Fhnolgs,, (1987) I, 109 116 c 19X7 Longman Group UK Ltd
Regulation of Plasminogen Activator and Plasminogen Activator-inhibitor Production by Tissue Culture Cells: Evidence for Independent Induction and Regulation
A. Rehemtulla, R. Smith, D. A. Hart
SUIMMARY. Human bladder carcinoma cells (T-24) were found to secrete plasminogen
activator activity when cultured under serum-free conditions. The activity co-migrated with high molecular weight human urokinase when analysed by SDS-PAGE followed by zymography and it could be inhibited by anti-human urokinase. Treatment of the cells with phorbol myristate acetate led to enhanced secretion of the urokinase-like enzyme as well as induced the appearance of an 85 000 dalton plasminogen activator in the conditioned medium. The 85 000 dalton species of activity could also be inhibited by anti-human urokinase antibodies and was shown to consist of an SDS-stable complex between the 52 000 dalton high molecular weight urokinase-like plasminogen activator and a 33 000 dalton inhibitor. The urokinase-like enzyme was always produced in excess compared with the 33000 dalton inhibitor. Phorbol myristate acetate induced stimulation of plasminogen activator synthesis occurred rapidly (1 h) while the appearance of the 85 000 enzyme-inhibitor complex was not evident until 5 h after addition of the phorbol myristate acetate. Addition of several compounds (LiCI, dexamethasone, gamma-interferon) to cultures of T-24 cells lead to a depression of plasminogen activator secretion. In contrast, addition of cyclic AMP to the cultures did not inhibit enzyme production but completely suppressed complex formation. Dexamethasone inhibited both PA secretion and inhibitor secretion while LiCI and gamma interferon only inhibited enzyme secretion. Thus, some biologically active molecules can regulate the production of the two components co-ordinately or independently. KE Y WORDS. Plasminogen hibitors in tumour cells.
There are two functionally and immunologically distinct classes of plasminogen activators, the urokinase like (UK-like) and tissue plasminogen activator-like (tPA-like). Both forms of PA serve many normal functions such as vascular fibrinolysis, tissue remodelling and in the function of cells such as macrophages. l-’ In addition to normal cells, many cancer cells or transformed cells express PA, usually in great excess compared to their normal counterparts.3V4 The presence of high PA levels has been correlated with the processes of invasion and metastasis.“-6 Several proteinase inhibitors with specificity for PA have been described from a variety of cells, fluids and tissues. Inhibitors which form SDS-stable complexes with PA have been described from plasma,7v8 urine,‘-” placenta,l* endothelial cells,‘3-‘s human
activator inhibitor synthesis. Proteinases
HEp-3 cells, I6 human renal carcinoma cells,17 fibroblasts,‘** l9 and from monocyte-macrophages.20-22 During the activation df PA negative monocytes, the sequence of activation appears to be cell associated PA expression, PA secretion and then PA + PAinhibitor secretion. 2o Treatment of cell lines with the tumour promoter, phorbol myristate acetate (PMA), has been shown to enhance PA production and in some cases, also enhance PA-inhibitor production.19,21,23-26 Thus in several systems PA production and PA-inhibitor production by cell lines appears to be regulated in a somewhat co-ordinate fashion. The present experiments were designed to investigate the production of PA and PA-inhibitors in tissue culture cell lines to gain more insight into the mechanisms governing the regulation of these elements. We report that PMA treatment of human T-24 bladder carcinoma cells leads to enhanced PA production and the induction of a 33000 dalton inhibitor and that further treatment of the cells with biologically active compounds can regulate production of the enzyme and inhibitor co-ordinately or independently.
