THROMBIN INDUCES APOPTOSIS IN HUMAN TUMOR CELLSRasheed AHMAD, Laurent KNAFO, Jingwu XU, Sardar T.A.K. SINDHU, Jose´ MENEZES and Ali AHMAD*

Laboratory of Immunovirology, Pediatric Research Center and Department of Microbiology and Immunology, University ofMontreal and Sainte-Justine Hospital, Montreal, Quebec, Canada

Thrombin is a serine protease that is produced during thecoagulation process and plays an essential role for hemosta-sis, thrombosis and wound healing. It is a potent activator ofplatelets, induces proliferation of a wide variety of normaland malignant human cells, and enhances their invasivenessand metastatic potential. We studied the effect of thrombinon the proliferation of a wide variety of human tumor cellsand report here that, at low concentrations, thrombin in-duces proliferation of these cells. However, at higher concen-trations, thrombin inhibited their proliferation. We showthat this inhibition of cell proliferation was due to apoptosisof the tumor cells. The thrombin-mediated apoptosis wasinhibited significantly by its specific inhibitor, hirudin. Fur-thermore, no consistent pattern of induction and/or modu-lation of p53, p21 and bcl-2 was observed in the thrombin-mediated apoptosis. To our knowledge, this is the first reportto describe the pro-apoptotic effects of thrombin on humantumor cells and may have implications for chemotherapy incancer patients and for the pathogenesis of AIDS as well. Int.J. Cancer 87:707–715, 2000.© 2000 Wiley-Liss, Inc.

Thrombin is a multifunctional serine protease that is generatedin vascular injury as part of the coagulation process (reviewed byHou et al.,1998; Brass, 1995). It is the most abundant product ofthis process. Thrombin is a potent activator of platelets and inducestheir aggregation and secretion (Brass, 1995). In addition to its rolein hemostasis and coagulation, thrombin has numerous other bio-logical activities which affect inflammation, immune responses,tissue repair and wound healing (Houet al.,1998; Brass, 1995).These biological effects of thrombin are mediated via so-called“thrombin receptors”. At least, three such receptors on varioushuman cells have been identified and their genes cloned (Ishiharaet al., 1997; Kahnet al., 1998; Xu et al.,1998; Vu et al.,1991),which constitute a specialized group of seven transmembranedomain-containing G-protein-coupled receptors that carry a teth-ered peptide ligand in their extracellular N-terminal part. Thesereceptors become activated by proteases (e.g., thrombin) thatcleave in the extracellular region and release the tethered ligandpeptide that is at the N-terminus of the cleaved receptor andinteracts with the receptor and transduces the signal (reviewed byDery et al.,1998; Coughlin, 1998; Donovan and Cunningham,1998; Coughlinet al.,1992). For this reason, these receptors arealso called protease-activated receptors (PAR). At present, fourPARs,i.e., PAR1-PAR4 have been identified (Ishiharaet al.,1997;Kahn et al.,1998; Xuet al.,1998; Vuet al.,1991). Three of thesei.e., PAR1, 3 and 4 are activated by thrombin, and serve as itsreceptors whereas PAR2 is activated by trypsin or trypsin-likeproteases. Each PAR has distinct tethered ligand peptide (Houetal., 1998). It is noteworthy, however, that most of the cellulareffects of thrombin can be explained by activation of PARs.

Thrombin has several effects on human cells. It is mitogenic andinduces proliferation of several cell types,e.g., smooth muscles,fibroblasts, endothelial cells, monocytes and certain tumor cells(Reilly et al.,1993; Derianet al.,1997; Guttridgeet al., 1997;Anratheret al., 1997). Thrombin-treated tumor cells have at leasttwofold higher lung colonizing ability and the expression ofthrombin receptors has been correlated with both the physiologicaland malignant invasive processes (Even-Ramet al., 1998; Ni-erodzik et al.,1998; Wojtukiewiczet al.,1993; reviewed in Walzand Fenton, 1995). Thrombin induces adhesion of tumor cells,neutrophils, and monocytes to endothelial cells by increasing theexpression of the integrina11bb3 in these cells (Wojtukiewiczet

al.,1992; Nierodziket al., 1998; Klepfishet al.,1993). On endo-thelial cells, thrombin induces expression of ICAM-1 (CD54) andVCAM-1 (CD106) via activation of NF-kB (Kaplanskiet al.,1998;Rahmanet al.,1999). It also increases the cytolytic potential of NKcells and induces the secretion of several cytokines and chemo-kines from monocytes, endothelial cells and fibroblasts (Naldiniand Carney, 1996; Soweret al.,1995). Despite these diverse bio-logical effects of thrombin on a variety of human cells, its apop-tosis-inducing effects on human tumor cells have not been re-ported. Apoptosis or programmed cell death is a well-regulatedphysiological process of cell death, which is essential for mainte-nance of normal cellular homeostasis during embryonic develop-ment, thymic selection and immune regulation (Rathmell andThompson, 1999; Raff, 1999). In recent years, significant progresshas been made in understanding the mechanism and regulatorycontrol of apoptosis. We report here the induction of apoptosis bythrombin in a wide variety of human tumor cell lines, which mayhave important implications for cancer patients.

MATERIAL AND METHODS

Cell culture, lines and reagentsThe different human tumor cell lines used in this study and their

origins are listed in Table I. These tumor cells were cultured in theculture medium,i.e., RPMI-1640 containing 10% heat-inactivatedfetal bovine serum (FBS), penicillin G (100 U per ml; NovapharmLtd., Toronto, Canada), gentamycin (1mg per ml; Schering Can-ada, Pointe Claire, Canada), and fungizone (2mg per ml; SquibbCanada, Montreal, Canada), unless indicated otherwise. Alphathrombin and its inhibitor hirudin were obtained from BoehringerMannheim (Laval, Quebec, Canada) and Sigma (St. Louis, MO),respectively.

