Enhancement of the Expression of Urokinase-Type Plasminogen Activator from PC-3 Human Prostate Cancer Cells by Thrombin'

Home , Hirudin, Thrombin, Urokinase

ICANCERRESEARCH54,3300-3304,June15,1@94I Enhancement of the Expression of Urokinase-Type Plasminogen Activator from PC-3 Human Prostate Cancer Cells by Thrombin'

Etsuo Yoshida,2 Elaine N. Verrusio, Hisashi Mihara,2 Doyeun Oh, and Hau C. Kwaan3

Department of Medicine, Hematology/Oncology Division, Northwestern University Medical Schoo4 and VA Lakeside Medical Ce,Uer, Chicago, Illinois 6@11 [E. V., E. N. V., D. 0., H. C. K./, and Department ofPhysiology, Miyazaki Medical College, Miyazaki 889-16, Japan [H. M.J

ABSTRACT extracellular matrix. This is supported by the fact that anti-uPA could reduce tumor invasion and metastatic ability (7). uPA has been also The presence of procoagulants and fibrin deposition have been dem found to have a mitogenic effect on several cell lines (8, 9). onstrated in malignant tumors. Although thrombin, a key enzyme in In view of these findings, it is possible that thrombin may affect coagulation, has other various biological functions, the significance of its tumor cell behavior by modulating the expression of the components presence in tumors is not known. We studied the effects of thrombin on of the plasminogen-plasmin system. In the present study, we focused the expression of urokinase-type plasminogen activator (uPA) which is on the effect of thrombin on uPA expression in cancer cells and known to play a role in tumor invasion, using a human prostate cancer cell line PC-3. Human a-thrombin added to cultures of PC-3 produced a examined three prostate cancer cell lines: PC-3, a high uPA producer; dose-dependent and time-dependent increased secretion of uPA that was DU-145, which is a low uPA producer; and LNCaP, a non-uPA greatest at 3â€”6h after exposure to thrombin. Increase in uPA antigen producer. We demonstrate here that thrombin induced uPA production paralleled the Increase in mRNA level, which reached a maximum at 4 h. by PC-3 cells through the stimulation of a thrombin-specific receptor. Thrombin showed the maximum effect on uPA expression at a concen tratlon 1â€”2units/mL Zymography showed that transient exposure to thrombin induced an increase in fibrinolytic activity which could be MATERIALS AND METHODS quenched by anti-uPA antibody. The thrombin receptor-activating pep tide also caused an increase in uPA protein and mRNA level, indicating Materials. Human a-thrombin (3250 NIH units/mg) was purchased from the presence of the same thrombin specific receptor on PC-3 cells as on Sigma Chemical Co. (St. Louis, MO). Cycloheximide and actinomycin D were platelets and endothelial cells. Thrombin did not affect the expression of purchased from Sigma Chemical Co., and heparin was purchased from Elkins other components of the plasminogen activation system, tissue-type plas Simm,Inc. (Cherry Hill, NJ). Native hirudin was purchased from American minogen activator and type-i plasminogen activator inhibitor, and uPA Diagnostics, Inc. (St. Louis, MO), and [32P]dCTP was purchased from Am receptor. These results Indicate that thrombin increases uPA expression ersham (Arlington Heights, IL). Antithrombin III was kindly provided by Dr. selectively by the stimulation of a functional thrombin receptor on PC-3 T. Yin, Haemachem,Inc.(St. Louis, MO).RPMI-1640andfetalbovine serum cells. Since uPA is known to play a role in periceflular proteolysis of were obtained from Central facility, Northwestern University Cancer Center. extracellular matrix, thrombin may be involved In the regulation of tumor PPACKwas purchasedfromChemicaAlta,Ltd.