A. Rehemtulla, R. Smith, D. A. Hart, Immunological Sciences Research Group, University of Calgary, Health Sciences Centre, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4Nl Canada. 109
The T-24 human bladder carcinoma cell line (ATCC: HTB-4) was obtained from the laboratory of Dr L. M. Jerry (Tom Baker Cancer Centre, Foothills Hospital, Calgary, Alberta). The cells were maintained in tissue culture medium RPMI-1640 (Gibco) containing lop/, fetal bovine serum (Hyclone). The cells were grown as adherent monolayers in 75cm2 tissue cultures flasks (Corning). Immediately prior to treatment, the confluent monolayers were washed 2x with serum-free medium and then were placed in culture with serumfree RPMI-1640. Incubation of the cells under serumfree conditions for up to 36 h did not affect cell viability (trypan blue exclusion) and did not induce detachment from the flask. Reagents
Phorbol myristate acetate (PMA) was obtained from Sigma Chemical Co. The reagent was dissolved in DMSO (Sigma) at a concentration of 100 ug PMA/ml and stored at - 20°C. When added to cells, an equivalent volume of DMSO was added to control cells. Dibutyryl cyclic AMP (DBcAMP) and dexamethasone were also obtained from Sigma Chemical Co. DBcAMP was solubilised in RPMI-1640 and dexamethasone was dissolved in ethanol. Both reagents were freshly prepared for each experiment. An equivalent volume of ethanol was added to control cultures. Lithium chloride and KC1 were purchased from Mallinckordt Chemicals as reagent grade compounds. Recombinant human gamma interferon (gammaIFN), beta interferon (beta-INF) and tissue necrosis factor (TNF) were obtained from Dr Y. H. Tan (Department of Microbiology and Infectious Diseases, University of Calgary HSC). The reagents were titered by Dr Tan and maintained at - 70°C until use. High molecular weight urokinase (HMW-UK, 52 000 daltons), low molecular weight urokinase (LMW-UK, 33 000 daltons) and rabbit anti-human urokinase IgG were prepared by Green Cross Corp. and obtained through Alpha Therapeutics. The rabbit anti-human urokinase was that used previously.” Tissue PA was obtained from American Diagnostica. Human urine was obtained and prepared as described previously. 9,1o All reagents for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were from Bio-Rad. Plasminogen Activator Inhibitor Analysis
Plasminogen activators and plasminogen activator inhibitor were analysed by SDS-PAGE followed by followed by reverse zymography,27 SDS-PAGE zymography14 or by fibrinolysis in gel assay performed as described by Saksela, et al.22 In the latter method the lysis area (mm*) was determined after incubation at 37°C for 24 h. Under these conditions
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1 IU of UK yielded a lytic zone of approximately 13s 140mm2. Urokinase standards and/or human urine samples were included in all assays as controls and reference points and human plasminogen was purified from DFP-treated plasma as described previously.” Iodination of Human UK and Autoradiography
Low molecular weight UK was labelled with 1251as described previously.” Following separation by SDSPAGE, gels containing lz51-UK were dried and placed under Kodak X0-MAT film for 2496 h.
Conditioned medium (CM) from cultures of T-24 cells was separated by SDS-PAGE and analysed for the presence of PA by zymography. As depicted in Figure 1, conditioned medium from untreated cells contained a single band of fibrinolytic activity which comigrated with HMW-UK. Additional experiments revealed that the activity in the CM was plasminogen dependent and it could be specifically inhibited by inclusion of rabbit anti-UK IgG into the indicator gel (data not shown). Treatment of the cells with DMSO for 18 h did not change the pattern of PA detected in the CM [Fig. 11. However, treatment of the cells with 1OOng PMA/ml for 18 h led to the detection of a new plasminogen-dependent activity which migrated with an apparent M, = 85 000 [Fig. 11. Additional analyses revealed that the 85000 dalton species migrated slightly faster than the 90000 dalton species of PA detected in human urine concentrates.‘, lo Both the
Fig. 1 Zymographic analysis of conditioned media (CM) demonstrating the presence of plasminogen activator species. Cells were incubated in serum free media as indicated and then 100 ul of CM were analysed by SDS-PAGE and zymography as described in the Materials and Methods. Lane A: Purified HMW-UK and LMM-UK; Lane B: Untreated cells; Lane C: Cells treated with 0.1% DMSO; Lane D: Cells treated with lOOng/ml PMA in DMSO.
a cn z > 1
Fig. 2 Fibrinolytic activity of conditioned media (CM) from PMA treated and untreated cells. Duplicate cultures were incubated for the indicated times and then 15 ul of the CM were applied to a fibrin plate. After 24 h the plates were stained and the lysis area determined. PMA induced cultures (0); Untreated cultures (0).
