Cytochemical and biochemical evidence of cathepsin B in malignant,

transformed and normal breast epithelial cells

E. KREPELA*, J. BARTER, D. SKALKOVA

Research Institute of Clinical and Experimental Oncology, Zluty kopec 7, 656 01 Brno, Czechoslovakia

j . VICAR

Department of Medical Chemistry, Medical Faculty, Palacky University, Olomouc, Czechoslovakia

D. RASNICK

Enzyme Systems Products, Dublin, California, USA

J. TAYLOR-PAPADIMITRIOU and R. C. HALLOWES

Imperial Cancer Research Fund, London, UK

• T o whom requests for reprints should be sent

Summary

Human breast cancer cell lines, as well as trans-formed mammary epithelial cells (HBL-100) andgrowth-stimulated normal breast epithelial cellsshowed positive cytochemical reaction with theproteinase substrate 2-(JV-benzyloxycarbonyl-L-arginyl - L - arginylamido) - 4 - methoxynaphtha-lene, in the presence of 5-nitrosalicylaldehyde.The reaction product, small fluorescent granules,was distributed throughout the cytoplasm, in theperinuclear zone, in some cytoplasmic projec-tions, and at the cell surface. Using a panel ofvarious proteinase inhibitors, we found that theformation of the reaction product was an enzy-mic function of a cysteine proteinase.

Using the substrate 7-(N-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methylcoumarin, we

evaluated some biochemical properties of thecysteine proteinase, including pH-activity pro-file, pH stability, apparent relative molecularmass and sensitivity toward various proteinaseinhibitors. We found that the proteinase from thestudied breast epithelial cells exhibited charac-teristics of a mature form of cathepsin B.

Taken together, the cytochemical and bio-chemical data provide evidence that humanbreast epithelial cells of cancer origin, as well asin the transformed or growth-stimulated stateexpress active cathepsin B and compartmentalizeit into specific subcellular sites.

Key words: breast cancer, epithelial cells, mammarygland, cathepsin B, cysteine proteinases.

Introduction

A secreted form of cathepsin B, cysteine proteinase,has been suggested as playing an important role in thedestruction of extracellular matrix by human breastcarcinoma cells (Recklies & Poole, 1982). This en-zyme is released in large amounts from breast carci-noma tissues maintained in organ culture (Recklies etal. 1980) and represents an alkali-stable high MT formof cathepsin B occurring in active (Mort et al. 1980;Recklies et al. 1982; Mort & Recklies, 1986) and latent

Journal of Cell Science 87, 145-154 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

(Mort & Recklies, 1986) molecular variants. Unlikethe human liver cathepsin B, the active stable high MT

cathepsin B variant was not bound by the plasmaproteinase inhibitor o^-macroglobulin, which indi-cated its limited proteolytic activity (Mort & Recklies,1986).

The cellular source of the high Mr cathepsin Bvariants secreted from human breast carcinoma tissuehas been not defined. It has been suggested that thestable active enzyme might be of tumour epithelial cellorigin, since an increased rate of its release was

145

observed in some breast tumour tissue specimens inresponse to estradiol 17-/J (Poole et al. 1980). How-ever, it has been pointed out that the culture mediafrom breast cancer cell lines contained barely detect-able activity of cathepsin B (Poole et al. 1980).

It has been already shown that human epithelialcells derived from primary breast carcinomas andbreast cancer effusion metastases actively invade anddestroy extracellular matrix (Russo et al. 1982; Smithet al. 1985). Recently, it has been demonstrated thatbreast cancer cell lines effectively degrade the endo-thelial basement membrane metabolically labelledwith radioactive amino acid precursors and that thisprocess requires direct cell contact with the basementmembrane (Yee & Shiu, 1986). The precise proteo-lytic mechanism of extracellular matrix degradation bybreast carcinoma cells has not been clarified at themolecular level to date.

