Antisense RNA inhibition of cathepsin L expression reducestumorigenicity of malignant cells

H. Kirschke a,b,*, R. Eerola b, V.K. Hopsu-Havu c, D. BroÈ mme d, E. Vuorio b

aInstitute of Physiological Chemistry, University of Halle, D-06097 Halle (Saale), GermanybDepartment of Medical Biochemistry and Molecular Biology, University of Turku, FIN-20520 Turku, Finland

cDepartment of Dermatology, University of Turku, FIN-20520 Turku, FinlanddDepartment of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA

Received 5 August 1999; received in revised form 17 November 1999; accepted 22 November 1999

Abstract

Several tumour-forming cell lines are known to overproduce the lysosomal cysteine peptidase cathepsin L. We have used anantisense approach to investigate whether inhibition of cathepsin L overexpression in two malignant cell lines (myeloma SP cellsand L cells) reduces their tumorigenic potential. Two di�erent cDNA fragments of murine cathepsin L were inserted in the anti-

sense direction into the pcDNA3 vector, and SP and L cells were stably transfected with these plasmid constructs. Several of theselected clones expressing the antisense transcript showed speci®c reduction of the mRNA level and the intracellular activity ofcathepsin L, and a greatly diminished amount of secreted procathepsin L. When tested in Balb/c nu/nu mice, the cell lines with low

cathepsin L activity exhibited a signi®cantly decreased potential for tumour growth when compared with control cells expressingwild-type levels of cathepsin L activity. This observation suggests that cathepsin L is a critical factor in tumour growth. # 2000Published by Elsevier Science Ltd. All rights reserved.

Keywords: Cathepsin L; Antisense RNA; Tumour growth; Procathepsin L; Cathepsin B; Myeloma SP cells; L cells

1. Introduction

The activity of proteolytic enzymes is required for allsteps of tumour growth, such as local invasion, intra-vasation, extravasation, angiogenesis and metastasis [1].Elevated levels of serine peptidases [2], metallopepti-dases [3], aspartic peptidases [4] and cysteine peptidases[5±7] have been shown to correlate with the malignancyof tumour cells. However, little is known about thefunction of individual proteolytic enzymes, their possi-ble interaction in activation cascades [8], and proteaseredundancy in tumour progression and metastasis.Several studies have demonstrated that tumour growthand metastasis can be in¯uenced by modulating theactivity of individual peptidases and their endogenous

inhibitors by synthetic inhibitors [9], antibodies [10,11]and antisense inhibition [12,13].Our own results [10] and the results by Jean and col-

leagues [11] have shown that in the presence of anti-bodies to cathepsin L tumour cells lose, to a greatextent, their potential to induce tumours after implan-tation into mice. It remains unclear, however, whetherthis was caused by the inhibition of cathepsin L activityor by the phagocytic removal of tumour cells loadedwith the antigen±antibody complex at the surface bymacrophages, i.e. by an unspeci®c defence mechanism.To examine directly the importance of the elevated

cathepsin L concentration in tumours, we decided tosuppress the overexpression of this enzyme by antisenseRNA inhibition. Here we describe the e�ect of cathepsinL antisense RNA on the mRNA levels of cathepsins Land B, glyceraldehyde 3-phosphate dehydrogenase(GAPDH) and b-actin, on the catalytic activities of thelysosomal enzymes and on the malignant phenotype ofmurine myeloma cells and L cells.

0959-8049/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.

PI I : S0959-8049(00 )00014-9

European Journal of Cancer 36 (2000) 787±795

www.ejconline.com

* Corresponding author. Fax: +49-33200-409042.

E-mail address: hkirschke@web.de (H. Kirschke).

2. Materials and methods

2.1. Preparation of plasmid constructs

Total RNA was isolated from murine myeloma cellsas described earlier [14], and used as a template for theampli®cation of two di�erent cDNA fragments (insert 1and insert 2) of murine cathepsin L using the reversetranscription±polymerase chain reaction (RT-PCR)technique.Insert 1 contained a 628 bp cDNA corresponding to

nucleotides 402±1029 of cathepsin L cDNA [15] andwas RT-PCR ampli®ed (Pfu polymerase, Stratagene, LaJolla, CA, USA) using synthetic primers (50 CTGCGGCCGC GTC CAC AGT GGT TGT CCC GG 30

and 50 CT TCTAGA CCC AAG TCT GTG GAC TGGAG 30). The 600 bp insert 2, corresponding to nucleo-tides 6±605, was ampli®ed similarly (AmpliTaq1, Per-kin Elmer, Branchburg, NJ, USA) using syntheticprimers: 50 CT AAGCTT CAG GCC TCC GTT ACAGCC CTG 30 and 50 CT TCTAGA GGA TCC GAGTTT GCA GAC TTC 30. The primers contained NotIand XbaI (insert 1) and HindIII and XbaI (insert 2)recognition sites (underlined) for subsequent cloning ofthe puri®ed inserts in the antisense orientation inpcDNA3 vectors digested with the correspondingenzymes (Invitrogen, San Diego, CA, USA). The con-structs were referred to as pcDNA-catL1 (insert 1) andpcDNA-catL2 (insert 2).The ®re¯y luciferase gene (1710 bp) was cloned into

the same pcDNA3 vector between BamHI and ApaIsites and named pcDNA-luc.