Cell proliferation assayThe cell proliferation was measured by3H-thymidine uptake as

described in our earlier publications (Ahmadet al.,1993; Ahmadand Menezes, 1997). Briefly, aliquots of 23104 cells were incu-bated in the wells of a 96-well microculture plate in 200ml volumeof the culture medium with or without the addition of thrombin. Insome experiments, the microcultures were pre-incubated with asignal transduction pathway inhibitor with appropriate controls.After 15 hours’ incubation at 37°C in 5% CO2, the microcultureswere pulsed with 1mCi of 3H-thymidine (specific activity 20Ci/nmol; ICN, Montreal, Canada) in 20ml volume. The 3H-thymidine uptake was measured 6 hours later by cell harvestingand liquid scintillation counting as described earlier (Ahmadet al.,1993). For cells that grow in monolayers, microculture plates withflat bottom wells were used. The procedure was the same asdescribed above for cells growing in suspension except that fol-lowing pulsing with3H-thymidine, the wells were washed exten-sively with cold PBS. Then cells, in individual wells, were lysed

Grant sponsor: Medical Research Council of Canada (MRC).

*Correspondence to: Ali Ahmad, D.V.M., Ph.D., Laboratory of Immu-novirology, Center of Research, Hopital Sainte-Justine, 3175 Cote Ste-Catherine, Montreal, H3T 1C5, Canada. Fax: 514-345-4801.E-mail: ahmada@justine.umontreal.ca

Received 19 November 1999; Revised 6 March 2000

Int. J. Cancer:87, 707–715 (2000)© 2000 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

with 150 ml of 2% NaOH and 100ml of each lysate was mixedwith 3 ml of a scintillation cocktail (Cytoscint; ICN) and radioac-tivity counted in a liquid scintillation counter as described (Ahmadand Menezes, 1997).

Detection of DNA fragmentationInternucleosomal DNA fragmentation, which occurs in apopto-

tic cells, was detected as described earlier (Luet al.,1994). Briefly,23106 cells were cultured with or without the presence of throm-bin for 24 or 48 hours at 37°C in 5% CO2. At the end of this cultureperiod, cells were washed with PBS and their DNA was extractedusing a commercial DNA extraction kit (Catalog no. D5500;Gentra Systems, Minneapolis, MN) and run on 1.8% agarose gel.The gels were stained with ethidium bromide and photographed.The appearance of the characteristic “DNA ladder” representingmono- and oligo-nucleosomal DNA fragments (discrete multiplesof 180-bp subunits) indicated the occurrence of apoptosis in thesecultures.

We also used a commercial photometric sandwich-enzyme im-munoassay kit (Cell Death Detection Kit; Boehringer Mannheim)for the quantitative determination of cytoplasmic histone-associ-ated mono- and oligonucleosomal DNA fragments that accumulatein the cytoplasm of the apoptosed cells. For this purpose, 53104

control and thrombin-treated cells were lysed in 500ml of the lysisbuffer (provided in the kit) and centrifuged at 20,000g for 109 at4°C. Four hundred microliters of the clear upper cytoplasmicfraction was removed and used for measuring DNA fragmentsfollowing the manufacturer’s recommendations. Two antibodiesare used in this assay: the first antibody is a mouse monoclonalantibody (mAb) that recognizes histones, and the second antibodyis a horseradish peroxidase (HRPO)-conjugated mouse anti-DNAmAb. The DNA fragments are quantitated by measuring HRPOincorporated into the immune complexes by the chromogenicsubstrate ABT (2-29-azino-di-[3-ethylbenzthiazoline sulfate]. Thefinal reaction product in this procedure is quantitated by measuringabsorbance at 405l. The results are expressed as the enrichmentfactor (for mono- or oligonucleosomal DNA), which are deter-mined by the following formula:

Enrichment factor

5OD of the sample~dying or dead cells!

OD of the corresponding control~viable cells!

Western blotsIn order to determine whether thrombin treatment of the human

tumor cells had any effect on apoptosis-related proteins p53, bcl-2or p21, Western blots were performed as described earlier (Khyattiet al.,1998). Briefly, 53106 cells (with or without incubation with

thrombin) were lysed 24 to 30 hours later in 500ml of the lysisbuffer containing Tris HCl (pH 6.8; 10 mM), and SDS (2%), andsonicated for 15 seconds on ice. The lysates were clarified bycentrifugation at 4°C (15,000g for 209). Protein concentrations ofthe lysates were determined using a commercial kit (Biorad, Rich-mond, VA) and 50–70mg of the lysate proteins were resolved on15% SDS-PAGE. After electroblotting onto nylon membranes,unbound sites were blocked with PBS containing 5% skim milkpowder and 0.05% Tween 20. The membranes were then incu-bated with primary antibodies specific for bcl-2 (mouse monoclo-nal of IgG1 isotype; Catalog no. SC-509), for p53 (polyclonalrabbit antibodies; Catalog no. SC-6243), or for p21 (goat poly-clonal antibodies; Catalog no. SC397) at the manufacturer’s rec-ommended dilutions. All these three antibodies were purchasedfrom Santa Cruz Biotech. Inc. (Santa Cruz, CA). Protein bandswere revealed using alkaline phosphatase (AP-) conjugated anti-mouse (Sigma), anti-rabbit or anti-goat antibodies (both fromPromega, Madison, WI) and chromogenic substrates NBT andBCIP (Promega) following the manufacturer’s recommendations.

Ethidium bromide stainingThe apoptotic cells can be stained with ethidium bromide, which

can be detected by flow cytometry as a function of forward scatter.The apoptotic cells shrink in size and consequently have decreasedforward scatters as compared to the viable or necrotic cells. Forthis staining, thrombin-treated and untreated cells were tested forethidium bromide staining as described earlier (Luet al., 1994).Briefly, 13106 cells were washed with cold PBS, and resuspendedin 1 ml of PBS containing 1mg/ml of the dye and 1mg/ml ofRNAse A. After 309 incubation on ice, the cells were washed andresuspended in 1% paraformaldehyde and examined by flow cy-tometry. A total of 10,000 cells were analyzed by FACScan withlogarithmic amplification of red fluorescence (X-axis) and linearamplification of the forward scatter (Y-axis).

Statistical analysisThe comparison between group means (thrombin-treated and

untreated cells) was made using Student’st-test as described in ourearlier publication (Ahmadet al.,1993). Differences were deemedsignificant atp # 0.05.

