 |
|
Urokinase-Type Plasminogen Activator Expression and Proliferation Stimulation in Head and Neck Squamous Cell Carcinoma In Vitro and In Situ
Marianne Schmidt, PhD;
Petra Grünsfelder
Arch Otolaryngol Head Neck Surg. 2001;127:679-682.
ABSTRACT
| |
Background Stimulation of proliferative activity by urokinase-type plasminogen
activator (uPA) has been demonstrated in vitro for cultured primary and carcinoma
cells.
Objective To examine the effect of uPA stimulation on cultured squamous cell carcinoma
cell lines of the head and neck in vitro and to compare the results with the
situation in tumor tissue specimens.
Design The uPA-mediated growth stimulation of 2 head and neck squamous cell
carcinoma cell lines after suppression of endogenous uPA production was monitored
by measuring 3H-thymidine uptake into cellular DNA. Alternatively,
applications of antibodies against the uPA-binding domain of the urokinase
receptor were used to suppress autostimulation. To analyze the situation in
situ we performed Western blot and zymographic studies on tissue homogenates
of 25 squamous cell carcinoma specimens. We tested the expression of proliferating
cell nuclear antigen (PCNA), a marker for proliferative activity, and uPA
in tissue lysates and correlated uPA and PCNA expression by regression analysis.
Results High-molecular-weight urokinase had a proliferation stimulative effect
on both cell lines in vitro. The uPA autostimulation was decreased by blocking
the uPA-binding domain of urokinase receptor with antibodies. Regression analysis
of zymographic and Western blot data of tumor tissue lysates revealed no significant
coherency between PCNA and uPA expression. Immunohistochemical stainings frequently
showed different sublocalization of uPA and PCNA within tumors.
Conclusion In vitro uPA-mediated growth stimulation is not necessarily transferable
to the in situ situation.
INTRODUCTION
UROKINASE-TYPE plasminogen activator (uPA) is a multidomain serine protease
secreted by a variety of cell types. Proteolytic cleavage of extracellular
matrix proteins occurs via proteolytic cleavage of plasminogen into plasmin.
Urokinase-type PA itself is secreted as a single-chain proenzyme, which has
to become activated by proteolytic cleavage to the 2-chain form of uPA. Activation
is catalyzed by plasmin and other proteinases (for a review see Andreasen
et al1). Active uPA can also occur cleaved
in the form of the low-molecular-weight uPA, which possesses the complete
catalytic domain. The amino-terminal part of uPA contains a growth factor
domain, which binds to the cell surface urokinase receptor.1
In 1987, Kirchheimer et al2 reported an in
vitro proliferation stimulation by uPA in human epidermal tumor cells and
in primary and malignant renal cells3 in vitro.
During the past decade, increasing evidence for growth stimulation caused
by urokinase has accumulated. The growth factor–like activity of urokinase
has been shown to be independent of a plasmin-mediated process.4
Concerning head and neck cancer, uPA has been demonstrated to be increased
in carcinoma tissue compared with normal mucosa.5, 6
The aim of this study was to examine the effect of exogenous urokinase
stimulation on cultured squamous cell carcinoma cell lines of the head and
neck in vitro. Furthermore, we tried to relate the in vitro results to the
situation in situ and examined expression of uPA and the proliferation marker
proliferating cell nuclear antigen (PCNA) in head and neck tumor tissue extracts
using Western blot, zymography, or immunohistochemical analysis.
RESULTS
IN VITRO STIMULATION OF HEAD AND NECK CARCINOMA CELL LINES
The head and neck carcinoma cell lines HLaC79 and HSmC78 were tested
for secretion of urokinase and expression of its receptor using fibrin zymography
and Northern blot, respectively. Urokinase and its receptor were detected
in both cell lines (Figure 1) in
different amounts. To suppress endogenous urokinase production, we incubated
the cell lines before 3H-thymidine incorporation experiments with
cycloheximide14 and afterwards with increasing
concentrations of urokinase or equivalent amounts of cell culture medium as
controls. Incubation with cycloheximide decreased 3H-thymidine
uptake considerably and decreased uPA secretion in vitro (data not shown).
Increasing concentrations of high-molecular-weight uPA were able to partially
reverse the growth inhibitory effect of cycloheximide (Figure 2).
|
|
|
|
Figure 1. Expression of urokinase-type plasminogen
activator (uPA) receptor and uPA in the cell lines HLaC79 and HSmC78 tested
by Northern blot (uPA receptor) and fibrin zymography (uPA; culture supernatants).