(Edmonton,Alberta,Canada). invasion and metastasis. PPACK-thrombin was prepared by dissolving 50 p@gofPPACK and 3.3 @gof thrombin in 5 ml of 0.3 M sodium phosphate buffer (pH 7.4) and dialyzing against 0.1 Msodium phosphate buffer-0.15 MNaCI (pH 7.4) to eliminate free PPACK. The activity of PPACK-thrombin was less than 1% of that of original INTRODUCTION thrombin by clotting time. The presence of the coagulation process in malignant tumors has The human uPA probe was prepared by PCR from pRSV-uPA (10), which is a gift from Dr. F. Blasi (University of Milano, Italy). uPA gene fragment been well recognized. Fibrin deposition has been demonstrated in the (from +5433 to +6148 in exon XI) was amplified by PCR with a set of stroma around the invading front of the tumor (I, 2) and on circulating oligonucleotide primers, and then the products were used as a template for cancer cells in blood (3). The presence of procoagulants has also been second PCR. The final product showed a single 0.7-kilobase band in agarose demonstrated in cancer tissues (4). In the process of coagulation, gel and was used as uPA probe. Human GAPDH complementary DNA probe thrombin is generated which, in addition to fibrin formation, has other was a gift from Dr. 0. Soff(Northwestern University). TRAP (SFLLRNPND various biological functions such as an increase in vascular perme KYEPFWE) and a CP (FSLLRNPNDKYEPFWE) in which the first two ability, stimulation of adherence of platelet and endothelial cells, amino acids, sermneandphenylalanine, are transposed were synthesized, based chemoattraction of monocytic cells, and mitogenic activity on endo on the report by T. K. Vu et aL (11) at the Biotechnology Facility in Northwestern University, Chicago, IL. thelial cells and fibroblasts (5). Thus, it is likely that thrombin gen Cell Culture. The PC-3cell line is a prostatecancercell line derivedfrom crated in the vicinity of cancer cells may affect tumor cell behavior. a lumbar vertebral metastasis of a poorly differentiated human prostatic ade However, the significance of thrombin or other components of the nocarcinoma. These cells are androgen independent, readily metastasize when coagulation system is not fully understood. inoculated into nude mice, and are biologically aggressive (12). Cells were On the other hand, the plasminogen-plasmin system has been grown to confluence on 24-well plates (Corning, Corning, NY) in RPMI-1640 shown to play a role in tumor growth, invasion, and metastasis (6). with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 uPA4 is believed to be an initiator of pericellular proteolysis of the g.@g/ml).Studies were performed by washing confluent cultures twice with RPMI-1640andincubatingtheculturesat37Â°Cin1 ml RPMI-1640containing the indicated concentration of human a-thrombin and 0.1% bovine serum Received 2/28/94; accepted 4/20/94. The costs of publication of this article were defrayed in part by the payment of page albumin to exclude nonspecific protein effect. After incubation, the condi charges. This article must therefore be hereby marked advertisement in accordance with tioned medium was collected, centrifuged at 8000 X g to remove cell debris, 18 U.S.C. Section 1734 solely to indicate this fact. and stored at â€”70Â°Cuntilassayed. The cell number was counted using a I This work is supported in part by a VA Merit Review Research Grant. hemocytometer. 2 Present address: Department of Physiology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-16, Japan. Measurement of uPA Antigen. The level of uPA antigen in the condi 3 To whom requests for reprints should be addressed, at Hematology/Oncology Divi tioned medium was measured by an ELISA (TintElize uPA kit, Biopool; sion, VA Lakeside Medical Center, 333 East Huron, Chicago, IL 60611. 4Theabbreviationsusedare:uPA,urokinase-typeplasminogenactivator;tPA,tissue Meditech, Ventura, CA) according to the manufacturer's description. tPA and type plasminogen activator; scuPA, single-chain uPA; tcuPA, two-chain uPA; PPACK, phenylalanyl-prolyl-arginyl-chloromethyl ketone; PCR, polymerase chain reaction; ing peptide; CP, control peptide; EUSA, enzyme-linked immunosorbent assay; PAl-i, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRAP, thrombin receptor activat type-i plasminogen activator inhibitor; SDS, sodium dodecyl sulfate. 3300