2.2. Transfection and selection

Non-adherent murine myeloma cells (SP cells, SP2/0-Ag14) were cultured in RPMI-1640 medium (supple-mented with glucose, NaHCO3, HEPES, pyruvate, suc-cinate, insulin, l-glutamine, b-mercaptoethanol)containing 10% (v/v) fetal bovine serum and gentamy-cin (50 mg/ml).Aliquots of 2.5�106 cells were washed and then sus-

pended in 0.8 ml of RPMI medium without fetal bovineserum and gentamycin in 40 mm tissue culture dishes.Two micrograms of each plasmid (pcDNA-catL1,pcDNA-catL2 and pcDNA-luc) were mixed individu-ally with 10 ml Lipofectin1-Reagent in 200 ml mediumexactly as described by the manufacturer (GIBCO, NY,USA) for stable transfection. The Lipofectin±DNAcomplexes were gently mixed with the cells and incu-bated for 6 h at 37�C in a CO2 incubator. The incuba-tion was continued for another 48 h after addition of 2ml RPMI medium to each plate without gentamycin butsupplemented with the normal percentage of serum.After centrifugation, the cells of each dish were resus-pended in 30 ml RPMI medium (without gentamycin)

containing 10% (v/v) fetal bovine serum and 1 mg/mlGeneticin1 (G418) (GIBCO) for selection of the trans-fected cells. After 14 days in selection medium, thestable G418-resistant transformants were cloned bydilution. Several clones of the SP cells transfected withpcDNA-catL1 (SP1-1, SP1-2, SP1-3...), clones trans-fected with pcDNA-catL2 (SP2-1, SP2-2, SP2-3...) andclones transfected with pcDNA-luc (SPluc-1, SPluc-2,SPluc-3...) were cultured in RPMI medium withoutgentamycin containing 500 mg/ml of G418 for furthercharacterisation.Adherent murine L cells (NCTC clone 929) were cul-

tured in Dulbecco's modi®ed Eagles medium (DMEM)containing 10% (v/v) fetal bovine serum, penicillin G(100 U/ml) and streptomycin (50 mg/ml). Aliquots of2�105 cells in 5 ml complete medium were seeded in 60mm tissue culture dishes and incubated until the cellswere approximately 50% con¯uent (approximately 20h). Two micrograms of plasmids pcDNA-catL1 andpcDNA-luc were mixed with 10 ml Lipofectin1-Reagentas described by the manufacturer for stable transfectionof adherent cells. This was followed by the same cul-turing procedure, selection in 1 mg/ml G418 and clon-ing as described above. Several clones of L cellstransfected with pcDNA-catL1 (L1-1, L1-2, L1-3...) andclones transfected with pcDNA-luc (Lluc-1, Lluc-2,Lluc-3...) were characterised.

2.3. Cell lysates and NH4Cl-medium

Samples of subcon¯uent cells (2±5�107) were washedtwice with phosphate-bu�ered saline (PBS) and homo-genised in 1 ml 0.1 M acetate bu�er, pH 5.0, containing1 mM EDTA and 0.01% (w/v) Triton X-100. Afterthree cycles of freeze-thawing, the homogenate wascentrifuged for 10 min in a microcentrifuge at 4�C. Thesupernatant was stored at ÿ75�C and used for activityand protein determinations.Aliquots of approximately 1�106 SP cells were

washed with serum-free medium and seeded in 2 ml ofserum-free medium containing 8 mM NH4Cl in thewells of a 12-well culture plate. After 24 h in a CO2

incubator, the cells were centrifuged, counted, and thesupernatant was used for the assay of procathepsin L.L cells (about 2�105) were cultured in 2 ml of com-

plete DMEM for 24 h in 12-well plates. The cells werewashed three times with serum-free medium and thencovered with 2 ml of serum-free DMEM containing 8mM NH4Cl. After 24 h in a CO2 incubator, the super-natant was removed for the assay of procathepsin L andthe cells were released with trypsin and counted.

2.4. Enzyme assays

The activities of cathepsins L and B were determinedusing published methods [16]: 100 ml enzyme solution

788 H. Kirschke et al. / European Journal of Cancer 36 (2000) 787±795

and 200 ml bu�er with or without inhibitor were pre-incubated for 10 min at 37�C. After addition of 200 mlof substrate solution, the incubation (10 min) was ter-minated by addition of 500 ml of chloroacetate. Pro-cathepsin L was activated according to proceduresdescribed by Mason and colleagues [17]. The ®nal con-centrations of the bu�ers were 50 mM containing 3 mMdithiothreitol and 3 mM EDTA. The following con-centrations of the 7-(4-methyl)coumarylamide (NHMec)substrates (Bachem, Bubendorf, Switzerland) are indi-cated for each enzyme, those of the inhibitors refer tothe 10 min pre-incubation assay. Cathepsin L (EC3.4.22.15): 5 mM Z-Phe-Arg-NHMec, sodium acetatebu�er pH 5.5, inhibitor 0.5 mM Z-Phe-Phe-CHN2. Pro-cathepsin L: activation of the latent precursor at pH 3.0for 2 min, incubation with 5 mM Z-Phe-Arg-NHMec,sodium acetate bu�er pH 5.5 in the presence of 1.5 mMcathepsin B inhibitor, CA-074 (N-(L-3-trans-propyl-carbamoyloxirane -2 - carbonyl) -l - isoleucyl -l - proline,Peptide Institute, Osaka, Japan). Cathepsin B (EC3.4.22.1): 10 mM Z-Arg-Arg-NHMec, sodium phos-phate bu�er pH 6.0.The ¯uorescence of free aminomethylcoumarin

(NHMec) was determined in a Perkin-Elmer LS-2B¯uorimeter (excitation: 375 nm, emission: 460 nm). Oneunit of enzyme activity corresponds to 1 mmol of sub-strate degraded per min. The activity of luciferase wasqualitatively measured using the GenGlow-100 kit (Bio-Orbit, Turku, Finland).

