeted proteolysis, amplified through Pg activation, to allow the cellular migration and proliferation needed for angiogenesis to occur. However, PAI-1 can provide a control to allow this process to occur in a highly regulated manner. In the absence of PAI-1, unregulated proteolysis may be detrimental to angiogenesis. Furthermore, the absence of PAI-1 may inhibit endothelial cell motility via effects on the uPAR-vitronectin interaction. Additionally, we show that the PAI-1-deficient state may also inhibit neovascularization by affecting VEGF levels, which are needed for this process to occur. Thus, PAI-1 is a complex regulator of neovascularization, and the influence of these different mechanisms likely depends on the nature of the particular angiogenic process that is occurring in different cell types.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported in part by NIH Grant
HL13423 (to F. J. C.) and Grant HL63682 (to V. A. P.), a grant from
the W. M. Keck Foundation, and the Kleiderer-Pezold family endowed
professorship (to F. J. C.). Presented in part at the 41st Annual
Meeting of the American Society of Hematology, New Orleans, December
3–7, 1999. 
2 To whom requests for reprints should be
addressed, at University of Notre Dame, 229 Nieuwland Science Hall,
Notre Dame, IN 46556. Phone: (219) 631-6456; Fax: (219) 631-8017;
E-mail: castellino.1{at}nd.edu
3 The abbreviations used are: Pg, plasminogen; Pm,
plasmin; bFGF, basic fibroblast growth factor; BrdUrd,
bromodeoxyuridine; DAB, 3',3-diaminobenzidine-HCl; ECM, extracellular
matrix; MMP, matrix metalloprotein; PAI, plasminogen activator
inhibitor; RT-PCR, reverse transcription-PCR; tPA, tissue plasminogen
activator; TUNEL, terminal dUTP nick end labeling; MVC, microvessel
count; uPA, urokinase plasminogen activator; uPAR, urokinase
plasminogen activator receptor; VEGF, vascular endothelial growth
factor; vWF, von Willebrand factor; WT, wild type.
Received 5/ 1/00. Accepted 8/29/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C.M. Maillard, C. Bouquet, M.M. Petitjean, M. Mestdagt, E. Frau, M. Jost, A.M. Masset, P.H. Opolon, F. Beermann, M.M. Abitbol, et al. Reduction of brain metastases in plasminogen activator inhibitor-1-deficient mice with transgenic ocular tumors Carcinogenesis, November 1, 2008; 29(11): 2236 - 2242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-C. Chen, D. O. Henry, P. R. Reczek, and M. K.K. Wong Plasminogen activator inhibitor-1 inhibits prostate tumor growth through endothelial apoptosis Mol. Cancer Ther., May 1, 2008; 7(5): 1227 - 1236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Jost, C. Maillard, J. Lecomte, V. Lambert, M. Tjwa, P. Blaise, M.-L. Alvarez Gonzalez, K. Bajou, S. Blacher, P. Motte, et al. Tumoral and Choroidal Vascularization: Differential Cellular Mechanisms Involving Plasminogen Activator Inhibitor Type I Am. J. Pathol., October 1, 2007; 171(4): 1369 - 1380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Sternlicht, A. M. Dunning, D. H. Moore, P. D.P. Pharoah, D. G. Ginzinger, K. Chin, J. W. Gray, F. M. Waldman, B. A.J. Ponder, and Z. Werb Prognostic Value of PAI1 in Invasive Breast Cancer: Evidence That Tumor-Specific Factors Are More Important Than Genetic Variation in Regulating PAI1 Expression. Cancer Epidemiol. Biomarkers Prev., November 1, 2006; 15(11): 2107 - 2114. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Balsara, F. J. Castellino, and V. A. Ploplis A Novel Function of Plasminogen Activator Inhibitor-1 in Modulation of the AKT Pathway in Wild-type and Plasminogen Activator Inhibitor-1-deficient Endothelial Cells J. Biol. Chem., August 11, 2006; 281(32): 22527 - 22536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chen, R. C. Budd, R. J. Kelm Jr, B. E. Sobel, and D. J. Schneider Augmentation of Proliferation of Vascular Smooth Muscle Cells by Plasminogen Activator Inhibitor Type 1 Arterioscler Thromb Vasc Biol, August 1, 2006; 26(8): 1777 - 1783. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Alfano, I. Iaccarino, and M. P. Stoppelli Urokinase Signaling through Its Receptor Protects against Anoikis by Increasing BCL-xL Expression Levels J. Biol. Chem., June 30, 2006; 281(26): 17758 - 17767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Madsen, E. I. Deryugina, S. Niessen, B. F. Cravatt, and J. P. Quigley Activity-based Protein Profiling Implicates Urokinase Activation as a Key Step in Human Fibrosarcoma Intravasation J. Biol. Chem., June 9, 2006; 281(23): 15997 - 16005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. W.M. van Hinsbergh, M. A. Engelse, and P. H.A. Quax Pericellular Proteases in Angiogenesis and Vasculogenesis Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 716 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Grum-Schwensen, J. Klingelhofer, C. H. Berg, C. El-Naaman, M. Grigorian, E. Lukanidin, and N. Ambartsumian Suppression of Tumor Development and Metastasis Formation in Mice Lacking the S100A4(mts1) Gene Cancer Res., May 1, 2005; 65(9): 3772 - 3780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yoshida, K. Matsuzaki, S. Mori, Y. Tahashi, H. Yamagata, F. Furukawa, T. Seki, M. Nishizawa, J. Fujisawa, and K. Okazaki Transforming Growth Factor-{beta} and Platelet-Derived Growth Factor Signal via c-Jun N-Terminal Kinase-Dependent Smad2/3 Phosphorylation in Rat Hepatic Stellate Cells after Acute Liver Injury Am. J. Pathol., April 1, 2005; 166(4): 1029 - 1039. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Paavonen, N. Ekman, M. Wirzenius, I. Rajantie, M. Poutanen, and K. Alitalo Bmx Tyrosine Kinase Transgene Induces Skin Hyperplasia, Inflammatory Angiogenesis, and Accelerated Wound Healing Mol. Biol. Cell, September 1, 2004; 15(9): 4226 - 4233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mayasundari, N. A. Whittemore, E. H. Serpersu, and C. B. Peterson The Solution Structure of the N-terminal Domain of Human Vitronectin: PROXIMAL SITES THAT REGULATE FIBRINOLYSIS AND CELL MIGRATION J. Biol. Chem., July 9, 2004; 279(28): 29359 - 29366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Noel, C Maillard, N Rocks, M Jost, V Chabottaux, N E Sounni, E Maquoi, D Cataldo, and J M Foidart Membrane associated proteases and their inhibitors in tumour angiogenesis J. Clin. Pathol., June 1, 2004; 57(6): 577 - 584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Xu, R. D. Balsara, N. V. Gorlatova, D. A. Lawrence, F. J. Castellino, and V. A. Ploplis Conservation of Critical Functional Domains in Murine Plasminogen Activator Inhibitor-1 J. Biol. Chem., April 23, 2004; 279(17): 17914 - 17920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. A. Ploplis, R. Balsara, M. J. Sandoval-Cooper, Z. J. Yin, J. Batten, N. Modi, D. Gadoua, D. Donahue, J. A. Martin, and F. J. Castellino Enhanced in Vitro Proliferation of Aortic Endothelial Cells from Plasminogen Activator Inhibitor-1-deficient Mice J. Biol. Chem., February 13, 2004; 279(7): 6143 - 6151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Palumbo, K. E. Talmage, H. Liu, C. M. La Jeunesse, D. P. Witte, and J. L. Degen Plasminogen supports tumor growth through a fibrinogen-dependent mechanism linked to vascular patency Blood, October 15, 2003; 102(8): 2819 - 2827. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chuang-Tsai, T. H. Sisson, N. Hattori, C. G. Tsai, N. M. Subbotina, K. E. Hanson, and R. H. Simon Reduction in Fibrotic Tissue Formation in Mice Genetically Deficient in Plasminogen Activator Inhibitor-1 Am. J. Pathol., August 1, 2003; 163(2): 445 - 452. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Lambert, C. Munaut, P. Carmeliet, R. D. Gerard, P. J. Declerck, A. Gils, C. Claes, J.-M. Foidart, A. Noel, and J.-M. Rakic Dose-Dependent Modulation of Choroidal Neovascularization by Plasminogen Activator Inhibitor Type I: Implications for Clinical Trials Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2791 - 2797. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Collen, R. Hanemaaijer, F. Lupu, P. H. A. Quax, N. van Lent, J. Grimbergen, E. Peters, P. Koolwijk, and V. W. M. van Hinsbergh Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix Blood, March 1, 2003; 101(5): 1810 - 1817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Gutierrez, M. Suckow, J. Lawler, V. A. Ploplis, and F. J. Castellino Thrombospondin 1--a regulator of adenoma growth and carcinoma progression in the APCMin/+ mouse model Carcinogenesis, February 1, 2003; 24(2): 199 - 207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Han, J. Leng, D. Bian, C. Mahanivong, K. A. Carpenter, Z. K. Pan, J. Han, and S. Huang Rac1-MKK3-p38-MAPKAPK2 Pathway Promotes Urokinase Plasminogen Activator mRNA Stability in Invasive Breast Cancer Cells J. Biol. Chem., December 6, 2002; 277(50): 48379 - 48385. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Palumbo, J. M. Potter, L. S. Kaplan, K. Talmage, D. G. Jackson, and J. L. Degen Spontaneous Hematogenous and Lymphatic Metastasis, but not Primary Tumor Growth or Angiogenesis, Is Diminished in Fibrinogen-deficient Mice Cancer Res., December 1, 2002; 62(23): 6966 - 6972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R. Binder, G. Christ, F. Gruber, N. Grubic, P. Hufnagl, M. Krebs, J. Mihaly, and G. W. Prager Plasminogen Activator Inhibitor 1: Physiological and Pathophysiological Roles Physiology, April 1, 2002; 17(2): 56 - 61. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. DEVY, S. BLACHER, C. GRIGNET-DEBRUS, K. BAJOU, V. MASSON, R. D. GERARD, A. GILS, G. CARMELIET, P. CARMELIET, P. J. DECLERCK, et al. The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent FASEB J, February 1, 2002; 16(2): 147 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Providence, L. A. White, J. Tang, J. Gonclaves, L. Staiano-Coico, and P. J. Higgins Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene J. Cell Sci., January 10, 2002; 115(19): 3767 - 3777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Rowland-Goldsmith, H. Maruyama, K. Matsuda, T. Idezawa, M. Ralli, S. Ralli, and M. Korc Soluble Type II Transforming Growth Factor-{beta} Receptor Attenuates Expression of Metastasis-associated Genes and Suppresses Pancreatic Cancer Cell Metastasis Mol. Cancer Ther., January 1, 2002; 1(3): 161 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C.Y. Chan, D. A. Duszczyszyn, F. J. Castellino, and V. A. Ploplis Accelerated Skin Wound Healing in Plasminogen Activator Inhibitor-1-Deficient Mice Am. J. Pathol., November 1, 2001; 159(5): 1681 - 1688. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Pepper Role of the Matrix Metalloproteinase and Plasminogen Activator-Plasmin Systems in Angiogenesis Arterioscler Thromb Vasc Biol, July 1, 2001; 21(7): 1104 - 1117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. A. McMahon, E. Petitclerc, S. Stefansson, E. Smith, M. K. K. Wong, R. J. Westrick, D. Ginsburg, P. C. Brooks, and D. A. Lawrence Plasminogen Activator Inhibitor-1 Regulates Tumor Growth and Angiogenesis J. Biol. Chem., August 31, 2001; 276(36): 33964 - 33968. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