30 cellsfl6h, while thrombin StimUlatiOnenhanced more than a 100% increase in uPA secretion. Nonstimulated DU-145 cells produce only 0.05 ng/1f9 cdlls/16h, and this pmduction was not affected by thrombin stimuIatio@t LNCaP did not secrete detectable levels of uPA with or without thrombm U Northern blot analysis also showed that thrombin had no effect on uPA )20 mRNA level in DU-145 and LNCaP. Therefore, we chose PC-3 ceHs for further study of the detailed mechanism involved in thmmbin regulation of uPA. Effect of Thrombin on uPA Secretion. Fig. 1 shows the time course of thrombin stimulation of uPA antigen level in medium %10 conditioned by PC-3 cells. Preliminary experiments of the dose dependent effect of thrombin on uPA production for 24 h showed a maximal effect at a concentration of 1â€”2units/ml. Thus, we used 2 units/ml thrombin for the time-course study. PC-3 cells produce increasing amounts of uPA in the medium over the course of 24 h of 0 the experiment. The addition of thrombin resulted in more than 100% 0 4 8 12162024 increase in the secretion of uPA antigen. During the first 3 h, there Time (h) was no difference between the thrombin-stimulated cells and the control. Over the next 3 h between 3 and 6 h, the average rate of secretion, Fig. 1. Time course of the effect of thrombin on uPA secretion by PC-3 cells. as indicated by the slope, increased to 2.43 ng/1O@cells, which is 3-fold Confluentcellswereincubatedwith(0) or without(â€¢)2units/mlthrombinforvarious periods of time. uPA antigen levels in the medium were measured by ELISA. Each point greater than control, and then it declined to 0.97 ng/1O@cells between 12 and represents the mean of quadruplicate determinations from a single experiment. The result 24 h, as shown in the legend. This 3-h time lag suggests that thrombin represents three separate experiments. The average secretion rate calculated from the induced uPA protein synthesis and not the release of stored uPA. The other above results is components ofthe plasminogen-plasmin system, Le, tPA, PAl-i, and PAI-2, cells)Oâ€”3h3â€”6h6.-12h12â€”24hThrombin0.672.43i.120.97None0.500.870.440.43Av. rate of uPAsecretion (ng/bJ10@ were not detectable by EUSA. Thrombin has been known to stimulate cell proliferation in several cell types (5). To select an optimum incubation time for further study, we studied the growth effect of thrombin on PC-3 cells. No significant increase in cell numbers were found, nor were there any morpholog ical changes between thrombin-stimulated cells and nonstimulated cells observed within 24 h. Prolonged serum-free conditions reduce PAl-i antigen levels were also measured by TintElize kits. TintElize PAl-i kit the viability of PC-3 cells. Samples were collected at 6 h after detects PA/PM-i complexes with the same efficiency as free PAl-i. Zymography. SDS-polyacrylamide gel electrophoresis was performed thrombin stimulation, when the maximum increase rate was observed with 12% acrylamide gels according to Laemmli (13). Human high molecular (Fig. 1). Since the cell numbers remained constant during this period weight uPA (American Diagnostics, Greenwich, Cr) and single chain tPA in both the thrombin-treated and control groups, we have chosen to (American Diagnostics) were run in parallel for a standard marker. The fibrin use the end point as uPA antigen/mI in the following experiments. agar indicator gels contain 1% agarose, 20 @gfmlplasminogen,0.5 Nifi Fig. 2 shows the concentration dependence of the thrombin stimu units/nil thrombin, and 4 mg/nil fibrinogen. After electrophoresis, the SDS gels lation of uPA secretion. The effect of thrombin on uPA secretion was were soaked in 2.5% Triton X-100 for 1 h to remove the SDS and applied to the surface of the fibrin agar indicator gel. The gel was allowed to develop at 37Â°C. RNA Isolation and Northern Blot Analysis. Total RNA was isolated 15 from PC-3 cells by treatment with guanidinium isothiocyanate-sarcosyl solu lion followed by phenol extractionand ethanol precipitation.For Northern analysis, 12 g@gRNAper lane was electrophoresed through 1.2% formalde hyde agarose gels, transferredto nylon membrane(BoehringerMannheim, Mannheim, Germany), and cross-linked with UV light. The blots were prehy E 10 bridized in 30% formamide, 5 X Denhardt's solution, 0.1% SDS, and 100 @Lg/mlof salmon sperm DNA for 4 h at 42Â°C. The prehybridization mixture C was replaced with fresh solution containing labeled uPA probe prepared by nick translation using [32P]dCl'P. Hybridization was performed overnight at a. 42Â°Candfollowed by a wash. The ifiters were exposed to Fuji Imaging plate 3 BAS m for 3 h and analyzed by Fujix Bio-Imaging Analyzer BAS 2000 (Fuji, 5, Kanagawa, Japan). After autoradiography, the filter was treated with 50% formamide-1 mM EDTA-O.i M Tris for 30 mm at 85Â°Cto remove uPA probe and then rehybridized with GAPDH probe under the same experimental conditions. 0 - - -