2.5. Protein assay

Protein was determined by a micromodi®cation of theLowry method [18] using bovine serum albumin asstandard.

2.6. RNA preparation and Northern hybridisation

Total RNA was isolated from approximately 5�107cells (washed with PBS) by the guanidinium iso-thiocyanate method and CsCl ultracentrifugation [14].The yield of RNA was 100±200 mg. Aliquots (10 mg) oftotal RNA were fractionated on 1% (w/v) glyoxal±agarose gels, transferred to Pall Biodyne membranesand hybridised as recommended by the supplier (PallEurope, Portsmouth, UK).For screening of the cell lines, total RNA was isolated

by the rapid method with guanidinium isothiocyanateand phenol±chloroform according to the instructions ofPromega in 1996. Subsequently, 10 mg aliquots of totalRNA were separated on 1% (w/v) formaldehyde gelsand transferred to Pall membranes.To detect the mRNAs of cathepsins L, B, H and S,

GAPDH and b-actin, 32P-labelled murine cDNA probespMCatL-1, pMCatB-1, pMCatH-1, pMCatS-1 [19],pMGAPDH-1 and pgAct-1, produced by RT-PCR

based on published sequences, were labelled by randompriming (Boehringer, Mannheim, Germany). A probe formurine 28S rRNA was constructed similarly and labelledby nick translation (Boehringer, Mannheim, Germany).Blots were washed three times with 2�SSC (1.5 mM

trisodium citrate dihydrate, 150 mM sodium chloride),0.1% (w/v) sodium dodecyl sulphate (SDS) at roomtemperature and twice with 0.1�SSC, 0.1% (w/v) SDSat 55�C for 30 min each, exposed to X-ray ®lms atÿ75�C, and quanti®ed on a Molecular Imager Phos-phoImager (Bio-Rad, Hercules, CA, USA). The scan-ned screens were processed as to lightness, saved as abmp ®le, and ®nally produced in Harvard Graphics.For rehybridisation of the ®lters, the probes wereremoved by boiling in 0.1% (w/v) SDS.For the synthesis of sense and antisense cRNA

probes, plasmids pcDNA-catL1 and pcDNA-catL2were linearised by digestion with NotI (sense), XbaI(antisense), HindIII (sense), and XbaI (antisense),respectively. The transcription assays (20 ml) contained1 mg linearised plasmid, 4 ml transcription bu�er, 2 ml100 mM DTT, 0.5 ml RNasin, 1 ml each of 10 mM ATP,GTP and UTP, 2 ml 100 mM CTP, 5 ml [a-32P] CTP (800Ci/mmol) (Amersham, Buckinghamshire, UK), and therespective RNA polymerase SP6 or T7. The riboprobeswere labelled to high speci®c activities, using 100 mCi32[P]-CTP per reaction instead of the recommended 50mCi (Promega, 1996).The labelled riboprobes were puri®ed by gel ®ltration

(Sephadex G-50) and used for hybridisation of theNorthern blots at 65�C for 16 h according to Srivastavaand Schonfeld [20]. The hybridisation bu�er contained0.75 M NaCl, 50 mM sodium phosphate, pH 7.4, 50mM EDTA, 0.2% (w/v) skimmed milk powder, 0.5%(v/v) deionised formamide and 250 mg/ml denatured calfthymus DNA. The membranes were washed 4 timeswith 1�SSC, 0.2% (w/v) SDS at 65�C for 20 min andanalysed as described above.

2.7. Tumour formation in nude mice

Samples of 6�106 cells of each of the normal cell linesand those transfected with luciferase and antisensecathepsin L constructs were injected subcutaneouslyinto the back of nude mice, 4±5 mice in each group. Allmice of the same group received 0.2 ml of cells sus-pended in PBS from the same culture ¯ask. Fourteendays after injection, the tumours were excised andweighed and the organs inspected for metastases.Although skin is not the `natural' growth site of SP

and L cells, we did not inoculate the cells into the peri-toneal cavity, because the mice developed numeroussmall solid tumours in the peritoneum and in this casequanti®cation by weight was impossible.Balb/c nu/nu male mice (age 6±8 weeks) were main-

tained in the experimental animal facility of the Medical

H. Kirschke et al. / European Journal of Cancer 36 (2000) 787±795 789

Faculty, University of Turku. The experiments wereapproved by the committee for animal welfare of theUniversity of Turku.