Equivalent loading for Northern hybridization is demonstrated by total RNA
lanes (upper panel, left side). kb indicates kilobase.
|
|
|
|
|
|
|
Figure 2. Growth stimulation by urokinase-type
plasminogen activator (uPA) expression in the cell lines HSmC78 and HLaC79.
A, 3H-thymidine uptake after application of culture medium (n.t.),
cycloheximide (cy), or cy + uPA. Data are mean values; error bars represent
SD. B, Proliferating cell nuclear antigen expression after treatment with
cy or cy + uPA.
|
|
|
In addition, we carried out cycloheximide treatment and subsequent addition
of urokinase (2.8 nmol/L) and tested PCNA expression in cell extracts using
Western blot. Applying 30 µg of total cell protein to Western blots,
we observed an up-regulation of PCNA in the cell lines (Figure 2). In a second series of experiments, we compared untreated
cells under serum-free conditions with cells incubated with an antibody against
the uPA-binding site of the urokinase receptor, preventing uPA receptor binding.9 Cells incubated with the antibody reacted with a decrease
in 3H-thymidine uptake, which probably represents the elimination
of the growth-stimulating effect of endogenously produced urokinase. HLaC79
and HSmC78 reacted to antibody treatment with average decreases of 64.98%
and 3.78%, respectively. The weaker growth inhibition in HSmC78 might be explained
by the much higher expression of uPA receptor in these cells.
QUANTITATIVE EXPRESSION ANALYSIS OF uPA AND PCNA IN TISSUE HOMOGENATES
Expression of the proliferation marker PCNA and urokinase was quantitatively
determined by Western blot and fibrin zymography, respectively. Figure 3 shows the results of fibrin zymography and Western blot
for 11 tumor samples. Densitometric evaluation and statistical regression
analysis revealed no trend toward increasing PCNA levels in tumors with high
uPA contents. Figure 3 shows the
regression analysis of uPA level vs PCNA expression. Although not statistically
significant, there was a trend toward lower PCNA expression in tumor tissue
with higher uPA contents.
|
|
|
|
Figure 3. A, Urokinase-type plasminogen
activator (uPA) and proliferating cell nuclear antigen (PCNA) expression in
11 tumor samples tested by fibrin zymography or Western blot. Co indicates
culture supernatant (uPA) or cell lysate of HT1080 fibrosarcoma cells. B,
Regression analysis of uPA and PCNA expression in 25 tested lysates. IOD indicates
integrated optical density.
|
|
|
IMMUNOHISTOLOGICAL LOCALIZATION OF PCNA AND uPA
To investigate sublocalization of urokinase and the proliferation marker
PCNA in tumor tissue, we performed immunohistological staining on frozen sections
of tumor specimens. Immunohistological localization showed that in many cases
uPA and PCNA were sublocalized in distinct tumor compartments. Figure 4 shows a sample with uPA and PCNA expression in the same
tumor specimen but in different subareas.
|
|
|
|
Figure 4. Immunohistochemical staining of
the same tumor specimen with proliferating cell nuclear antigen (A), urokinase-type
plasminogen activator antibody (B), and control (culture supernatant) (C).
Brown staining indicates antibody staining, counterstaining with Meyer's Håmalaun
solution. Bar represents 100 µm.
|
|
|
COMMENT
High-molecular-weight urokinase has been shown to stimulate cell proliferation
in many in vitro cell systems.2, 3, 4
In 1989, Kirchheimer et al14 tested the in
vitro proliferation stimulatory effect of uPA on a melanoma cell line. Because
the cell line secreted uPA itself, the authors carried out antibody inhibition
experiments and also treated the cells with cycloheximide before exogenous
application of uPA. In their experiments, uPA secretion decreased and the
number of uPA-occupied uPA receptors decreased, but the total number of uPA
receptors remained almost the same. Using cycloheximide inhibition experiments
and blocking of uPA-uPA receptor binding by specific antibodies, we demonstrated
that head and neck carcinoma cell lines also react to urokinase stimulation
in vitro. However, there are only a few speculations about the significance
of these uPA-associated properties in situ or in vivo. The first evidence
for an in vivo–relevant importance of urokinase expression was provided
by Jensen and Lavker,15 who showed decreased
proliferation of epidermal cells in mice with targeted uPA deletion. However,
in complex cellular systems such as tumor tissue, there is still a lack of
understanding concerning the functions of uPA. Urokinase has been shown to
be overexpressed in head and neck cancer,5, 6
although the significance of uPA overexpression for metastatic and invasive
behavior of head and neck squamous cell carcinoma is still controversial.