RESULTS

Induction of DNA fragmentation in thrombin-treated cellsIn the course of studying effects of thrombin-activated human

platelets on tumor cells, we incubated these cells in the presence ofthrombin as controls. After 48 hours’ incubation, we found thatthrombin treatment induced mono-and oligonucleosomal DNAfragmentation in these cultures. These fragments were visible as

TABLE I – CELL LINES USED IN THIS STUDY AND THEIR ORIGIN

Tumor cell line Origin Reference

HL-60 Promyelocytic Collinset al. (1997)U937 Histocytic (monocytoid) Ralphet al. (1976)K562 Erythroleukemic Lozzio and Lozzio (1975)BL41 Burkitt lymphoma EBV genome-negative Cherneyet al. (1998)E 6-1 T cell leukemic Weisset al. (1984)CA 46 Burkitt lymphoma EBV genome-negative Cherneyet al. (1998)1301 ALL, null cell line Khelifa and Menezes (1982)CEM-NKr T-lymphoblastoid Howellet al. (1985)Jurkat ALL, T cell origin Walkeret al. (1987)LCL In vitro EBV transformed B cells Cherneyet al. (1998)Raji Burkitt lymphoma EBV genome-positive Epsteinet al. (1966)Daudi Burkitt lymphoma EBV-genome-positive Kleinet al. (1968)HeLa Epithelioid carcinoma Joneset al. (1971)Caco-2 Adenocarcinoma (epithelial-like) Foghet al. (1973)MCF-7 Breast adenocarcinoma Souleet al. (1973)MDA 231 Breast adenocarcinoma Cailleauet al. (1974)KSY-1 Kaposi’s Sarcoma, endothelial origin Lunardi-Iskandaret al. (1985)

708 AHMAD ET AL.

multiples of 180 bp (typical ladders) on ethidium bromide-stainedagarose gels. Following this surprising result, we sought to deter-mine the minimum concentration of thrombin that was needed toinduce apoptosis in these cells. For this purpose, we incubatedCEM.NKr cells with different concentrations of thrombin andexamined these cells 48 hours later for apoptosis. As shown inFigure 1, 0.3 U/ml and higher concentrations of thrombin inducedapoptosis in these cells. Using this concentration of thrombin (i.e.0.3 U per ml), we investigated whether this phenomenon wasspecific to CEM.NKr cells or other human tumor cells were alsosusceptible to the thrombin-induced apoptosis. As shown in Figure2, a wide variety of human tumor cells underwent apoptosis whencultured in the presence of thrombin for 48 hours. We found thatit was necessary to incubate cells with thrombin for more than 24hours for the induction of apoptosis. At 24 hours, only a few celllines (BL41, CA46, K562) underwent apoptosis (data not shown).Interestingly, some cell lines (MCF-7, Daudi, Raji, HeLa, andMDA 231, LCL, PBMC with or without prior activation with IL-2)did not show signs of apoptosis even after 48 hours incubation asdetermined by the DNA fragmentation assay. However, most ofthese cell lines showed apoptosis when tested by other methods(see below).

Inhibition of thrombin-induced apoptosis by hirudinHirudin is a naturally-occurring principal anticoagulant pro-

duced by the medicinal leech Hirugo medicinalis and is a specificinhibitor of thrombin (Kaplanskiet al., 1998; Vergnolleet al.,1999). In order to see that the thrombin-induced apoptosis wasspecific to thrombin and not due to any accompanying impurity inthis product, we pre-incubated thrombin with hirudin (0.3 U ofthrombin with 10.0 U of hirudin) before its addition to the cellcultures. As tested by DNA fragmentation assay (Fig. 3), hirudinsignificantly abrogated the apoptotic effect of thrombin. These datastrongly suggest that DNA fragmentation in human tumor celllines is specifically induced by thrombin.

Detection of apoptosis in tumor cells by ELISAAs the DNA fragmentation assay for the detection of apoptosis

described above is not very sensitive, we used a photometricenzyme assay based commercial kit (Death Detection Kit; Boehr-inger Mannheim) to quantitate the enrichment of histone-associ-

ated mono- and oligonucleosomal DNA fragments in the cyto-plasm of thrombin-treated cells. The enrichment factor for varioushuman tumor cell lines is shown in Table II.For viable cells, thisfactor does not exceed 1. It is noteworthy that several tumor celllines (e.g., Daudi, Raji, HeLa) that did not show apoptosis in DNAfragmentation assay (described above) were positive for apoptosisin this assay. Furthermore, these cell lines also showed positivityfor apoptosis by ethidium bromide staining (see below). Onlyhuman PBMC (with or without prior activation with IL-2) andEpstein-Barr virus (EBV)-transformed human lymphoblastoid celllines (LCL) showed no apoptosis (which was further confirmed bythe ethidium bromide staining; see below).

Ethidium bromide stainingIt is well known that cells undergoing apoptosis retain ethidium

bromide (Luet al.,, 1994). As an additional test for apoptosis, westained thrombin-treated and untreated cells with this nuclear dyeas described in Material and Methods. The thrombin-treated tumorcell lines show a clear shift of red fluorescence to the right with aconcomitant decrease in forward scatter (due to cell shrinkage; Fig.4). It is clear from these data that most of the cell lines undergoapoptosis after 48 hours of incubation with thrombin. Consistentwith the results from the commercial apoptosis detection kit (TableII), all cell lines, except for LCL and human PBMC, showedapoptosis upon incubation with thrombin. These data further con-firm that thrombin induces apoptosis in a wide variety of humantumor cells.

Thrombin-induced morphological changes in the tumor cellsTumor cells cultured in the presence of thrombin showed mor-

phological changes suggestive of apoptosis. The adherent cellsgrowing in monolayers became gradually rounded, detached andshrank in size (Fig. 5).