3. Results

3.1. Transfected cell lines

We ®rst tested the e�cacy of the two di�erent anti-sense RNA molecules to inhibit endogenous cathepsinL expression in SP and L cells. Insert 1 (catL1) corre-sponded to nearly the full coding sequence of themature enzyme from nucleotide 402 (Pro-2) to 1029(Gly-210) [15], whereas insert 2 (catL2) contained 600bp of the 50 portion of the cathepsin L cDNA(nucleotides 6±605) which included the ATG codon(60±62).All clones which were stably transfected with pcDNA-

catL1 and pcDNA-catL2 were screened for their levelsof cathepsin L mRNA and activity. One SP cell clonecontaining insert 1 (SP1-52), two L cell clones withinsert 1 (L1-29, L1-34), and three SP cell clones con-taining insert 2 (SP2-1, SP2-5, SP2-20) were chosen forfurther characterisation.Clones transfected with the luciferase construct were

screened for their levels of luciferase activity. Threeclones were selected (SPluc-3, SPluc-6, Lluc-3) to serveas additional controls in all experiments.Only those clones which showed the same growth

properties as normal cells were selected for the tumourgrowth experiment. The doubling time was calculatedduring the exponential growth phase, and was 20 h fornormal SP cells, the antisense clones SP1-52, SP2-1,SP2-5, SP2-20 and the luciferase clones (SPluc-3, SPluc-6). L cells and the clones L1-29, L1-34 and Lluc-3 grewfaster. The doubling time was 13 h.

3.2. Levels of mRNA

Northern blot analyses (Fig. 1) revealed an approxi-mately 42% reduction of Cat L mRNA in two clonestransfected with antisense insert 2 (SP2-5, SP2-20). Thelevels of the other mRNAs were only slightly reduced(5±14%) compared with 285 rRNR (denoted as 1). Inclone SP2-1 the level of Cat L mRNA was reduced(45%) concomitantly with cathepsin B mRNA (49%).Only slight reduction (20%) of Cat L mRNA in cloneSP1-52 could be detected. This result has been con-®rmed by several di�erent experiments. The othermRNA levels of the antisense clone SP1-52 and of theluciferase transfected cell lines were insigni®cantlya�ected. In transfected L cells (L1-29, L1-34, Lluc-3)the mRNA levels including the cathepsin L mRNA alsoremained unchanged (data not shown).

No cathepsin S mRNA was detected in the cells andthe level of cathepsin H mRNA was too low for thesemiquantitative determination.

3.3. Expression of antisense transcripts

Special conditions such as a high speci®c activity ofsense cRNA probes, extended exposure times, andstringent hybridisation and washing conditions allowedthe detection of antisense RNA produced in some of thetransfected SP cell clones, but not in the L cell clones.The antisense RNA was visible in clones SP1-38 andSP1-52 (Fig. 2) and faintly detectable in clones SP2-1,SP2-5 and SP2-20 (Fig. 3). The sense cRNA probes also

Fig. 1. Northern analysis of transfected clones. (a) Total RNA sam-

ples (10 mg) of SP cells, antisense clones SP2-1, SP2-5, SP2-20, lucifer-

ase clone SPluc-6 and SP cells, antisense clone SP1-52, luciferase clone

SPluc-3 were separated by electrophoresis, blotted on Nylon mem-

branes and hybridised successively with radiolabelled cDNA probes of

28S, cathepsin L (Cat L), cathepsin B (Cat B), GAPDH and b-actin.This is a representative expt chosen from several for demonstration

purposes. (b) Hybridisation intensity was analysed using a phospho-

imager. The mRNA levels were calculated as relative hybridisation

units per 28S rRNA. The columns represent cathepsin L mRNA &,

cathepsin B mRNA , GAPDH mRNA & and b-actin mRNA .

790 H. Kirschke et al. / European Journal of Cancer 36 (2000) 787±795

hybridised to an unknown RNA migrating in the sameposition as the 28S rRNA. The antisense cRNA probeshybridised with cathepsin L mRNA as expected (Fig. 4).

3.4. Catalytic activity of cathepsins L and B

The activities of cathepsins L and B in cell lysateswere most probably due to the mature enzymes locatedin lysosomes. Activities of cathepsins H and S could notbe detected in the cell lysates. The concentration ofsecreted procathepsin L was determined in conditionedNH4Cl-medium after activation of the inactive pre-cursor. Before activation, the medium (pH 7.2) did notcontain any cathepsin L or cathepsin B activity. Weattributed the increase in enzyme activity measured inthe presence of a cathepsin B inhibitor to procathepsinL. This activity was completely inhibited by 0.5 mM Z-Phe-Phe-CHN2, a speci®c inhibitor of cathepsin L at 0.5mM [21].Higher concentrations of NH4Cl (usually 10 mM)

caused approximately 50% cell death in some of thetransfected cell lines after 24 h incubation, thus secre-tion of procathepsin L was enhanced in all experimentsby 8 mM NH4Cl in serum-free medium. Protein deter-mination revealed that 107 SP cells contained 1 mg oftotal protein and 107 L cells 0.4 mg of protein. Cellswere washed twice with PBS before lysis and cen-trifugation. The intracellular concentration of cathepsinL (2 munits (mU)/107 cells) approximately equalled the

Fig. 2. Northern hybridisation of sense catL1 riboprobe to RNA of

antisense transfectants. Total RNA samples (10 mg) of SP cells, anti-

sense clones SP1-1, SP1-11, SP1-38, Sp1-52 and luciferase clones

SPluc-3 and SPluc-6 were denatured by formaldehyde, separated by

electrophoresis, blotted on a Nylon membrane and hybridised with

radiolabelled sense cRNA under stringent conditions. The arrow

indicates the size of 28S rRNA. SP1-1, SP1-11 and SP1-38 were not

characterised further as they showed decreased growth properties.

Fig. 3. Northern hybridisation of sense catL2 riboprobe to RNA of

antisense transfectants. Total RNA samples (10 mg) of SP cells, anti-

sense clones SP2-1, SP2-5, SP2-20 and luciferase clone SPluc-6 were

separated by electrophoresis, blotted on a nylon membrane and

hybridised with radiolabelled sense cRNA under stringent conditions.