RESULTS 0 01 .1 1 10

In order to examine the cell-specific expression and its responsiveness to Thrombin (U/mi) thrombin, we determined the uPA pmtein levels in the medium conditioned Fig.2. Concentrationdependenceofthe thrombinstimulationofuPA secretionby by three prostatecancer cell lines, PC-3, DU-145, and LNCaP cells, after PC-3 cells. Confluent cells were incubated for 6 h with various concentrations of human a-thrombin. The uPA antigen level in the medium was measured by EUSA. Points, mean incubation with various concentrations of thmmbin for 16 h in serum-free of quadruplicate determinations from a single experiment, representative of four separate conditions. NOnStImUIatedcontrol PC-3 cells produce about 8 ng uPN1O@ experiments. Bars, SD. 3301

faint lytic zones were observed. These two zones were confirmed to be tPA and probably tPA/PAI complex since these two lytic zones disappeared when anti-tPA antibody (final concentration, 10 @Wml) was incorporated in the indicator gel. This finding indicates that the continuous presence of thrombin is not necessary to stimulate uPA production, suggesting that thrombin stimulation triggers directly or I.o.O indirectly intracellular signal transduction followed by RNA synthe sis, protein synthesis, and secretion. @ . ,. . As shown in Table 1, when cultures were treated with cyclohexi mide, a protein synthesis inhibitor, the effect of thrombin on uPA secretion was completely blocked. Treatment with actinomycin D, an RNA synthesis inhibitor, also blocked the thrombin effect completely. t U 3612 3612h These findings and the 3-h time lag before the increase in uPA control thrombin secretion (Fig. I) indicate that the stimulation of uPA release by thrombin requires concomitant RNA and protein synthesis. Fig. 3. Zymography of the medium conditioned by PC-3 cells in the presence of hirudin after transient exposure to active thrombin. Media samples from the same Effect of Thrombin on uPA mRNA Level. To confirm the above experiment as Fig. 5 were analyzed by zymography as described in â€œMaterialsand result, we carried out Northern blot analysis. Fig. 4 shows the effect Methods.â€•Loading from left to right: (t) tPA; (u) uPA; Control, no thrombin treatment, of various concentrations of thrombin on uPA mRNA expression. 3, 6, and 12 h; 1-h exposure to thrombin (2 units/mI), 3, 6, and 12 h. Increased mRNA level was detectable at 0.01 unit/ml thrombin, and 1 unit/ml thrombin produced the strongest signal for uPA. Thrombin (10 Table I Effect of cycloheximide and actinomycin D on the thrombin induction of units/ml) resulted in a lesser message level. This down-regulation of uPA from PC-3 cells mRNA level is in agreement with the uPA protein level as shown in Confluent cells were incubated for 1 h with 25 @.LMcycloheximide,0.2 p.Mactinomycin D, or no inhibitor, and then for an additional 6 h with 2 units/mI thrombin. uPA antigen Fig. 2. Fig. 5 shows a transient increase in uPA mRNA levels in in the medium was measured by ELISA and is presented as the mean Â±SE of quadru response to thrombin stimulation. The message level was increased plicate determinations from a single experiment and are representative of three separate during the first 4 h and then declined. The maximal mRNA level at 4 experiments. h was in good agreement with an increased rate of protein secretion to (ng/ml)None6.2 uPA the medium between 3 and 6 h, as shown in Fig. 1. Moreover, we 2.30Thrombin Â± examined the mRNA level of uPA receptor and PAl-i before and after i.04+ (2 units/mI)11.6 Â± thrombin stimulation. Thrombin did not affect the mRNA levels of 0.57+Cycloheximide (25 @LM)2.2 Â± uPA receptor or PAl-i in PC-3 cells. To test whether protein synthesis Actinomycin D (0.2 @.LM)3.8 Â±1.99