Gohring et al16 found a significant correlation
between uPA and PCNA expression in primary breast cancer specimens. Volm et
al17 analyzed 137 non–small cell lung
carcinoma specimens immunohistochemically for expression of urokinase and
further determined the proliferative activity of the tumors using a flow cytometric
approach. The authors detected uPA expression in 77% of the tumors. However,
they did not show any relationship between urokinase expression and cell cycle
phase distribution (proliferative activity). This is in agreement with our
observations in head and neck carcinoma specimens. Immunohistochemical analysis
with PCNA and uPA antibodies revealed distinct sublocalization of uPA-positive
and highly proliferative areas in many tumor specimens. Therefore, it seems
likely that in a highly deregulated system such as tumor tissue, urokinase-mediated
autostimulation does not necessarily take place. There are several possible
explanations for the lack of endogenous urokinase proliferation stimulation.
Probably a certain balance of uPA production and secretion of plasminogen
activator inhibitors (PAIs) can affect the behavior and proliferation of cells.
In vitro uPA cleaves the amino-terminal fragment of urokinase in an autocatalytic
process.18, 19 The release of aminoterminal
fragment (aminoterminal fragment of uPA with proliferation stimulatory activity)
by uPA cleavage has been shown to be inhibitable by PAIs. Thus, in hepatocellular
carcinoma cells it has been demonstrated that PAI-1 can decrease proliferation
and invasiveness,20 and Hibino et al18 eliminated the growth stimulatory effect of uPA in
keratinocytes by PAI-2. Furthermore, overexpression of PAI-1 (together with
uPA) in head and neck cancer has been demonstrated.21
Plasminogen activator inhibitor–1 might at least in part abolish the
growth stimulatory effect of urokinase.
Another question concerns sublocalization of urokinase and its receptor
in carcinoma tissue. In a previous study,13
we investigated uPA and uPA receptor expression in head and neck carcinomas.
We did not show a relationship between uPA and uPA receptor content in tumor
homogenates or a compelling colocalization within the tumors. The different
sublocalization might prevent signal transmission by uPA receptor.
Furthermore, distinct components of uPA-mediated signaling cascades
might be missing because of chromosomal deletions occurring frequently in
cancer cells. As long as the uPA-mediated signaling pathway has not been identified
in detail, a failure of signal transmission in certain cell types might explain
the lack of autostimulation in tumors and the lacking proliferation stimulation
by uPA in certain cell lines (eg, U 937 lymphoma cells22).
Previously, Schmidt and Hoppe23 showed
that patients with head and neck carcinomas can be recognized by significantly
increased levels of soluble uPA receptor in blood plasma, which suggests "shedding"
of uPA receptor by tumor cells. Soluble uPA receptor within the tumor tissue
might be a further suppressor of autostimulation by binding uPA and thus competing
with cell-associated uPA receptor.
Fischer et al9 stimulated OV-MZ-6 ovarian
cancer cells with high-molecular-weight urokinase. Considering the concentration-dependent
growth stimulation in these cell cultures, it is conspicuous that maximal
growth stimulation reached a peak at about 1-nmol/L uPA concentration and
decreased again with increasing uPA concentrations. Certain effective uPA
concentrations are probably also necessary in vivo.
In summary, we showed an in vitro growth stimulative effect of urokinase
in head and neck carcinoma cell lines. We did not extend these observations
to the situation in native tumor tissue comparing uPA and PCNA expression,
neither quantitatively nor qualitatively.
AUTHOR INFORMATION
Accepted for publication January 17, 2001.
We thank Ute Reuning, MD, PhD, Frauenklinik der Technischen Universität
Munich, Munich, Germany, for providing the monoclonal antibody IIIF10.
From the Department of Otorhinolaryngology, University of Wuerzburg,
Wuerzburg, Germany.
Corresponding author: Marianne Schmidt, PhD, Department of Otorhinolaryngology,
University of Wuerzburg, Josef-Schneider-Strasse 11, D-97080 Wuerzburg, Germany.
REFERENCES
| |
1. Andreasen PA, Kjoller L, Christensen L, et al. The urokinase-type plasminogen activator system in cancer metastasis:
a review. Int J Cancer. 1997;72:1-22.
FULL TEXT
|
ISI
| PUBMED
2. Kirchheimer JC, Wojta J, Christ G, et al. Proliferation of a human epidermal tumor cell line stimulated by urokinase. FASEB J. 1987;1:125-128.
ABSTRACT
3. Kirchheimer JC, Wojta J, Christ G, et al. Mitogenic effect of urokinase on malignant and unaffected adjacent
human renal cells. Carcinogenesis. 1988;9:2121-2123.