The arrow indicates the size of 28S rRNA.

Fig. 4. Northern hybridisation of antisense catL2 riboprobe to RNA

of antisense transfectants. Total RNA samples (10 mg) of SP cells,

antisense clones SP2-1, SP2-5, SP2-20 and luciferase clone SPluc-6

were separated by electrophoresis, blotted on a Nylon membrane and

hybridised with radiolabelled antisense catL2 cRNA under stringent

conditions. The arrows indicate the size (1.5 kb) of cathepsin L mRNA

(bottom arrow). The top arrow indicates the position of 28S rRNA

(which is not visible).

H. Kirschke et al. / European Journal of Cancer 36 (2000) 787±795 791

concentration of procathepsin L secreted by the sus-pended SP cells in 24 h (2.55 mU/107 cells) (Table 1). Incontrast, the concentration of procathepsin L secretedby adherent L cells in 24 h was approximately 28 timeshigher (9.99 mU/107 cells) than the intracellular con-centration of the enzyme (0.36 mU/107 cells) (calculatedwith Lluc-3).The speci®c reduction in cathepsin L activity by the

antisense transcript of insert 1 was visible in clone SP1-52 (Table 1). We measured 80% and 83% inhibition ofthe speci®c activities of mature cathepsin L and pro-cathepsin L, respectively, and only 17% inhibition ofcathepsin B in comparison with normal cells or clonestransfected with the luciferase construct.L cell clones transfected with insert 1 also showed a

reduction of cathepsin L, but not of cathepsin B activ-ity. The speci®c activities of mature cathepsin L and

procathepsin L were inhibited by 44 and 29%, respec-tively, in L1-29 cells and by 67 and 53%, respectively, inL1-34 cells (Table 1).The speci®c inhibition of cathepsin L and pro-

cathepsin L was demonstrated in two transfectants withinsert 2: SP2-5 and SP2-20 (Table 1). Cathepsin Bactivity was only slightly a�ected. In addition todecreased activities of cathepsin L and procathepsin L,clone SP2-1 showed a strong inhibition of cathepsin B(Table 1).In addition to the activity measurements, the expres-

sion of secreted procathepsin L was analysed by Wes-tern blotting. Very low levels of procathepsin L inclones SP1-52 and SP2-5 in comparison to wild-type SPcells were seen. Clones SP2-1 and SP2-20 also showedreduced levels of the secreted precursor of cathepsin L(data not shown).

3.5. Tumour growth

Only those antisense clones which showed a reducedexpression of cathepsin L and normal levels of cathepsinB were selected for inoculation into nude mice. Thetumours were removed 14 days after injection andweighed (Table 2). Three mice (injected with clones SP1-52 or L1-29) did not develop tumours. All tumours grewat sites of injection and no metastases were detected.Tumours induced by the highly malignant myeloma

cells reached approximately double the size of thoseinduced by L cells during the 14-day follow-up. Luci-ferase-transfected cell lines (SPluc-6, Lluc-3) exhibitedapproximately the same potential for tumour growth asthe respective normal cell lines. The tumour cells pre-served the pcDNA-luc plasmid, because luciferaseactivity was detected in the tumour homogenates.Transfected cell lines SP1-52, SP2-5, SP2-20, L1-29

and L1-34 showed signi®cantly reduced tumour growthcompared with normal and luciferase transfected cells(Table 2).

Table 1

Speci®c activities of cathepsins L (Cat L) and B (Cat B) in normal,

antisense and luciferase transfected cellsa

Cell line Cat L mU/mg Procat L mU/106 cells Cat B mU/mg

SP 2�0.23 255�25.4 2.4�0.2

SPluc-3 2�0.25 241�25.7 2.1�0.36

SPluc-6 1.8�0.06 267�16.9 2.5�0.42

SP1-52 0.4�0.26 44�4.4 2�0.26

SP2-1 0.7�0.06 80�7.2 0.7�0.17

SP2-5 0.5�0.25 92�18.5 2.1�0.2

SP2-20 0.8�0.1 148�8.2 1.9�0.23

L 0.9�0.2 n.d. 4.1�0.5

Lluc-3 0.9�0.27 999�12 4.2�0.46

L1-29 0.5�0.15 714�116.5 3.9�0.2

L1-34 0.3�0.19 468�53.2 4�0.31

a The data represent speci®c activities (munits (mU) per mg of

protein) of cathepsins L and B in cell lysates and of secreted pro-

cathepsin L (Procat L) (in 24 h) after activation (munits per 106 cells)and are given as an average and standard deviation of three di�erent

lysates and NH4Cl-supernatants, respectively, of independently cul-

tured cells.

n.d., not determined.

Table 2

Tumour weightsa

Cells injected No. of mice with tumour

per no. of mice injected

Weights (g) g (average)�S.D. P value

SP 4/4 4.36, 2.76, 1.91, 1.50 2.63�1.26

SPluc-6 5/5 4.78, 2.71, 1.75, 1.58, 0.98 2.36�1.49 n.s.

SP1-52 3/5 1.10, 0.32, 0.03, 0.00, 0.00 0.29�0.43 <0.01

SP2-5 5/5 1.35, 0.74, 0.71, 0.43, 0.15 0.68�0.45 <0.01

SP2-20 5/5 2.35, 1.26, 1.20, 0.80, 0.47 1.22�0.71 <0.05

L 5/5 1.68, 1.55, 0.95, 0.82, 0.68 1.14�0.45

Lluc-3 4/4 2.63, 1.07, 0.62, 0.60 1.23�0.96 n.s.