90000 dalton species of PA from urine and the analogous species of PA detected in CM from PMAtreated T-24 cells could be inhibited by inclusion of anti-UK into the indicator plates.‘@ and data not shown Therefore, the 85000 dalton species in the CM was UK-like. The antisera did not inhibit tPA activity when included in the same gel. Previous investigations have revealed that the 90000 dalton species detected in urine concentrates was due to the formation of an SDS-stable complex between UK and a 40000 dalton binding protein.” Therefore, additional experiments were performed to determine whether the 85000 dalton species of PA in the CM was due to a complex between the 52000 dalton UK and a PMA-induced 33 000 dalton inhibitor. The first experiment was designed to determine whether PMA induced the synthesis of the new species of PA or induced the secretion of cell-associated molecules. Detergent lysates of untreated T-24 cells contained only small amounts of the 52 000 dalton species of PA. Addition of exogenous UK to the cell lysates did not induce the appearance of very high molecular weight species of PA (data not shown). Therefore no evidence for a cell-associated 85 000 dalton PA species or a 33000 dalton PA-binding protein could be detected. If PMA induced the synthesis of the 85000 dalton PA or a 33 000 dalton PA-binding protein, then it was important to determine the concentration dependence of this observation. Therefore, cultures of T-24 cells were incubated with O-500ng PMA/ml for 18 h and then the CM analysed for PA activity. The 85000 dalton species of activity could be detected after incubation with 25ng PMA/ml and reached maximum activity when incubated with 1OOng PMA/ml. Concentrations of PMA in excess of 200ng/ml were toxic to the cells.
Utilising the optimal concentration of PMA, lOOng/ml,’ individual flasks of T-24 cells were incubated for 0.5-18 h and the CM was analysed by zymography and the fibrinolysis in gel assay. By the fibrinolysis in gel assay, PA activity could be detected in the CM by 0.5 h and the activity accumulated with time until 6 h at which point the PA activity remained constant throughout the remainder of the experiment [Fig. 21. No change in activity was observed between lo-18 h post-induction (data not shown). When the same samples were assayed by zymography, the 52 000 dalton PA could be detected at the early time points while the 85 000 dalton PA could first be detected after 5 h of induction and reached a maximum percentage of the total activity at approximately 8 h postinduction [Fig. 31. Thus, there is a correlation between the appearance of the 85000 dalton species of PA by zymography (Fig. 31 and the plateau in PA activity observed by the fibrinolysis in gel assay [Fig. 21. These findings are consistent with the hypothesis that the 85 000 dalton PA is a complex between the 52 000 dalton UK and a 33000 dalton inhibitor. The complex would be inactive in the fibrinolytic assay but reactivated by SDS to yield a band of activity by zymo-
graphy. Attempts to determine the presence of uncomplexed material in the CM capable of inhibiting UK (reverse zymography) or complexing with 1251-UK (SDSPAGE and autoradiography) were unsuccessful. Such methods have been successfully used to determine the 40 000 dalton PA-binding protein in urine.lO; and data not shownTherefore, if the 85 000 dalton PA is formed as a UK + Inhibitor complex, all of the inhibitor must be complexed to the excess PA in the medium. Parallel experiments with uninduced T-24 cells revealed that the 52 000 dalton PA could first be detected in the CM 6 h after initiation of the culture (corresponding to the
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52 KD Fig. 3 Zymographic OSh.2h.4h.6h,8h,9h,llh
media from PMA treated T-24 cells incubated
Fig. 4 Zymographic analysis of conditioned media (100 ul) from cells treated as follows; Lane A: Treated with PMA (lOOng/ml) for 5 h only; Lane B: Conditioned media from cells treated with PMA for 5 h and then cultured for an additional 5 h in the absence of PMA; Lane C: Conditioned media from cells treated with PMA for 5 h and which was then replaced with fresh media containing PMA; Lane D: Conditioned media from cells treated with PMA for 10h continuously.