Effect of thrombin on cell proliferationSince it is known that thrombin is mitogenic and induces pro-

liferation in several types of cells, especially at low doses, westudied the effect of different concentrations of thrombin on theproliferation of these human tumor cell lines. The results fromseveral experiments are shown in Figure 6. It is noteworthy that at

FIGURE 1 – Effect of different thrombin concentrations on DNA fragmentation. Two million CEM.NKr cells were cultured with differentconcentrations of thrombin (0–0.5 U/ml) and 48 hours later DNA was extracted from these cells and run on 1.8% agarose, stained with ethidiumbromide and photographed. The cell cultures containing 0.3 U per ml or higher concentration of thrombin show a clear “ladder-like” pattern ofinternucleosomal DNA cleavage. Shown here are cell cultures without serum (positive control; Lane 2), with 10% FCS (negative control; Lane3), and cell cultures with 0.5, 0.3, 0.15, 0.08, 0.04 and 0.02 units per ml of thrombin (Lanes 4-9, respectively). Lane 1 shows DNA size markers.

709THROMBIN INDUCES APOPTOSIS IN HUMAN TUMOR CELLS

lower doses, thrombin tends to induce cell proliferation while athigher doses, it suppresses cell proliferation by inducing apoptosis.

Effect of thrombin on apoptosis-related cellular proteinsSeveral cellular proteins,e.g., p53, bcl-2, p21/WAF-1/Cip1 are

known to be induced and/or modulated in the process of apoptosis(Rathmell and Thompson, 1999; Raff, 1999). Therefore, we stud-ied the expression profiles of these proteins by Western blots inthrombin-treated and untreated cells. As shown in Figure 7a, 1301cells constitutively express p53 and thrombin treatment did notincrease its expression in these cells. On the contrary, p53 levelswere clearly decreased in thrombin-treated cells. The cyclohexi-mide treatment reduced these levels in both thrombin-treated anduntreated cells (Fig. 7a). The p21 expression was induced inKSY-1 cells upon treatment with thrombin (Fig. 7b). However, itcould not be detected in both thrombin-treated and untreatedCEM.NKr cells (which are highly susceptible to the thrombin-induced apoptosis; data not shown). Finally, the expression of theanti-apoptotic protein bcl-2 was somewhat increased in U937 and1301 cells upon thrombin treatment (Fig. 7c). In U937 cells, thethrombin treatment (at 0.3 U per ml and higher concentrations)induced the appearance of a protein band migrating at approxi-mately 15 kDa (see Fig. 7c, Lane 4), which is reactive to theanti-bcl-2 mAb. The exact identity of this protein is under inves-

tigation. Similar results were obtained in Western blots whenthrombin was used at higher concentrations,i.e., (0.3 U per ml;data not shown). These data suggest that these proteins may not bedirectly involved in the thrombin-induced apoptosis of humantumor cells.

TABLE II – DETECTION OF THROMBIN-INDUCED APOPTOSIS BY ELISA1

Cell lineEnrichment factor

24 hours 48 hours

Hela 1.63 2.27Caco2 1.32 2.22KSY-1 3.38 2.23CA46 2.46 1.751301 6.45 8.42Raji 5.70 5.65LCL 1.68 1.37Daudi 6.94 3.22B41 4.16 1.67U937 2.12 13.69E6.1 1.95 2.43HL60 15.00 3.58PBMC 0.99 0.98IL-2 activated PBMC 0.87 0.891Apoptosis was detected using a commercial kit (Cell Death Detec-

tion ELISA; Boehringer Mannheim). Cell were lysed 24 and 48 hoursafter culture in the presence or absence (controls) of thrombin, and celllysates equivalent to 103 cells were used following the manufacturer’srecommendations. The enrichment factor, shown here, denotes theaccumulation of mono- and oligo-nucleosomal DNA fragments in thecytoplasm of thrombin-treated cells as compared to the untreated cells.The value of this factor for the apopotosing cells should exceed 1.00depending upon the extent of DNA fragmentation.

FIGURE 2 – Induction of DNA fragmentation by thrombin in differ-ent human tumor cells. Different human tumor cell lines (indicatedbelow) were cultured for 48 hours in the presence of 0.3 U per ml ofthrombin. After this incubation period, DNA was extracted from cells,run on 1.8% agarose, stained with ethidium bromide and photo-graphed. Each number on top of a panel designates a cell line. The firstlane for each cell line shows DNA from the culture without thrombinwhile the second lane shows DNA from cells cultured in the presenceof thrombin (0.3 U/ml). (a) The numbers represent cell lines as 2:E6.1, 3: CA46, 4: 1301, and 5: CEM.NKr. (b) These numbers represent2: HL60, 3: U937, 4: K562, 5: BL41, and 6: Jurkat. The first panel(numbered 1) in both these figures show DNA size markers, positiveand negative controls as described in the Figure 1 legend.

FIGURE 3 – Inhibition of thrombin-induced apoptosis by hirudin. Inorder to see that thrombin-induced apoptosis in human tumor cells wasdue to thrombin and not due to any accompanying impurity in theproduct, thrombin was pre-incubated with hirudin (a specific inhibitorof thrombin; 0.3 U of thrombin with 10.0 U of hirudin) on ice for 15minutes and then added to the cell culture. After 48 hours, the cellcultures were terminated and processed for the DNA fragmentationassay as described in the Figure 1 legend. Lane 1 shows DNA sizemarkers. Lanes 2 and 3 represent negative and positive controls,respectively, as described in the legend to Figure 1. Lane 4: cellscultured in the presence of thrombin (0.3 units per ml); Lane 5: in thepresence of hirudin-neutralized thrombin, and Lane 6: in the presenceof hirudin.

710 AHMAD ET AL.

FIGURE 4 – Detection of thrombin-induced apoptosis in human tumor cells by ethidium bromide staining. Two million cells were cultured for24 or 48 hours with or without thrombin (0.3 U per ml). After this incubation, the cells were stained with ethidium bromide and examined forred fluorescence and forward scatter, which are shown on Y-axis (linear scale) and X-axis (legarithmic scale), respectively. For each cell line,the left and right panels indicate cells cultured in the absence, and in the presence of thrombin, respectively. (a) Cells stained 24 hours afteraddition of thrombin. (b) Cells stained after 48 hours. The apoptosing cells show a decrease in cell size (forward scatter on Y-axis and increasein red fluorescence on X-axis), and consequently tend to move in the B gate.