0.01.1 1 10 u/mI detectable at 0. 1 units/ml and was maximal at 1â€”2units/ml, where a 100% increase in uPA concentration was observed. However, higher concentrations of thrombin resulted in a decrease in uPA secretion. Thus, we had chosen the concentration of 2 units/ml for the following .@1@28S @ experiments. On the other hand, tPA and PAl-i were not detected by U @j@.â€”@ ELISA at any concentration of thrombin. When the conditioned medium of thrombin-stimulated cells was @uIÃ¸ 18S subjected to zymography, we observed a decrease in the fibrinolytic activity (data not shown) in contrast to an increase in the uPA protein level measured by ELISA. This is due to the conversion by thrombin @ of the scuPA secreted by cells to inactive tcuPA. Thrombin is known GAPDH to cleave scuPA at Arg 156 forming an inactive tcuPA, which is not activated by plasmin (14) but can be activated by cathepsin C (15). Fig. 4. Concentration dependence of the thrombin induction of uPA mRNA by PC-3 However, under physiological conditions, a prolonged presence of an cells. After incubation with various concentrations of thrombin for 4 h, total RNA was active enzyme, such as thrombin, is unlikely, because a number of isolated and analyzed for uPA mRNA. After stripping, the same filter was rehybridized protease inhibitors exist in the plasma and extracellular fluids. Thus, with GAPDH probe. The positions of the 285 and 18S ribosomal RNA are indicated to the right of the blot. the stimulation by an active enzyme is thought to be short-lived. To test whether transient exposure to thrombin can induce uPA expression, hirudin, a highly specific thrombin inhibitor, was added to 0 2 4 8 1624h block thrombin catalytic activity in the conditioned medium after the cultures were treated with thrombin for 1 h; then the medium were collected 3, 6, and 12 h later and analyzed by zymography. Under this uPA condition, thrombin activity was less than 1% of that of original thrombin as determined by clotting time. Hirudin alone had no effect on uPA secretion by PC-3 cells. Zymography showed clearly in GAPDH creased fibrinolytic activity in agreement with the increased uPA antigen level (Fig. 3). The major lytic zone was confirmed as uPA since it corresponds to the standard marker. Furthermore, the activity Fig. 5. Time course of thrombin induction of uPA mRNA in PC-3 cells. After cells was quenched when anti-uPA antibody (final concentration 10 @.tWml) were incubated with thrombin (2 units/mI) for the indicated time periods, total RNA was isolated and analyzed for uPA mRNA. After stripping, the same filter was rehybridized was added to the fibrin agar indicator gel. Besides the uPA band, two with GAPDH probe. 3302