observations in Fig. 2). Therefore, PMA rapidly induces elevated synthesis of UK-like PA and only later induces the 85000 dalton form of PA. Thus, in the induced state, a quantity of PA would accumulate in the medium prior to induction of the putative inhibitor, thereby favouring rapid complex formation. To minmise the influence of PA secreted early in the culture, cells were incubated with 100 ng PMA/ml for 5 h and then the medium was removed and replaced with fresh media k PMA. Cells cultured for 10h with PMA served as the positive control. As depicted in Figure 4, CM from cells treated with PMA for 5 h, washed, and recultured for an additional 5 h contained both the 52 000 dalton PA and the 85 000 dalton PA irrespective of whether PMA was included during the
times (as in Fig. 2). Lanes A to G:
last 5 h of culture. The activity in the 85 000 dalton PA in this situation was comparable to the level of activity detected in the CM from cells cultured continuously for 10h [Fig. 43. By reverse zymography or the ‘*‘I-UK assay, no uncomplexed 33000 PA inhibitor could be detected in any of the CM from these experiments. Therefore, even when the putative inhibitor is being maximally produced, the 52000 dalton UK is being secreted in excess. In order to alleviate the interference of excess 52 000 dalton UK on the detection of a 33 000 dalton inhibitor, two additional experiments were performed. First, cultures of T-24 cells were treated with 1OOng PMA/ml for 5 h and then the medium was removed and replaced with either medium alone or 5ml medium containing 600 IU LMW-UK. Since no LMW-UK was present in the PMA induced CM [Fig. 11, exogenous LMW-UK could complex with any native 33 000 dalton inhibitor to yield a 66 000 dalton complex, analogous to the 70000 dalton complex detected in urine.‘, lo As depicted in Figure 5A, when LMW-UK is added to PMA induced cells, a new species of VHMW-PA activity is detected with an apparent M, = 65-67000 [Lane 41. This activity migrates slightly faster than the analogous 70000 dalton complex detected in urine [Lane 51. Interestingly, when LMW-UK is added to the induced cells the 85 000 dalton PA activity is diminished compared with the CM obtained when LMW-UK was omitted [Lane 4 us Lane 31. To support the conclusion that the 66000 dalton PA activity is a complex between LMW-UK and a 33 000 inhibitor, the above described experiment was repeated with [‘251]-LMW-UK instead of unlabelled UK. Cells were again induced with 1OOng PMA/ml for 5 h and then the medium was replaced with fresh medium containing [‘251]-LMWUK. After an additional 6 h, the 125T-medium was removed and analysed by SDS-PAGE followed by autoradiography. As depicted in Figure 5B, incubation of [’ 251]-LMW-UK under such conditions led to the appearance of a new band of [’ 251] migrating with an apparent M, = 66000 [Lane I]. This band of
92 85 70 66
KD KD KD KD
Fig. 5 Analysis of conditioned media from T-24 cells cultured in the presence of low molecular weight urokinase. [A] Zymographic analysi Lane 1: Purified HMW-UK and LMW-UK; Lane 2: Purified LMW-UK; Lane 3: Conditioned media from PMA treated cells; Lane 4: Conditioned media from PMA treated cells that were induced in the presence of 600 IU LMW-UK; Lane 5: Dialyzed human urine. IBl Radioautomaohv ofconditioned media from PMA treated cells incubated in the presence of [ ‘ZSI]-LMW-UK. Lane I: Conditioned medium; Lane 2: [“‘I]-LMW-UK.