711THROMBIN INDUCES APOPTOSIS IN HUMAN TUMOR CELLS

DISCUSSION

We have shown here that at lower concentrations, thrombin ismitogenic and induces proliferation of a wide variety of humantumor cell lines but, at relatively higher concentrations, it inhibitstheir proliferation by inducing apoptosis in these cells. Apart frombeing the most important enzyme of the coagulation cascade,thrombin is known to exert a wide range of biological effects onboth normal as well as on malignant human cells (Houet al.,1998;Brass, 1995). However, to our knowledge, this is the first report todescribe pro-apoptotic effects of thrombin on a wide variety ofhuman tumor cells. Previously, the apoptosis-inducing effects ofthrombin have been shown only in neurons and astrocytes (Don-ovan and Cunnigham, 1998; Smirnovaet al.,1998; Donovanetal.,1997; Akiyamaet al.,1992; Choiet al.,1995). In these studies,the pro-apoptotic effect of thrombin was only visible at muchhigher concentrations (40–100 U/ml) than those used in our study(0.5 U/ml). At lower concentrations, thrombin is also mitogenic forthese nervous tissue cells and protects them from oxidative stress,cytotoxicity of b-amploid, hypoxia and withdrawal of growthfactors (Debeiret al.,1996a,b). Schafberget al., (1997) recentlyreported quite opposite effects of thrombin on a rat glioma cell lineC6, i.e. it was mitogenic at high concentrations and apoptotic atlower concentrations. These effects of thrombin may be unique tothis rat cell line as we could not find any other report corroboratingthese results. Our results are also in agreement with those reportedfor neurons and astrocytes (Donovan and Cunningham, 1998;Smirnovaet al.,1998; Donovanet al.,1997; Debeiret al.,1996a,b).

In human cancer patients, disorders of thrombosis due to higherthrombin activities have been well documented (reviewed in Walz

and Fenton, 1995). Since thrombin is also known to enhance tumormetastasis and proliferation, such patients are usually treated withanti-clotting (thrombin-neutralizing) agents,e.g., heparin, warfa-rin, synthetic hirudin analogs, etc. (Walz and Fenton, 1995). Thesetreatments, however, do not show consistent beneficial effects(Walz and Fenton, 1995). The results presented here suggest thatenhanced thrombin activities in cancer patients may be beneficialfor them as thrombin may cause apoptosis of tumor cells unless thepatient faces immediate threat from intravascular thrombosis.These results also explain the earlier observations that prolongedexposure (.16 hours) of tumor cells to thrombin decreased theirinvasiveness (Wojtukiewiczet al.,, 1993). However, further workwill be needed to determine whether thrombin can also induceapoptosis of tumorsin vivo.

It is noteworthy that our results show that thrombin can mediateapoptosis of tumor cells at much lower levels,i.e. 80–200-foldlower than those needed for the induction of apoptosis in neurons(0.3 U/ml for tumor cells compared with 40–100 U/ml for neu-rons). These thrombin concentrations required to induce apoptosisof tumor cells are the ones needed for most of its physiologicalactivities (Brass, 1998).

We found that prolonged exposure of tumor cells to thrombin (atleast for 24 hours) was necessary to induce apoptosis. At 24 hours,only a few cell lines showed apoptosis whereas most of the celllines showed apoptosis at 48 hours. Interestingly, similar observa-tions were recently reported for neurons and astrocytes (Smirnovaet al., 1998; Donovanet al.,1997). The reasons for the need ofprolonged exposure to thrombin for the induction of apoptosis arenot clear. Since thrombin-activated receptors have tethered peptide

FIGURE 5 – Thrombin-induced morphological changes in human tumor cells. Shown here are the morphological changes in human tumor cellswhen grown in the presence of thrombin (0.3 U per ml). Addition of thrombin to the cells growing in monolayers causes them to becomerounded, detached, shrunk in size and vesiculated. (a,c) KSY-1, and HeLa cells, respectively, cultured in the absence of thrombin. (b,d) Samecells respectively cultured in the presence of thrombin. The pictures were taken 24 hours after the addition of thrombin.

712 AHMAD ET AL.

ligands, they do not require exogenous ligands for signal trans-duction (Deryet al., 1998). These tethered ligands theoreticallymay continuously transduce signals and mimic thrombin effectseven if thrombin is removed or becomes degraded in the medium.To avoid this continuous signaling, cells have evolved novelmechanisms of desensitization to thrombin (Hammes and Cough-lin, 1999, reviewed in Deryet al.,1998). During this desensitiza-tion period, cells become refractory to the effects of thrombin.Thrombin may essentially be acting as a growth factor for thesetumor cells (as it does for myoblasts; Chinniet al.,1999) andprolonged desensitization periods may deprive cells of this sur-vival factor prompting them to undergo apoptosis. Another possi-bility is that thrombin may be inducing apoptosis independently ofits effects through activation of PARs. It may be important tomention here that the peptide SFLLRN was not able to induceapoptosis in the tumor cells at up to 250mM concentrations (ourunpublished data). Although most of the physiological effects ofthrombin can be explained through activation of PARs, somebiological effects of thrombin can not be explained by PARactivation (reviewed in Houet al.,1998). Although thrombin re-ceptor activating peptide (or TRAP; SFLLRN) can induce apopto-sis in neurons (Donovanet al.,1997), it does not exclude thepossibility of a direct PAR-independent effect of thrombin or of itsbreak down products. Thrombin contains an RGD peptide (Walzand Fenton, 1995) and it was shown recently that RGD peptides(break down products from extracellular matrix proteins) can entercells and cause direct activation of caspases by interacting withRGD-motifs of these caspases (Buckleyet al.,1999). The longerexposures for thrombin-mediated apoptosis may simply be neededfor enough thrombin degradation to generate an effective pool ofRGD peptides. Further studies are needed to test these hypotheses.Our results also show that there is no induction of p53 and/orconsistent pattern of induction or modulation of bcl-2 and p21 incells undergoing thrombin-induced apoptosis. Many apoptoticstimuli, e.g., anti-cancer drugs, UV light and viral transforming

proteins mediate and/or modulate apoptosis by their effects on oneor more of these proteins (Rathmell and Thompson, 1998; Raff,1999). However, it should be noted that bcl-2 belongs to a largefamily of proteins with either pro-apoptotic or anti-apoptotic ef-fects and it is the balance between these two kinds of proteins thatdetermines cell survival or apoptosis. Many models of apoptosisdo not involve changes in the expression of bcl-2 (reviewed inYang and Korsmeyer, 1996). Further studies will be needed todetermine the role of other members of this family in the thrombin-induced apoptosis.