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. ThROMBIN INDUCtiON OF uPA FROMCANCER is required for a thrombin effect on the uPA mRNA level, PC-3 cells TRAP CP were incubated for 4 h with cycloheximide in combination with 05205010050100 uM thrombin. A similar increase in the uPA mRNA level as when incu bated with thrombin alone was observed, indicating that de novo protein synthesis is not required. Effect of Thrombin Inhibitors on uPA Secretion. To determine @@@28S whether enhanced expression of uPA by thrombin was actively site dependent, several kinds of thrombin inhibitors were used: (a) anti uPA thrombin III and heparin, which are physiological inhibitors; (b) hirudin, which inhibits both active site and receptor binding site of -uI@ 18S thrombin; and (c) PPACK, which blocks the active site but not binding to thrombin receptors (16). As shown in Fig. 6, the thrombin effect was inhibited by antithrombin Ill-heparin complex or hirudin, and PPACK-thrombin did not stimulate uPA release from PC-3 cells. This result indicates that intact thrombin is necessary to exert the GAPDH effect on uPA secretion by PC-3 cells. Fig. 8. Effect of TRAP on uPA mRNA expression by PC-3 cells. After cells were Effect of TRAP on uPA Expression. To determine whether this incubated with various concentrations of TRAP and CP for 4 h, total RNA was isolated thrombin effect is mediated directly by a thrombin receptor or mdi and analyzed for uPA mRNA. After stripping, the same filter was rehybridized with the GAPDH probe. The positions ofthe 285 and 18S ribosomal RNA are indicated to the right rectly via other protease actions, we used a new concept for the of the blot. activation of the thrombin receptor proposed by Vu et a!. (1 1). They cloned a thrombin-specific receptor, which is a member of a seven transmembrane receptor family, and the presence of this receptor was by the fact that a synthesized peptide, mimicking the putative new shown in platelets and endothelial cells. From the deduced amino acid amino terminus which is exposed by proteolysis of an extracellular sequence, a novel proteolytic mechanism through which thrombin amino terminal extension of the receptor, could activate thrombin activates the receptor was proposed. This hypothesis was confirmed receptors. Based on their report, we synthesized two peptides. One is a new amino terminal peptide, i.e., TRAP, the other is a CP in which the first two amino terminal amino acids were transposed. (The sequence is shown in â€œMaterialsand Methods.â€•) thrombin After 6 h incubation with various concentrations of TRAP and CP, control the uPA protein level in the media conditioned by PC-3 cells was measured by ELISA. Fig. 7 shows the effect of TRAP on uPA ATIII/heparin+thrombln secretion by PC-3 cells. TRAP produced a dose-dependent increase in hirudin+thrombln uPA secretion. On the other hand, CP had no effect, even at higher concentrations of 50 and 100 @m. PPACK-thrombln To confirm further the effect of TRAP on uPA expression, we 0 10 20 carried out Northern blot analysis (Fig. 8). After 4 h incubation with uPA(ng/mI/6h) the peptides, total RNA were extracted and analyzed for uPA mRNA. Fig. 6. Effect of thrombin inhibitors on the thrombin induction of uPA from PC-3 cells. Again, we observed the increased expression of uPA mRNA by TRAP Confluent cells were incubated with the specified amount of thrombin or thrombin inhibitors for 6 h. uPA antigen levels in the medium were measured by ELISA. PPACK in a dose-dependent manner but not by CP. thrombin was prepared as described in â€œMaterialsandMethodsâ€•andadded at equivalent These results clearly indicate that PC-3 prostate cancer cells have the protein concentrations (2 units/mI). Hirudin (2 units/mI), antithrombin III (0.2 unit/ml), same thrombin receptor as endothelial cells and platelets, and the TRAP and heparin (0.2 unit/ml) were incubated with 2 units/mI thrombin for 30 mm at 37Â°C before addition to the medium. Points, mean of quadruplicate determinations from a single could bypass the protease action of thrombin to activate the thrombin experiment, representative of four separate experiments. Bars, SD. receptor on PC-3 cells, leading to an increased uPA expression.

DISCUSSION

Tumor cells and associated mononuclear phagocytes have been found to possess procoagulant activity (4, 17). These facts imply that >. thrombin could be generated in the tumor microenvironment through their procoagulant activity. The generated thrombin can convert fi C., brinogen to fibrin, which helps stromal formation and angiogenesis (18) in tumortissue,andstimulatetumorcellproliferation;alsoin the present study, we demonstrated that thrombin could stimulate uPA expression by cancer cells through thrombin-specific receptor activa tion. Although PC-3 cells are high uPA producers, a further increase in uPA production by thrombin would overwhelm its inhibitors, thus enhancing the breakdown of the extracellular matrix. Our findings indicate that thrombin showed cell-type specificity. 5 20 50 50 lOOUM L I j Thrombin enhances uPA production by PC-3 cells but did not affect SFLL FSLL uPA expression by low or non-uPA producers, DU-145 and LNCaP. Fig. 7. Effect of TRAP on uPA secretion by PC-3 cells. TRAP (SFLL) and CP (FSLL) A cell type-specific and enhancer-dependent silencer was identified of indicated concentrations were added to the culture, and 6 h later, the uPA antigen level in the medium was measured by ELISA. iOO%is defined as the uPA level induced by 2 and found to be active in some cell lines which produce little uPA but units/mI thrombin. 0% is the uPA level produced by nonstimulated PC-3 cells. was not active in the high uPA producer, PC-3 (19). It is assumed that 3303