which was not detectable in samples of [‘251] [““II-UK incubated with medium alone [Lane 21. Analysis of cell lysates obtained with induced cells + ““I-UK failed to reveal the presence of the 66000 dalton material. Thus, these experiments performed with exogenous LMW-UK support the conclusion that a 33 000 dalton UK-binding protein is secreted from PMA induced cells which subsequently forms complexes with UK in the medium. Additional experiments were performed in order to further assess the regulation of PA and PA-Inhibitor secretion by T-24 cells. Therefore, cells were cultured in the absence or presence of PMA in addition to the compounds to be tested. Enzyme release was assessed in the CM by the fibrinolysis in gel assay and the influence of the compounds on complex formation was assessed qualitatively by zymography. Addition of LiCl (10_20mM), Dexamethasone (10-8~10-6M) or gamma-IFN (l-50 u/ml) led to dose dependent inhibition of PA release from both uninduced and PMA induced cells [Table 11. Maximal inhibition ranged from 40~50’1;, (LiCl) to 6OG70”; (Dexamethasone), depending on the compound tested. Interestingly, gamma-IFN was very effective in inhibiting PA release from both uninduced and induced cells (approximately 50”,(; inhibition). In comparison, PA production was not inhibited by either beta-IFN or TNF [Table I]. Similarly, PA production was very resistant to inhibition by CAMP (0.01&l mM). Addition of cAMP + LiCl to cells did not lead to a reversal of Li-induced inhibition [Table 11. When aliquots of CM from the above described experiments were analysed for the presence of PA-I complexes (85 000 daltons), the results depicted in Figure 6 were obtained. Lithium [Fig. 61 and gamma-IFN
(data not shown) did not diminish the detection of the 85 000 dalton species of PA and therefore did not inhibit PA-Inhibitor synthesis. In contrast, CAMP, which did not inhibit PA secretion, and Dexamethasone both dramatically inhibited the detection of the 85 000 dalton species of activity [Fig. 61 and therefore inhibit the production of the inhibitor. From these results one can conclude that the enzyme and inhibitor can be regulated independently.
DISCUSSION The results presented in this report demonstrate that treatment of T-24 cells with PMA leads to the induction and secretion of a protein capable of forming an SDS-stable complex with UK. The T-24 cell inhibitor exhibits an apparent M, = 33 000. The relationship of this PA-Inhibitor to those previous reported from other sources (urine, placenta, protease nexin, endothelial cells, etc.) must await further investigation. However, there are several interesting points raised by this study concerning the regulation of PA and PAInhibitor production by cells. First, the observation that T-24 carcinoma cells produce PA but not detectable levels of PA-I constitutively, implies that cells can produce the enzyme without concomitant production of the inhibitor. In this respect, T-24 cells differ from U937 cells2’ activated monocyte-macrophageszo~22 as well as the renal carcinoma cell lines, Cur and Caki-1” which produce both PA and PA-Inhibitor without treatment. Secondly, the observation that treatment of T-24 cells with a tumor promoter, PMA, enhances PA production and induces a unique PAInhibitor implies that these cells are programmed to
Table I Regulation Compounds
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Cells were grown to sub-confluency, washed and then cultured for 12-16 h under serum-free conditions in the presence of the indicated compounds k 1OOng PMA. Plasminogen activator activity in the conditioned medium was assessed by the fibinolysis in gel assay of Saksela, et al. ” None of the compounds tested inhibited the activity of purified human urokinase. The mean values obtained from 24 experiments are reported. Variation from the reported means was less than 10%.
Experiment No addition (Control) Ethanol control DMSO control 20 mM KC1 LiCl IOmM 20mM DBcAMP I ‘mM 0.1 mM 0.01 mM 20mM LiCl + 1mM DBcAMP De.~amethasonr (Dex) IO-‘M IO-‘M IO-*M 20mM LiCl + lo-‘M Dex 2OmM LiCl + 10msM Dex Interferon Gamma (Units/&) 0.1 1.0 5 50 500 Beta (Unit.s/ml) 5 50 500 Tissue necrosis factor 2000 Units/ml 5000 Units/ml
respond to this promoter in a coordinate fashion. The basis for the enhanced production of UK-like PA in the presence of PMA is not known, but in other studies with human carcinoma cells, PMA enhanced the synthesis of UK mRNA.23-2s The enhanced rate of mRNA synthesis could be detected within 30min after initiation of PMA treatment.25 Such events could account for the present observations with T-24 cells (Fig. 2). In contrast to the rapid enhancement of PA production by PMA, the induction of PA-Inhibitor production required approximately 5’h before the PA-Inhibitor complex could be detected in the CM. These results lead one to conclude that the sequence of events leading to the induction by PMA of PA-Inhibitor synthesis is somewhat different from the enhancement of PA synthesis which was already induced. Thus, while the T-24 cells are programmed to produce a PA-Inhibitor, this protein is regulated separately from the enzyme. This conclusion is supported by the results obtained with DBcAMP, dexamethasone and gamma-IFN (Table 1