In conclusion, we have demonstrated here for the first time thatthrombin induces apoptosis of a wide variety of lymphoid andnon-lymphoid human tumor cells at physiologically relevant con-centrations. Further studies to investigate whether thrombin canmediate these anti-tumor effectsin vivo would be highly desirableand may have important implications for chemotherapy of cancers.

ACKNOWLEDGEMENTS

We thank Medical Research Council of Canada (MRC) forfinancial support. A.A. is a recipient of the MRC Scholar Award.The excellent secretarial assistance from Micheline Patenaude andtechnical assistance in FACS analysis from Mary Blagdon aregratefully acknowledged.

FIGURE 6 – Effect of thrombin on cell proliferation. 23105 cellswere cultured in triplicate in the wells of a 96-microculture plate withdifferent concentrations of thrombin (indicated on the x-axis). After 16hours of incubation at 37°C, the microcultures were pulsed for 8 hourswith 1 mCi/well of 3H-thymidine. The microcultures were harvestedand3H-thymidine uptake was determined by liquid scintillation count-ing as described in Material and Methods. The average cpm6 stan-dard error of a triplicate microculture for each cell line at differentthrombin concentrations are shown. Similar results were observed withKSY-1, HL60, BL41, HeLa, E6.1 and MCF-7 cells.

FIGURE 7 – Expression of p53, bcl-2 and p21 in thrombin-treatedtumor cells. Five million cells were cultured for 24–30 hours with (0.3U per ml) or without thrombin. The cells were then washed with PBS,lysed in the lysis buffer (50 mM Tris-HCl, pH 6.8 and 2% SDS) andsonicated on ice for 15 seconds. Equal amount of proteins (50–75mg)were resolved on SDS-PAGE (12%) and electroblotted onto nylonmembranes. After blocking, blots were developed using protein-spe-cific primary antibodies and AP-conjugated secondary antibodies asdescribed in Material and Methods. (a) Expression of p53 in 1301cells. Lanes: 1, untreated; 2, thrombin-treated; 3, cycloheximide-treat-ed; and 4, thrombin-and cycloheximide-treated. (b) Expression of p21in KSY-1 cells. The lanes represent: 1, cells cultured without throm-bin; 2, with 0.1 units per ml of thrombin; 3, with 0.3 units per ml ofthrombin; and 4, cells without thrombin but exposed to UV for 1 hour.(c) Expression of bcl-2 in 1301 and U937. The lanes represent U937cells cultured in the absence of serum (1), in the presence of 10% FBS(2), in 0.1 U per ml of thrombin (3), in 0.3 U per ml of thrombin (4),1301 cells in 10% FBS (5), in 0.1 U per ml thrombin (6) and in 0.3 Uper ml thrombin (7). Note the induction of a band of approximately 15kDa in Lane 4.

713THROMBIN INDUCES APOPTOSIS IN HUMAN TUMOR CELLS

REFERENCES

AHMAD, A. and MENEZES, J., Binding of Epstein-Barr virus to humanplatelets causes the release of transforming growth factor-b1. J. Immunol.159, 3984–3988 (1997).

AHMAD, A., LADHA, A., COHEN, E.A. and MENEZES, J., Stable expression ofthe transfected HIV-1 env gene in a human B cell line: Characterization ofgp120-expressing clones and immunobiological studies.Virology 192,447–457 (1993).

AKIYAMA , H., IKEDA, K., KONDO, H. and MCGEER, P.L., Thrombin accu-mulation in brains of patients with Alzheimer’s disease.Neurosci. Lett.146, 152–154 (1992).

ANRATHER, D., MILLAN , M.T., PALMETSHOFER, A., ROBSON, S.C., GREEZY,C., RITCHIE, A.J., BACH, F.H. and EWENSTEIN, B.M., Thrombin activatesNF-kB and potentiates endothelial cell activation by TNF.J. Immunol.159, 5620–5628 (1997).

BRASS, L.F., The molecular basis for platelet activation.In R. Hoffman,E.J. Benz Jr, B. Furie, H. J. Cohen and L.E. Silberstein (eds.), Hematology:Basic Principles and Practice.2nd ed., pp.1536–1546, Churchill Living-stone, New York (1995).

BUCKLEY, C.D., PILLING , D., HENRIQUEZ, N.Y., PARSONAGE, G., THREL-FALL, K., SCHEEL-TOELLNER, D., SIMMONS, D.L., AKBAR, A.N., LORD andJ.M. and SALMON, M, RGD peptides induce apoptosis by direct caspase-3activation.Nature397, 534–539 (1999).

CAILLEAU , R., YOUNG, R., OLIVE, M. and REEVES, W.J. Jr., Breast tumorcell lines from pleural effusions.J. Natl. Cancer Inst. 53, 661–674 (1974).

CHERNEY, B.W., SAGADARI, C., KANEGANE, C., WANG, F. and TOSATO, G.,Expression of the Epstein-Barr virus protein LMP-1-mediated tumor re-gression in vivo.Blood 91, 2491–2500 (1998).

CHINNI, C., DE NIESE, M.R., TEW, D.J., JENKINS, A.L., BOTTOMLEY, S.P. andMACKIE, E.J., Thrombin, a survival factor for cultured myoblasts.J. Biol.Chem. 274, 9169–9174 (1999).

CHOI, B.H., KIM, R.C., VAUGHAN, P.J., LAU, A., VAN NOSTRAND, W.E.,COTMAN, C.W. and CUNNINGHAM, D.D., Decreases in protease nexins inAlzheimer’s diseases brain.Neurobiol. Aging16, 557–562 (1995).

COLLINS, S.J., GALLO, R.C. and GALLAGHER, R.D., Continuous growth anddifferentiation of human myeloid leukemic cells in suspension culture.Nature270, 347–349 (1997).

COUGHLIN, S.R., sol Sherry lecture in thrombosis: how thrombin “talks” tocells: molecular mechanisms and roles in vivo.Arteriosclerosis, Thromb.Vasc. Biol.,18, 514–518 (1998).