At the present time no data have been reportedabout the occurrence of active cathepsin B in humanbreast epithelial cells. Therefore, we designed thisstudy to clarify whether growth-stimulated normalbreast epithelial cells, the transformed mammaryepithelial cell line HBL-100 and different breastcancer cell lines contain active cathepsin B. We usedcytochemical and biochemical approaches to investi-gate the distribution and some properties of theenzyme, respectively.

Materials and methods

Culture conditions of cell lines and organoidsCancer cell lines MCF-7, ZR-75-1, T-47D, MDA-MB-1S7,MDA-MB-231 (reviewed by Lasfargues & Coutinho, 1981)and CAMA-1 (Dobrynin, 1963), and the transformed cellline HBL-100 (Polanowski et al. 1976; Gaffney, 1982;de Fromentel et al. 1985) were cultured in Dulbecco'smodified Eagle medium (Gibco, Paisley, Scotland, UK)supplemented with 15 % foetal calf serum (heat-inactivatedfor 30min at 56°C), 3 - 7 m g m r ' NaHCO3, 300/igmr1 L-glutamine, lOOunitsml"1 penicillin and 100^gml"1 strepto-mycin. Cancer cell lines BT-20 and BT-474 (reviewed byLasfargues & Coutinho, 1981) were grown in supplementedDMEM* containing 10/igml"1 bovine insulin. The cancercell line PMC42 (Whitehead et al. 1983) was grown insupplemented DMEM containing 1/Jgml"1 insulin andlOnmolml"1 2-mercaptoethanol.

The human mammary organoids were prepared by enzy-mic digestion of reduction mammoplasty tissues (histo-pathologically proven to be free of disease) as previouslydescribed (Stampfer et al. 1980) and separated according tosize by filtration through pore-size controlled polyester cloth(Hallowes et al. 1980). The organoids collected on 200-/imfilters were used (i.e. smaller ducts with attached terminalductal lobular units) and were cultured in supplementedDMEM containing 10/Jgml"1 insulin, S^gml"1 humantransferrin, 1/igml"1 hydrocortisone, 5 n g m r ' mouse epi-dermal growth factor (Collaborative Research, Waltham,

MA, USA) and 5/igml ' Fungizone. Only the organoid-derived normal breast epithelial cells in primary culture wereused in this study.

Unless indicated otherwise, all cultures were maintainedin 50 mm plastic tissue culture dishes (Nunc, Denmark) at37 °C in humidified atmosphere of 95% air and 5% CO2.The viability of cells before introduction into subsequentexperiments reached at least 90 % as determined by TrypanBlue exclusion.

Fluorescence cytochemical staining for cathepsin BA modification of the previously described two-step couplingmethod (Dolbeare & Smith, 1977) was used. The semicon-fluent cell cultures growing on glass coverslips were washedwith PBS, covered without prior fixation with a freshlyprepared staining solution (100/il per coverslip) and incu-bated in a humidified box at 37°C for 10-20min. Thestaining solution (osmolarity = 295 mosmoll"1) contained1 mM-Z-Arg-Arg-2-NNapOMe (a sensitive substrate forcathepsin B; from Sigma, St Louis, MI, USA), 0-25mM-DTT, 0-20mM-Na2EDTA, 1 mM-5-nitrosalicylaldehyde (anefficient coupling reagent for liberated 4-methoxy-2-naph-thylamine; from Merck, Darmstadt, FRG) and 100 mM-NaCl in 50mM-sodium Mes buffer, pH6-0. In someexperiments, the cultures were incubated with the stainingsolution for 10min (or for shorter time intervals), thenquickly washed with PBS, covered with substrate-freestaining solution containing 10 fJM cysteine proteinase inhibi-tor (mostly E-64; from Peninsula Laboratories, Belmont,California, USA) and further incubated for 10 min.

