TECHNICAL NOTE |
Correspondence to: Saverio Alberti, Institute Mario Negri Sud, 66030 S., Maria Imbaro (Chieti), Italy.
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Summary |
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The urokinase-type plasminogen activator (uPA) is a serine protease that plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA receptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA using a conjugation procedure that did not significantly alter its binding characteristics to the uPAR. Thirty nM FITC-uPA efficiently stains 2 x 105 uPAR-transfected mouse cells in suspension, as determined by flow cytometric analysis. One µg of FITC-uPA efficiently stains 2 x 105 uPAR transfectants grown on slides and analyzed by fluorescence optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde only slightly reduced the efficiency of staining of both transfectants and cell lines. These characteristics allow the use of FITC-uPA in both static and dynamic morphological studies of uPAR-expressing cells. (J Histochem Cytochem 45:1307-1313, 1997)
Key Words: urokinase-type plasminogen activator, fluorescein labeling, cell surface receptors, fluorescence microscopy, flow cytometry
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Introduction |
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The serine protease urokinase-type plasminogen activator (uPA) is synthesized as a soluble single-chain inactive precursor (pro-uPA) and is activated by proteolysis after binding to the uPA receptor (uPAR). Active uPA specifically proteolizes plasminogen into plasmin, which in turn degrades extracellular matrix proteins (fibronectin, laminin, and proteoglycans) or activates matrix metalloproteases and the pro-uPA itself (
Because uPA, together with other proteases, can play a major role in blood coagulation and tumor invasion, it would be of interest to be able to efficiently follow the uPA-uPAR complex, e. g., during its formation, internalization or shedding, and interaction with its substrate. Of further use would be detection of the uPA-uPAR complex on living cells and following it dynamically over time. To reach this goal, we decided to produce a directly labeled uPA that could be used with high efficiency in fluorescence microscopy and flow cytometry. The huPA has a Kd of 2-20 x 10-9 M for the uPAR (
The feasibility of direct labeling of uPA was also suggested by the efficient and nondestructive labeling of other proteic ligands, e. g., IL-2 (, TGFß (British Biotechnology; Oxford, UK), growth hormone (
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Materials and Methods |
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Tissue Culture
Murine LB6 cells co-transfected with the phuPAR (containing an expressable human uPAR cDNA) and the pRSVneo selectable vector or with the pRSVneo vector alone (LC) were obtained from Dr. F. Blasi through the courtesy of Dr. P. Mignatti (
FITC Conjugation of uPA
Purified mature, i. e., double-chain human uPA (Ukidan; Serono, Rome, Italy) was kindly supplied through the courtesy of Dr. B. Donati. uPA 400 µg in 0.5 ml PBS were salt-exchanged to carbonate buffer (1.7 g Na2CO3 and 2.8 g NaHCO3 in 100 ml H2O), pH 9.4, using a PD-10 column (Pharmacia; Uppsala, Sweden). FITC freshly dissolved in DMSO was added to uPA in bicarbonate buffer at 27 µg/ml per mg of protein. The FITC-uPA was incubated in the dark under continuous rocking for 3 hr at room temperature (RT). Free FITC was removed from the FITC-uPA containing solution by salt-exchanging on a PD-10 column (Pharmacia). The absorbance of FITC-uPA was measured at 280 nm and 495 nm to determine the fluorochrome to protein (F/P) ratio, using the following formula (
F = OD495nm/68,000 (FITC molar extinction coefficient)
P = [OD280nm - 31% OD495nm]/0.96 (uPA molar extinction coefficient) x 51,800 (uPA MW)
The molar extinction coefficient of uPA was determined from the absorbance of known amounts of purified uPA in PBS at 280 nm.
FITC-uPA Binding Assay in Flow Cytometry
Cells were collected with PBS 0.6 µM EDTA, centrifuged for 5 min at 1200 RPM at 4C, and resuspended in staining medium (SM). SM is 50% 1 x PBS, 50% 1 x HBSS, 20 mM HEPES, pH 8.1, supplemented with essential and nonessential amino acids, sodium pyruvate (Gibco BRL), 3% fetal calf serum (HyClone), and 0.1% NaN3. Preliminary assays allowed to determine conditions of staining that resulted in high signal on uPAR-expressing cells and no background on control cells (
uPAR Competition Assay in Flow Cytometry
The binding specificity and affinity of FITC-uPA were assayed in a competition assay using unlabeled huPA. Briefly, 0.3 µg of uPA conjugated to FITC was mixed with increasing amounts of unlabeled uPA, i. e., 0.3 µg, 2.7 µg or 29.7 µg. Each premixed solution was added to 2 x 105 living or fixed cells in 200 µl of SM and incubated for 1 hr on ice. After three washes with SM they were resuspended in 200 µl of SM-PI or SM, respectively, and analyzed by flow cytometry. The CV and net fluorescence values of the profiles obtained were compared with the expected values assuming unchanged affinity of uPA after FITC conjugation (see below).
Fluorescence Analysis and Cell Sorting
Fluorescence analysis was performed on a FACSTAR (Becton Dickinson; Sunnyvale, CA), used essentially as described (
arithmetic mean fluorescence of the stained uPAR transfected cells - arithmetic mean fluorescence of the stained pRSVneo transfected control cells
The net fluorescence is an indicator of the total amount of uPA bound to the cells. Importantly, it does not depend on a specific procedure for estimating positive cells and includes dimly expressing cells. The net fluorescence values presented in Table 1 were calculated on 5000 cells.