COUGHLIN, S.R., VU, T-K.H., HUNG, D.T. and WHEATON, V.I., Character-ization of a functional thrombin receptor: issues and opportunities.J. Clin.Invest. 89, 351–355 (1992).

DEBEIR, T., BENAVIDES, J. and VIGE, X., Dual effects of thrombin and a14-amino acid peptide agonist of the thrombin receptor on septal cholin-ergic neurons.Brain Res., 708, 159–166 (1996a).

DEBEIR, T., GUEUGNON, J., VIGE, X. and BENVIDES, J., Transduction mech-anisms involved in thrombin receptor-induced nerve growth factor secre-tion and cell division in primary cultures of astrocytes.J. Neurochem.,66,2320–2328 (1996b).

DERIAN, C.K., ECKARDT, A.J. and ANDRADE-GORDON, P., Differential reg-ulation of human keratinocyte growth and differentiation by a novel familyof protease-activated receptors.Cell Growth Differ.,8, 743–749 (1997).

DERY, O., CORVERA, C.U., STEINHOFF, M. and BUNNETT, N.W., Proteinase-activated receptors: novel mechanisms of signaling by serine proteases.Am J Physiol274, C1429–1452 (1998).

DONOVAN, F.M. and CUNNINGHAM, D.D., Signaling pathways involved inthrombin-induced cell protection.J. Biol. Chem. 273, 12746–12752(1998).

DONOVAN, F.M., PIKE, C.J., COTMAN, C.W. and CUNNINGHAM, D.D.,Thrombin induces apoptosis in cultured neurons and astrocytes via apathway requiring tyrosine kinase and RhoA activities.J. Neurosci.,17,5316–5326 (1997).

EPSTEIN, M.A., ACHONG, B.G., BARR, Y.M., ZAJAC, B., HENLE, G. andHENLE, W., Morphological and virological investigations on cultured Bur-kitt tumor lymphoblasts (strain Raji).J. Natl. Cancer Inst. 37, 547–559(1966).

EVEN-RAM, S., UZIELY, B., COHEN, P., GRISARU-GRANOVSKY, S., MAOZ,M., GINZBURG, Y., REICH, R., VLODAVSKY, I. and BAR-SHAVIT, R., Throm-bin receptor overexpression in malignant and physiological invasion pro-cess.Nature Med.,4, 909–914 (1998).

FOGH, J., WRIGHT, W.C. and LOVELESS, J.D., Absence of HeLa cell con-tamination in 169 cell lines derived from human tumors.J. Natl. CancerInst. 58, 209–214 (1977).

GUTTRIDGE, D.C., LAY, A., TRAN, L. and CUNNINGHAM, D.D., Thrombincauses a marked delay in skeletal myogenesis that correlates with thedelayed expression of myogenin and p21 cip1/WAF1.J. Biol. Chem. 272,24117–24120 (1997).

HAMMES, S.R. and COUGHLIN, S.R., Protease-activated receptor-1 can me-diate responses to SFLLRN in thrombin-densesitized cells: evidence for anovel mechanism for preventing or terminating signaling by PAR1’stethered ligand.Biochemistry,38, 2486–2493 (1999).

HOU, L., HOWELLS, G.L., KAPAS, S. and MACEY, M.G., The protease-activated receptors and their cellular expression and function in blood-related cells.Brit. J. Haematol. 101, 1–9 (1998).

HOWELL, D.N., ANDREOTTI, P.E., DAWSON, J.R. and CRESSWELL, P., Naturalkilling target antigens as inducers of interferon: Studies with an immunos-elected, natural killing-resistant human T lymphoblastoid cell line.J. Im-munol. 134, 971–976 (1985).

ISHIHARA, H., CONNOLLY, A.J., ZENG, D., KAHN, M.L., ZHENG, Y.W.,TIMMONS, C., TRAM, T. and COUGHLIN, S,R., Protease-activated receptor 3is a second thrombin receptor in humans.Nature386, 502–506 (1997).

JONES, H.W. JR, MCKUSICK, V.A., HARPER, P.S. and WUU, K.D., The HeLacell and a reappraisal of its origin.Obstet. Gynecol., 38, 945–949 (1971).

KAHN, M.L., ZHENG, Y-W., HUANG, W., BIGORNIA, V., ZENG, D., MOFF, S.,FARESE, R.V., JR., TAM, C. and COUGHLIN, S.R., A dual thrombin receptorsystem for platelet activation.Nature,394, 690–694 (1998).

KAPLANSKI, G., MARIN, V., FABRIGOULE, M., BOULAY, V., BENOLIEL, A.M.,BONGRAND, P., KAPLANSKI, S. and FARNARIER, C., Thrombin-activatedhuman endothelial cells support monocyte adhesion in vitro followingexpression of intercellular adhesion molecule-1 (1-CAM-1; CD54) andvascular cell adhesion molecule-1 (VCAM-1;CD106).Blood, 92, 1259–1267 (1998).

KHELIFA, R. and MENEZES, J., Use of fluoresceinated Epstein-Barr virus tostudy Epstein-Barr virus-lympoid cell interactions.J. Virol., 41, 649–656(1982).

KHYATTI , M., AHMAD, A., BLAGDON, M., FRADE, R. and MENEZES, J.,Binding of the endogenously expressed Epstein-Barr virus (EBV) envelopeglycoprotein 350 with the viral receptor masks the major EBV-neutralizingepitope and affects gp350/220-specific ADCC.J. Leukocyte Biol. 64,192–197 (1998).

KLEIN, E., KLEIN, G., NADKARNI , J.S., NADKARNI , J.J., WIGZELL, H. andCLIFFORD, P., Surface IgM-Kappa specificity on a Burkitt lymphoma cell invivo and in derived culture lines.Cancer Res.,28, 1300–1310 (1968).

KLEPFISH, A., GRECO, M.A. and KARPATKIN, S., Thrombin stimulatesmelanoma tumor cell binding to endothelial cells and subendothelial ma-trix. Int. J. Cancer, 53, 978–982 (1993).