L1-29 4/5 0.96, 0.52, 0.20, 0.06, 0.00 0.35�0.36 <0.05

L1-34 5/5 0.56, 0.37, 0.21, 0.05, 0.07 0.25�0.21 <0.05

a The average and standard deviations (S.D.) of tumour weights from identically treated mice were determined. Statistical analyses: general

factorial ANOVA and comparison of the normal with transfected treatment groups by simple contrasts.

n.s., non signi®cant.

792 H. Kirschke et al. / European Journal of Cancer 36 (2000) 787±795

3.6. Correlation between inhibition of cathepsin L andtumour growth

Table 3 gives a summary of the decreases in mRNA,cathepsin L and procathepsin L activities, and tumourweights in the antisense transfected clones in order tocompare the levels of inhibition.A positive correlation between all parameters can be

observed in clones SP2-5 and SP2-20. The clones trans-fected with insert 1 lack a signi®cant decrease in mRNAlevels, whereas the other parameters are correlated inantisense clones SP1-52 and L1-34. In some of theclones we observed a higher correlation between maturecathepsin L activity and tumour weight than betweenprocathepsin L activity and tumour weight.

4. Discussion

This study was designed to investigate antisenseRNA-mediated suppression of the cathepsin L geneoverexpression in murine tumour cells to determine thefunction of this enzyme in tumour induction andgrowth.The suppression of the cathepsin L gene expression

was checked at mRNA and protein levels, the latter byactivity determinations of intracellular mature cathepsinL and secreted procathepsin L and by immunologicaldetection. SP and L cells obviously do not contain anyof the peptidases of the papain family which show atissue-restricted expression pattern such as cathepsinsK, S, W and V (L2). Only cathepsin F is ubiquitouslydistributed and cleaves substrates such as Z-Phe-Arg-NHMec with high catalytic e�ciency comparable withcathepsin L [22]. We determined the activity of intra-cellular cathepsin L with the same substrate, but in thepresence of Z-Phe-Phe-CHN2 [21]. It is unknown yet,

whether cathepsin F is also inhibited under the specialconditions speci®c for the inhibition of cathepsin L. Theextracellular secreted procathepsin L was activated andincubated with the same substrate, but in the presenceof the cathepsin B inhibitor CA-074. Incubation with Z-Phe-Phe-CHN2 abolished completely the activityagainst Z-Phe-Arg-NHMec. The pattern of the Westernblot con®rmed our results on the inhibition of secretedprocathepsin L activity in the antisense SP cell clonesand proves the speci®city of cathepsin L determination(data not shown).Clone SP2-5 demonstrated the expected character-

istics of speci®c translational inhibition of cathepsin Lby antisense cathepsin L RNA. The construct (pcDNA-catL2) was designed to hybridise with the mRNA at its50 end covering the AUG codon. The antisense tran-script was produced in the cell and faintly detectable inthe RNA preparation from SP2-5 cells (Fig. 3). Cathe-psin L was inhibited at the mRNA level (42%) (Fig. 1)and at the protein level (intracellular activity by 75%,secreted procathepsin L by 64%) (Table 1), whereascathepsin B (mRNA and activity) and the mRNA levelsof two constitutive genes (GAPDH and �-actin) werenot signi®cantly a�ected. We suggest that the greatlyreduced tumour growth (Table 2) in nude mice by theclone SP2-5 was a consequence of the reduced cathepsinL expression.Clone SP2-20 showed similar properties Ð but the

inhibition of protein expression was not so pronouncedas in SP2-5 and tumour growth was not reduced to suchan extent as with SP2-5, although both clones SP2-20and SP2-5 exhibited approximately the same reductionin the mRNA levels of cathepsin L (Fig. 1).It is noticeable that we could not ®nd normally

growing antisense clones which showed more than 50%reduction of the cathepsin L mRNA.However, another transfectant revealed other kinds of

consequences of antisense cathepsin L RNA expression.Cell line SP1-52 transfected with the pcDNA-catL1construct expressing an antisense transcript com-plementary to the nucleotide sequence of the maturecathepsin L mRNA downstream of the ATG codon,exhibited the highest reduction (80±83%) (Table 1) inactivity of cathepsin L and the highest reduction intumour growth of all transfectants tested (Table 2) Ðbut, surprisingly, did not show a signi®cant reduction ofthe cathepsin L mRNA level (Fig. 1). A similar phe-nomenon was observed with L cells transfected withpcDNA-catL1 (L1-29, L1-34).We found only one paper dealing with growth inhibi-

tion of transformed tumour cells after treatment withantisense oligodeoxynucleotides directed to tax mRNA,where the target protein concentration was decreased,but the respective mRNA level was not a�ected [23].Usually RNA duplexes are rapidly degraded by RNasesand, therefore, a reduction in the level of the speci®c

Table 3

Decrease of mRNA, activities of cathepsin L (Cat L) and procathepsin

L (Procat L) and tumour weights in transfected cellsa

Cell line Cat L

mRNA

Cat L

activity

Procat L

activity

Tumour

weight

SP 0 0 0 0

SPluc-3 11 0 5 n.d.

SPluc-6 0 10 0 10

SP1-52 20 80 83 89

SP2-1 45 65 69 n.d.