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Fluorescence Microscopy
Two hundred thousand cells were plated on 20 x 20-mm glass coverslips in 6-well tissue culture plates. The next day the coverslips were washed twice in SM and the cells were incubated for 1 hr on ice with 1 µg of FITC-uPA in 50 µl SM. In competition assays, cells were stained with 1 µg of FITC-uPA together with either 1 µg, 9 µg, or 99 µg of unlabeled uPA. Stained coverslips were washed in SM and fixed in the dark for 10 min at RT in 4% paraformaldehyde (PFA) (Sigma Chemical) in PBS. Unreacted PFA was quenched with 50 mM NH4Cl for 10 min at RT. After two washes with SM the coverslips were mounted in Mowiol (Calbiochem-Novabiochem; La Jolla, CA) or in 90% glycerol-10% PBS, and were observed with a Zeiss Axiophot fluorescence microscope.
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Results |
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To reduce the chances of inactivating the binding region of uPA for uPAR, uPA was conjugated with FITC using a mild procedure adapted from a FITC conjugation protocol for antibody molecules (
The binding affinity and specificity of FITC-uPA towards the uPAR was assessed on mouse L-cells transfected with the human uPAR. Quantitation of binding was performed by flow cytometry. The Kd of uPA for uPAR transfectant LB6 cells is about 2-20 x 10-9 M (
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We also tested the efficiency of staining of U937 and HT-1080, i. e., human cell lines normally expressing uPAR (
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FITC-uPA efficiently stained both uPAR-transfected LB6 and U937 and HT-1080 cells fixed in 1% PFA (Figure 2A and Figure 2B). Competition of the FITC-uPA staining with unlabeled uPA confirmed the specificity of these findings (Table 1).
FITC-uPA can efficiently detect uPAR-expressing cells in fluorescence microscopy (Figure 3B). FITC-uPA staining is mostly distributed on the plasma membrane with a ring-like pattern. However, brighter spots in button-like structures are also evident, suggesting accumulation of FITC-uPA on potential cell-substrate adhesion areas (
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FITC-uPA was stored at -80C with no adverse effects for more than 2 years. Repeated freezing and thawing of the same aliquot of FITC-uPA did not affect binding or staining efficiency, and we did not observe reduction of the apparent titer of FITC-uPA over time due to autoproteolysis. An example is shown in Figure 1A vs 1B. The essentially overlapping profiles obtained on different batches of stably transfected cells, either freshly thawed or continuously kept in culture, confirm the reproducibility of the staining procedure presented and the stability of the FITC-uPA.
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Discussion |
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uPA and the uPAR play a crucial role in the coagulation cascade (
The main uPAR binding site of human uPA is between residues 12-32 (
In spite of the apparent simplicity of the production and use of labeled receptor ligands and of the theoretical advantages of their use, only relatively few of these have been produced to date (
We extended these results to human cell lines expressing the endogenous uPAR. Efficient and specific staining was obtained on the human monocytic U937 cells and on the HT-1080 ovarian carcinoma cells, albeit at lower levels, proving that FITC-uPA can detect the native human uPAR (data not shown). We also demonstrated efficient staining of PFA-fixed cells, indicating that FITC-uPA can be of potential use on histopathological samples.
FITC-uPA can efficiently detect uPAR-expressing cells in fluorescence microscopy. FITC-uPA staining is mostly distributed on the cell surface membrane with a ring-like pattern. However, brighter spots in button-like structures are also evident, suggesting accumulation of FITC-uPA on potential cell-substrate adhesion areas (
FITC-uPA is a stable conjugate and can be used for accurate quantitative comparisons over a considerable length of time. We have stored FITC-uPA aliquots at -80C with no adverse effects for over 2 years. Repeated freezing and thawing of the same aliquot of FITC-uPA did not affect binding and staining efficiency, and we did not observe reduction of the apparent titer of FITC-uPA over time due to autoproteolysis. An example is shown in Figure 1A vs 1B. The essentially overlapping profiles obtained on different batches of stably transfected cells, either freshly thawed or continuously kept in culture, confirm the high reproducibility of the staining procedure presented and the stability of the FITC-uPA. This is also an example of use of FITC-uPA to verify the stability of expression of the uPAR on transfectants. The same transfectants were also successfully sorted for predefined levels of uPAR by flow cytometry (unpublished observation). FITC-uPA maintains its physiological proteolytic activity after binding to uPAR (data not shown). Therefore, dynamic analysis by confocal microscopy of the redistribution and proteolytic activity of the FITC-uPA-uPAR complex on binding to a substrate matrix is also allowed by the use of labeled uPA.
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Acknowledgments |
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Supported by the Italian Association for Cancer Research and by the Italian National Research Council, Convenzione CNR-Consorzio Mario Negri Sud, ACRO contract no. 94.01319.39, and Progetto Bilaterale contract no. 93.00870.CT04.
We thank Drs P. Mignatti and B. Donati for material and support during the course of this work.
Received for publication December 9, 1996; accepted April 18, 1997.
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