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. THROMBIN INDUCtiON OF uPA FROM CANCER thrombin does not affect the transcriptional silencer of the uPA gene REFERENCES but stimulates the action of the enhancer. This may be a reason for the 1. Dvorak, H. F., Dickersin, G. R., Dvorak, A. M., Manseau, E. J., and Pyne, K. Human cell-type specificity of thrombin effect. Another reason may be that breast carcinoma: fibrin deposits and desmoplasia inflammatory cell type and DU-145 and LNCaP cells do not have a thrombin receptor. distribution. Microvasculature and infarction. J. Natl. Cancer Inst., 67: 335â€”345, In this study, 10 units/ml thrombin caused less uPA expression than 1981. 2. Zacharski, L R., Schned, A. R., and Sorenson, 0. D. Occurrence of fibrin and tissue 1â€”2units/ml thrombin. The reason for this is not fully understood. factor antigen in human small cell carcinoma of the lung. 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To our knowledge, cancer cells have amino-terminal fragment of urokinase isolated from a prostate cancer cell line (PC-3) not been shown to possess a thrombin receptor. In PC-3 cells, TRAP is mitogenic for osteoblast-like cells. Biochem. Biophys. Rca. Commun., 173: reproduced the same effect as intact thrombin on uPA expression, 1058â€”1064,1990. 10. Pedersen, N., Masuccin, M. T., Moller, L B., and Blasi, F. A noncatalytic human while CP in which the first two amino acids are transposed had no urokinase plasminogen activator derivative produced by mouse cells has full receptor effect. This successful effect of TRAP indicates that a cancer cell line, binding activity. Fibrinolysis, 5: 155â€”164,1991. PC-3, has a thrombin receptor similar to that on platelets and endo 11. Vu, T. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor thelial cells. Thus, our observations strongly suggest that thrombin activation. Cell, 64: 1057â€”1068,1991. generated in the vicinity of cancer cells can modulate cancer cell 12. Kozlowski, J. M., McEwan, R., Keer, H., Sensibar, J., Sherwood, E. R., Lee, C., behavior through stimulation of a thrombin-specific receptor. Grayhack, J. T., Albini, A., and Martin, G. R. Prostate cancer and the invasive Thrombin can convert scuPA to inactive tcuPA; but this phenom phenotype: application of new in vivo and in vitro approaches. In: L J. Fidler and G. Nicolson (eds.), Tumor Progression and Metastasis, pp. 189-231. New York: Alan R. enon may not be signfficant for these reasons: (a) prolonged presence Liss, Inc., 1988. of the active enzyme is unlikely during the time from thrombin 13. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of receptor stimulation to uPA secretion because of an excess amount of bacteriophage T4. Nature (Lund.), 227: 680â€”685,1970. 14. Ichinose, A., Fujikawa, K., and Suyama, T. The activation of prourokinase by protease inhibitors in plasma and extra cellular fluids; (b) even on the plasma kallikrein and its inactivation by thrombin. J. Biol. (1cm., 261: 3486â€”3489, surface of a single cell, the distribution of uPA receptor and of the 1986. tissue factor is thought to be different, thus a thrombin generation site 15. Nauland, U., and Rijken, D. C. Activation of thrombin-inactivated single-chain urok.inase-type plasminogen activator by dipeptidyl peptidase I (cathepsin C). could be different from a uPA functioning site; and (c) inactive tcuPA Thromb. Haemostasis, 69: 823, 1993. formed by thrombin can be reactivated by dipeptidylaminopeptidase 16. Tulinsky, T., and Qiu, X. Active site and exosite binding of athrombin. Blood Coagul. (15). In conclusion, our findings provide a new insight and implica Fibrinol., 4: 305â€”312,1993. tion of the role of thrombin in the regulation of pericellular proteolysis 17. Rambaldi, A., Alessio, G., Casali, B., Passserini, C., Donati, M. B., Mantovani, A., and Semeraro, N. Induction of monocyte macrophage procoagulant activity by of tumor cells through modulation of uPA expression. transformed cell lines. J. Immunol., 136: 3848â€”3855, 1986. 18. Zacharski, L R., Wojtukiewicz, M. Z., COstantini, V., Ornstein, D. L, and Memoli, V. A. Pathways ofcoagulation/flbrinolysis activation in malignancy. Semin. Thromb. ACKNOWLEDGMENTS Haemostasis, 18: 104â€”116,1992. 19. Cannio, R., Rennie, P. S., and Blasi, F. A cell-type specific and enhancer-dependent We are grateful to Dr. Gerald Soff and Deborah L. Cundiff for valuable silencer in the regulation of the expression of the human urokinase plasminogen advice and technical help and Bonnie Barone for manuscript preparation. activator gene. Nucleic Acids Res., 19: 2303â€”2308,1991.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. Enhancement of the Expression of Urokinase-Type Plasminogen Activator from PC-3 Human Prostate Cancer Cells by Thrombin

Etsuo Yoshida, Elaine N. Verrusio, Hisashi Mihara, et al.

Cancer Res 1994;54:3300-3304.

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