Plasminogen activator release (Percent ofcontrol) -PMA +PMA
114.6 95.8 100
85.4 92.3 109.3 59.1
91.5 94.0 94.6 66.9
30.3 26.5 43.1 45.8 45
41.5 41.8 51.5 41.7 41.5
loo 90 11.3 58.2 54.1
91.8 84 69.1 47.2 42.1
loo loo 89.6
89.1 100 12.2
and Fig. 6). DBcAMP (1 mM) completely inhibited the induction of PA-Inhibitor secretion but did not inhibit PA secretion either in the presence or absence of PMA. In contrast, gamma-IFN (immune interferon) inhibited PA production (approximately 50% inhibition) but did not have any observable effect on PA-Inhibitor induction. Interestingly, dexamethasone inhibited both PA production and the induction of PA-Inhibitor by PMA. Thus the enzyme and inhibitor can be regulated independently or coordinately. While the above discussion analyses the regulation of PA and PA-Inhibitor in a tumour cell line, it should be pointed out that other investigators have reported on the regulation of similar components in other cells and in many instances, expression of these molecules is regulated differently than what is observed with T-24 cells. For instance Saksela et alz2 have reported that dexamethasone inhibited PA production by differentiating macrophages but did not inhibit PA-Inhibitor synthesis. Dexamethasone has been reported to induce PA-Inhibitor production by rat hepatoma cellsz8
1 0m3M DBcAMP
Fig. 6 Zymographtc analysis of conditioned medium from T-24 cells cultured in the presence of PMA and biologically active compounds. Cells were incubated under serum-free conditions in the presence of IO0 ng PMA,‘ml i compounds to be tested. Panel A: Cells cultured in the presence of PMA alone (C), PMA + 10m3M DBcAMP, or PMA + 10-3M DBcAMP + 20mM LiCI. Panel B: Cells cultured in the presence of PMA alone (C)or PMA + 20mM LiCI. Panel C: Cells cultured in the presence of PMA alone (C) or PMA + indicated concentrations ofDexamethasonc.
and human fibrosarcoma cells29 while glucocorticoids have also been reported to induce PA-Inhibitor production by human fibroblasts.30 DMSO, which had no effect on T-24 cells, inhibited PA expression by HEp-3 cells but did not inhibit PA-Inhibitor expression.” There are other examples of differences between the findings presented in this report and those of other workers, but the main point is that different cells respond to various stimuli depending on both cellular programming as well as the expression of suitable receptors for various biologically active molecules. In addition to providing insights into the cellular regulation of PA and PA-Inhibitors, the results presented also have implications for the possibility of modulating the PA activity of cancer cells or inflammatory cells. As stated earlier, in many instances tumour cells express elevated levels of PA and high PA levels have been correlated with increased metastatic potential. 4-6 Recent reports have indicated that antibodies to PA can inhibit metastasis in an experimental in vivo mode131 and that inhibition of PA activity by hydrocortisone parallels inhibition of tumour growth in a mouse mammary carcinoma mode1.32 In addition it has recently been shown that exogenous protease nexin can alter the ability of tumour cells to degrade extracellular matrix in vitro.j3 Thus, if PA production by tumour cells could be inhibited and PA-Inhibitor synthesis by tumour cells or adjacent cells induced, one could generate a situation whereby the metastatic potential of a tumour could be decreased. In summary, the results presented provide new insights into the regulation of PA and PA-Inhibitor expression by tissue culture cell lines. Further investi-
gation into these regulatory mechanisms may provide the basis for future modulation of fibrinolysis in both normal and disease states such as cancer.
ACKNOWLEDGEMENTS The authors thank Judy Crawford for assistance in the preparation of the manuscript. This investigation was supported by the Alberta Heritage Foundation for Medical Research (AHFMR), the Alberta Cancer Board (H-277) and the Canadian Arthritis Society (18-261). AR was supported by an AHFMR Studentship and DAH is an AHFMR Scholar.
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Offprint orders to: Dr David A. Hart, Immunological Sciences Research Group, University of Calgary, Health Sciences Centre, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada. (403) 2204571.
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