FREE FULL TEXT
4. Kirchheimer JC, Wojta J, Hienert G, et al. Effect of urokinase on the proliferation of primary cultures of human
prostatic cells. Thromb Res. 1987;48:291-298.
FULL TEXT
|
ISI
| PUBMED
5. Schmidt M, Polednik C, Hoppe F. Proteolytic patterns in head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol. 1999;256:346-350.
FULL TEXT
| PUBMED
6. Clayman G, Wang SW, Nicolson GL, et al. Regulation of urokinase-type plasminogen activator expression in squamous-cell
carcinoma of the oral cavity. Int J Cancer. 1993;54:73-80.
ISI
| PUBMED
7. Zenner HP, Herrmann IF, Bremer W, et al. Head and neck carcinoma models. Acta Otolaryngol. 1983;95:371-381.
PUBMED
8. Zenner HP, Lehner W, Herrmann IF. Establishment of carcinoma cell lines from larynx and submandibular
gland. Arch Otorhinolaryngol. 1979;225:269-277.
FULL TEXT
| PUBMED
9. Fischer K, Lutz V, Wilhelm O, et al. Urokinase induces proliferation of human ovarian cancer cells: characterization
of structural elements required for growth factor function. FEBS Lett. 1998;438:101-105.
FULL TEXT
|
ISI
| PUBMED
10. Lowry OH, Rosebrough NNJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
FREE FULL TEXT
11. Heussen D, Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide
gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem. 1980;102:196-202.
FULL TEXT
|
ISI
| PUBMED
12. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without tank for
rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984;10:203-210.
FULL TEXT
|
ISI
| PUBMED
13. Schmidt M, Schler G, Grünsfelder P, Muller J, Hoppe F. Urokinase receptor up-regulation in head and neck squamous cell carcinoma. Head Neck. 2000;22:498-504.
FULL TEXT
|
ISI
| PUBMED
14. Kirchheimer JC, Wojta J, Christ G, et al. Functional inhibition of endogenously produced urokinase decreases
cell proliferation in a human melanoma cell line. Proc Natl Acad Sci U S A. 1989;86:5424-5428.
FREE FULL TEXT
15. Jensen PJ, Lavker RM. Urokinase is a positive regulator of epidermal proliferation in vivo. J Invest Dermatol. 1999;112:240-244.
FULL TEXT
|
ISI
| PUBMED
16. Gohring UJ, Scharl A, Thelen U, Ahr A, Titius BR. Prognostic value of immunohistochemical determination of urokinase
type plasminogen activator in primary breast cancers. Pathologe. 1995;16:398-403.
FULL TEXT
|
ISI
| PUBMED
17. Volm M, Mattern J, Koomägi R. Relationship of urokinase and urokinase receptor in non–small
cell lung cancer to proliferation, angiogenesis, metastasis and patient survival. Oncol Rep. 1999;6:611-615.
ISI
| PUBMED
18. Hibino T, Matsuda Y, Takahashi T, et al. Suppression of keratinocyte proliferation by plasminogen activator
inhibitor-2. J Invest Dermatol. 1999;112:85-90.
FULL TEXT
|
ISI
| PUBMED
19. Fishman DA, Kearns A, Larsh S, et al. Autocrine regulation of growth stimulation in human epithelial ovarian
carcinoma by serine-proteinase–catalysed release of the urinary-type
plasminogen-activator N-terminal fragment. Biochem J. 1999;341:765-769.
20. Morita Y, Hayashi Y, Kanamaru T, et al. Inhibitory role of plasminogen activator inhibitor-1 in invasion and
proliferation of HLE hepatocellular carcinoma cells. Jpn J Cancer Res. 1999;90:747-752.
FULL TEXT
|
ISI
| PUBMED
21. Wollenberg B, Jan NV, Jund R, Chaubal S, Untch M. Urokinase-type plasminogen activator and its inhibitor plasminogen
activator inhibitor-1: new functional risk factors in head and neck squamous
cell cancer. Oncol Rep. 1997;48:53-55.
22. Rabbani SA, Mazar AP, Bernier SM, et al. Structural requirements for the growth factor activity of the aminoterminal
domain of urokinase. J Biol Chem. 1992;267:14151-14156.
FREE FULL TEXT
23. Schmidt M, Hoppe F. Increased levels of urokinase receptor in plasma of head and neck squamous
cell carcinoma patients. Acta Otolaryngol (Stockh). 1999;119:949-953.
FULL TEXT
| PUBMED
RELATED ARTICLE
Archives of Otolaryngology–Head & Neck Surgery Reader's Choice: Continuing Medical Education
Arch Otolaryngol Head Neck Surg. 2001;127(6):725-726.
FULL TEXT
|