SP2-5 42 75 64 74

SP2-20 41 60 42 54

L 0 0 n.d. 0

Lluc-3 0 0 0 0

L1-29 �10 44 29 69

L1-34 �10 67 53 78

a The data (% inhibition) are taken from Fig. 1, Tables 1 and 2.

n.d., not determined.

H. Kirschke et al. / European Journal of Cancer 36 (2000) 787±795 793

mRNA is considered to be a good indicator of geneinhibition [24]. Lack of mRNA degradation may beexplained by formation of stable interactions betweenloop sequences of the target and antisense RNA withoutcomplete pairing, as has been described for prokaryoticsystems by Wagner and Brantl [25]. Another assumptionis that double-stranded RNAs formed downstream of theinitiation codon are not degraded by RNases as much asRNA duplexes at the 50 end. The ®ndings by Moroni andcolleagues [26] support, to some extent, this hypothesis.The inhibition of the epidermal growth factor receptor(EGF-R) expression by antisense constructs was moste�ective (at the level of protein expression and biologi-cal activity) with antisense RNA complementary to the30 end of the coding region Ð but showed a much lowerreduction of the EGF-R mRNA than antisense RNAcomplementary to the 50 untranslated and coding region.One transfectant (SP2-1) was characterised where the

inhibition of cathepsin L (mRNA and activity) wasconcomitant with a reduction of the mRNA and activityof cathepsin B, a related lysosomal enzyme. We canonly suggest that the antisense catL2 RNA hybridised invivo in this clone not only with cathepsin L mRNA, butalso with cathepsin B mRNA. But in vitro the antisensecatL2 riboprobe did not react with cathepsin B mRNA(2.2 kb) (Fig. 4).We describe here that speci®c antisense inhibition of

cathepsin L in myeloma SP cells and L cells resulted inreduced tumour growth in nude mice. Recently it hasbeen shown by Frade and colleagues [27] that over-expression of cathepsin L using constructs based on thesame pcDNA3 plasmid conferred tumorigenic andmetastatic properties to melanoma cells.However, cathepsin L is only one of the proteolytic

enzymes involved in tumour growth. Using the anti-sense technique, reduction of malignancy and metastaticpotential has also been achieved by inhibition of uroki-nase plasminogen activator [28] and matrilysin (MMP-7) [29], whereas antisense inhibition of tissue inhibitorsof metalloproteinases, TIMPs, increased tumorigenicity[12,30]. A diversity of other gene±protein systems, suchas receptors for urokinase plasminogen activator [13],and for endothelial growth factor [26], as well as vas-cular endothelial growth factor [31], the oncogene c-raf-1 [32], and matrix proteins, such as galectin-3 [33] andperlecan [34], have also been shown to mediate reducedtumorigenicity when inhibited with antisense probes.The present study adds cathepsin L to this list, asdownregulation of this peptidase activity led to sig-ni®cant reduction in tumour growth.

Acknowledgements

The authors are grateful to Liisa Peltonen, TuulaOivanen and Merja Lakkisto for expert technical assis-

tance. We thank Dr Claudia Berek (Deutsches Rheu-maforschungszentrum, Berlin) and Professor MartinIwig (Institut fuÈ r Physiologische Chemie, Halle) for thegifts of SP cells and L cells. This study has been ®nan-cially supported by grants from the Academy of Finland(project no 37311), the Turku University Foundation,and Arvo and Inkeri Suominen Foundation.

References

1. Mignatti P, Rifkin DB. Biology and biochemistry of proteinases

in tumor invasion. Physiol Rev 1993, 73, 161±195.

2. Danù K, Grùndahl-Hansen J, Eriksen J, Schnack Nielsen B,

Rùmer J, Pyke C. The receptor for urokinase plasminogen acti-

vator: stromal cell involvement in extracellular proteolysis during

cancer invasion. Portland Press Proc 1993, 6, 239±245.

3. Newell K, McDonnell S, Witty JP, Gaire M, Rodgers WH,

Matrisian LM. The stromelysin subclass of metalloproteinases in

tumour progression and metastasis. Portland Press Proc 1993, 6,

253±261.

4. Rochefort H, Capony F. Alterations of cathepsin D in human

breast cancer. Portland Press Proc 1993, 6, 233±238.

5. Denhardt DT, Greenberg AH, Egan SE, Hamilton RT, Wright

JA. Cysteine proteinase cathepsin L expression correlates closely

with the metastatic potential of H-ras-transformed murine ®bro-

blasts. Oncogene 1987, 2, 55±59.

6. Gottesman MM. Cathepsin L and cancer. Portland Press Proc

1993, 6, 247±251.

7. Kirschke H. Lysosomal cysteine peptidases and malignant

tumours. Adv Exp Med Biol 1997, 421, 253±257.

8. Kobayashi H, Ohi H, Sugimura M, Shinohara H, Fujii T, Terao

T. Inhibition of in vitro ovarian cancer cell invasion by modula-

tion of urokinase-type plasminogen activator and cathepsin B.

Cancer Res 1992, 52, 3610±3614.

9. Yagel S, Warner AH, Nellans HN, Lala PK, Waghorne C, Den-

hardt DT. Suppression by cathepsin L inhibitors of the invasion

of amnion membranes by murine cancer cells. Cancer Res 1989,

49, 3553±3557.

10. Weber E, GuÈ nther D, Laube F, Wiederanders B, Kirschke H.

Hybridoma cells producing antibodies to cathepsin L have

greatly reduced potential for tumour growth. J Cancer Res Clin

Oncol 1994, 120, 564±567.