LOZZIO, C.B. and LOZZIO, B.B., Human chronic myelogenous leukemiacell-line with positive Philadelphia chromosome.Blood, 45, 321–334(1975).

LU, Y-Y., KOGA, Y., TANAKA , K., SASAKI, M., KIMURA, G. and NOMOTO,K., Apoptosis induced in CD41 cells expressing gp160 of Human Immu-nodeficiency virus type 1.J. Virol. 68, 390–399 (1994).

LUNARDI-ISKANDAR, Y., GILL , P., LAM, V.H., ZEMAN, R.A., MICHAELS, F.,MANN, D.L., RETIZ, M.S., JR, KAPLAN, M., BERNEMAN, Z.N. and CARTER,D., Isolation and characterization of an immortal neoplastic cell line (KSY-1) from AIDS-associated Kaposi’s sarcoma.J. Natl. Cancer Inst., 87,974–981 (1995).

NALDINI , A. and CARNEY, D.H., Thrombin modulation of natural killeractivity in human peripheral lymphocytes.Cell. Immunol.172, 35–42(1996).

NIERODZIK, M.L., CHEN, K., TAKESHITA, K., LI, J-J., HUANG, Y-Q., FENG,X-S., D’ANDREA, M.R., ANDRADE-GORDON, P. and KARPATKIN, S., Pro-tease-activated receptor 1 (PAR-1) is required and rate-limiting for throm-bin-enhanced experimental pulmonary metastasis.Blood, 92, 3694–3700(1998).

RAFF, M., Cell suicide for beginners.Nature, 396, 119–122 (1999).

RAHMAN , A., ANWAR, K.N., TRUE, A.L. and MALIK , A.B., Thrombin-induced p65 homodimer binding to downstream NF-kB site of the promotermediates endothelial ICAM-1 expression and neutrohil adhesion.J. Immu-nol. 162, 5466–5477 (1999).

RALPH, P., MOORE, M.A. and NILSSON, K., Lysozyme synthesis by estab-lished human and murine histiocytic lymphoma cell lines.j. exp. med., 143,1528–1533 (1976).

RATHMELL , J.C. and THOMPSON, C.B., The central effectors of cell death inthe immune system.Ann. Rev. Immunol., 17, 781–828 (1999).

REILLY , C.F., CONNOLLY, T.M., FENG, D.M., NUTT, R.F. and MAYER, E.J.,

714 AHMAD ET AL.

Thrombin receptor agonist peptide induction of mitogenesis in CCL39cells.Biochem. Biophys. Res. Commun.,190, 1001–1008 (1993).SCHAFBERG, H., NOWAK, G. and KAUFMANN, R., Thrombin has a bimodaleffect on glioma cell growth.Brit. J. Cancer, 76, 1592–1595 (1997).SMIRNOVA, I.V., ZHANG, S.X., CITRON, B.A., ARNOLD, P.M. and FESTOFF,B.W., Thrombin is a extracellular signal that activates intracellular deathprotease pathways inducing apoptosis in model motor neurons.J. Neuro-biol., 36, 64–80 (1998).SOULE, H.D., VAZQUEZ, J., LONG, A., ALBERT, S. and BRENNAN, M., Ahuman cell line from a pleural effusion derived from a breast carcinoma.J.Natl. Cancer Inst., 51, 1409–1416 (1973).SOWER, L.E., FROELICH, C.J., CARNEY, D.H., FENTON, J.W. II and KLIMPEL,G.R., Thrombin induces IL-6 production in fibroblasts and epithelial cells:Evidence for the involvement of the seven-transmembrane domain (STD)receptor fora-thrombin.J. Immunol.,155, 895–901 (1995).VERGNOLLE, N., HOLLENBERG, M.D. and WALLACE, J.L., Pro- and anti-inflammatory actions of thrombin: a distinct role for proteinase-activatedreceptor-1.Brit. J. Pharmacol., 126, 1262–1268 (1999).VU, T-K. H., HUNG, D.T., WHEATON, V.I. and COUGHLIN, S.R., Molecularcloning of a functional thrombin receptor reveals a novel proteolyticmechanism of receptor activation.Cell, 64, 1057–1068 (1991).WALKER, B.D., KOWALSKI, M., GOH, W.C., KOZARSKY, K., KRIEGER, M.,ROSEN, C., ROHRSCHNEIDER, L., HASELTINE, W.A. and SODROSKI, J., Inhi-

bition of human immunodeficiency virus syncytium formation and virusreplication by castanospermine.Proc. Natl. Acad. Sci. U.S.A., 84, 8210–8124 (1987).

WALZ, D.A. and FENTON, J.W., The role of thrombin in tumor cell metas-tasis.Invasion Metast.,14, 303–308 (1995).

WEISS, A., WISKOCIL, R.L. and STOBO, J.D., The role of T3 surfacemolecules in the activation of human T cells: a two-stimulus requirementfor IL-2 production reflects events occurring at a pre-translational level.J. Immunol., 133, 123–128 (1984).

WOJTUKIEWICZ, M.Z., TANG, D.G., CIARELLI , J.J., NELSON, K.K., WALZ,D.A., DIGLIO, C.A., MAMMEN, E.F. and HONN, K.V., Thrombin increasesthe metastatic potential of tumor cells.Int. J. Cancer, 54, 739–806 (1993).

WOJTUKIEWICZ, M.Z., TANG, D.G., NELSON, K.K., WALZ, D.A., DIGLIO,C.A. and HONN, K.V., Thrombin enhances tumor cell adhesive and meta-static properties via increaseda11bb3 expression on the cell surface.Thromb. Res., 68, 233–245 (1992).

XU, W.F., ANDERSEN, S., WHITMORE, T.E., PRESNELL, S.R., YEE, D.P.,CHING, A., DAVIE, E.W. and FOSTER, D.C., Cloning and characterization ofhuman protease-activated receptor 4.Proc. Natl. Acad. Sci. U.S.A.,95,6642–6646 (1998).

YANG, E. and KORSMEYER, S.J., Molecular thanatopsis: A discourse on theBCL-2 family and cell death.Blood, 88, 386–401 (1996).

715THROMBIN INDUCES APOPTOSIS IN HUMAN TUMOR CELLS