11. Jean D, Bar-Eli M, Huang S, et al. A cysteine proteinase, which

cleaves human C3, the third component of complement, is

involved in tumorigenicity and metastasis of human melanoma.

Cancer Res 1996, 56, 254±258.

12. Khokha R, Waterhouse P, Yagel S, et al. Antisense RNA-

induced reduction in murine TIMP levels confers oncogenicity on

Swiss 3T3 cells. Science 1989, 243, 947±950.

13. Kook YH, Adamski J, Zelent A, Ossowski L. The e�ect of anti-

sense inhibition of urokinase receptor in human squamous cell

carcinoma on malignancy. EMBO J 1994, 13, 3983±3991.

14. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isola-

tion of biologically active ribonucleic acid from sources enriched

in ribonuclease. Biochemistry 1979, 18, 5294±5299.

15. Portnoy DA, Erickson AH, Kochan J, Ravetch JV, Unkeless JC.

Cloning and characterization of a mouse cysteine proteinase. J

Biol Chem 1986, 261, 14697±14703.

16. Barrett AJ, Kirschke H. Cathepsin B, cathepsin H, and cathepsin

L. Meth Enzymol 1981, 80, 535±561.

17. Mason RW, Gal S, Gottesman MM. The identi®cation of the

major excreted protein (MEP) from a transformed mouse ®bro-

blast cell line as a catalytically active precursor form of cathepsin

L. Biochem J 1987, 248, 449±454.

794 H. Kirschke et al. / European Journal of Cancer 36 (2000) 787±795

18. Langner J, Ansorge S, Bohley P, Kirschke H, Hanson H. Intra-

cellular protein breakdown. I. Activity determinations of endo-

peptidases using protein substrates. Acta Biol Med Ger 1971, 26,

935±951.

19. SoÈ derstroÈ m M, Salminen H, Glumo� V, Kirschke H, Aro H,

Vuorio E. Cathepsin expression during skeletal development.

Biochim Biophys Acta 1999, 1446, 35±46.

20. Srivastava RAK, Schonfeld G. Use of riboprobes for northern

blotting analysis. Biotechniques 1991, 11, 584±586.

21. Kirschke H, Shaw E. Rapid inactivation of cathepsin L by Z-

Phe-PheCHN2 and Z-Phe-AlaCHN2. Biochem Biophys Res Com-

mun 1981, 101, 454±458.

22. Wang B, Shi GP, Yao PM, Li ZQ, Chapman HA, BroÈ mme D.

Human cathepsin F. Molecular cloning, functional expression,

tissue localization, and enzymatic characterization. J Biol Chem

1998, 273, 32000±32008.

23. Kitajima I, Shinohara T, Bilakovics J, Brown DA, Xu X, Ner-

enberg M. Ablation of transplanted HTLV-I tax-transformed

tumors in mice by antisense inhibition of NF-kB. Science 1992,

258, 1792±1795.

24. Wagner RW. Gene inhibition using antisense oligodeoxynucleo-

tides. Nature 1994, 372, 333±335.

25. Wagner EGH, Brantl S. Kissing and RNA stability in antisense

control of plasmid replication. Trends Biochem Sci 1998, 23, 451±

454.

26. Moroni MC, Willingham MC, Beguinot L. EGF-R antisense

RNA blocks expression of the epidermal growth factor receptor

and suppresses the transforming phenotype of a human carci-

noma cell line. J Biol Chem 1992, 267, 2714±2722.

27. Frade R, Rodrigues-Lima F, Huang S, Xie K, Guillaume N, Bar-

Eli M. Procathepsin-L, a proteinase that cleaves human C3 (the

third component of complement), confers high tumorigenic and

metastatic properties to human melanoma cells. Cancer Res 1998,

58, 2733±2736.

28. Haeckel C, Krueger S, Roessner A. Antisense inhibition of uro-

kinase: e�ect on malignancy in a human osteosarcoma cell line.

Int J Cancer 1998, 77, 153±160.

29. Hasegawa S, Koshikawa N, Momiyama N, et al. Matrilysin-

speci®c antisense oligonucleotide inhibits liver metastasis of

human colon cancer cells in a nude mouse model. Int J Cancer

1998, 76, 812±816.

30. Castagnino P, Soriano JV, Montesano R, Bottaro DP. Induction

of tissue inhibitor of metalloproteinases-3 is a delayed early cellular

response to hepatocyte growth factor.Oncogene 1998, 17, 481±492.

31. Ellis LM, Liu W, Wilson M. Down-regulation of vascular endo-

thelial growth factor in human colon carcinoma cell lines by

antisense transfection decreases endothelial cell proliferation.

Surgery 1996, 120, 871±878.

32. Kasid U, Pfeifer A, Brennan T, et al. E�ect of antisense c-raf-1

on tumorigenicity and radiation sensitivity of a human squamous

carcinoma. Science 1989, 243, 1354±1356.

33. Bresalier RS, Mazurek N, Sternberg LR, et al. Metastasis of

human colon cancer is altered by modifying expression of the b-galactoside-binding protein galectin 3. Gastroenterology 1998,

115, 287±296.

34. Adatia R, Albini A, Carlone S, et al. Suppression of invasive

behavior of melanoma cells by stable expression of anti-sense

perlecan cDNA. Ann Oncol 1997, 8, 1257±1261.

H. Kirschke et al. / European Journal of Cancer 36 (2000) 787±795 795