Control experiments carried out in parallel included cellcultures incubated for 20 min with the staining solutioncontaining all the components (see above) and variousproteinase inhibitors: (1) 10/JM-E-64; (2) 10jUM-Z-Phe-AlaCH2F, synthesized as described previously (Rasnick,1985); (3) 10/ZM-leupeptin (Sigma); (4) 10/iM-antipain(Sigma); (5) 100/iM-pepatatin A (Sigma) in the presence of2-5%ethanol; (6) 0-5mM-PMSF (Serva, Heidelberg, FRG)in the presence of 2-5% ethanol; and (7) lmM-1,10-phenanthroline (Lachema, Brno, Czechoslovakia).

After the incubations were completed the cells werewashed with PBS, postfixed with 0 ' l % glutaraldehyde inPBS for 10 min, washed quickly in redistilled water and

•Abbreviations used: DMEM, Dulbecco's modified Eaglemedium; PBS, phosphate-buffered saline (containing137mM-NaCl, 2-68mM-KCl, l-47mM-KH2PO4, 645 mM-Na2HPO4, pH7-2); Z-Arg-Arg-2-NNapOMe, 2-(jV-ben-zyloxycarbonyl - L- arginyl - L- arginylamido) - 4- methoxynaph-thalene; Z-Arg-Arg-NHMec, 7-(/V-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methylcoumarin; NH2Mec, 7-amino-4-methylcoumarin; DTT, DL-dithiothreitol; E-64,N - [N - (L - 3 - trans - carboxyoxirane - 2 - carbonyl) - L-leucyl] -agmatine; Z-Phe-AlaCH2F, 3-(/V-benzyloxycarbonyl-L-phe-nylalanylamido)-DL-l-fluoro-2-butanone; Z-Phe-AlaCHN2,3-(iV-benzyloxycarbonyl-L-phenylalanylamido)-L-l-diazo-2-butanone; Z-Phe-PheCHN2, 3-(/V-benzyloxycarbonyl-L-phenylalanylamido)-L-l-diazo-4-phenyl-2-butanone; PMSF,phenylmethanesulphonyl fluoride; RFI, relative fluorescenceintensity.

146 E. Kfepela et al.

mounted in glycerol. The fluorescence microscopy andthe photomicrography were performed with an invertedOPTON ICM405 microscope (Oberkochen, FRG) fittedwith HBO mercury lamp, a Neofluar objective 63 X, and theexcitation BP 450-490 and barrier LP 520 niters.

Cell disintegration and fractionationCell lines growing in monolayers were washed three timeswith PBS and detached from culture dishes with 0-54 mM-Na2EDTA in PBS. The monolayers of normal mammaryepithelial cells were washed with PBS and gently scrapedwith a rubber policeman. The cells were pelleted and onceagain washed with PBS by centrifugation at 190^for 20minat room temperature. The cells were then resuspended in50mM-sodium acetate buffer, p H 5 5 , and exposed to threecycles of freezing (in liquid N2) and thawing (at 4°C). Allthe following manipulations were performed at 0-4°C. Anequal volume of 50mM-sodium acetate buffer, pH5-5,containing 200mM-NaCl was added and the suspensionswere sonicated by three bursts of 10s each. Samples of thehomogenate were taken for enzyme activity assays, andDNA and total protein determinations (see below). Theremaining homogenate was mixed with an equal volume of50mM-sodium acetate buffer, p H 5 5 , containing 100 mM-NaCl and 6 mM-Zwittergent® 3-12 detergent (Calbiochem,San Diego, CA, USA) (buffer S). The samples were slowlyagitated for l h and centrifuged at 120000# for 2h. Thesupernatant was carefully aspirated and the pellet wasthoroughly resuspended in buffer S. Before analysis thesamples were stored at — 70 °C.

Gel chromatographyThe experiments were carried out at 4°C. A column ofSephadex G-75 superfine (Pharmacia, Uppsala, Sweden)(75cmXl-6cm) was equilibrated with 50mM-sodium acet-ate buffer, pH5-5, containing 200mM-NaCl and lmM-Na2EDTA. The samples (0-5 ml) of the 120 000g cellhomogenate supernatant were loaded on the column via aperistaltic pump. The column was eluted with the equili-brating buffer (4-4mlh~1) and 0-5-ml fractions were col-lected. In the separated runs the column was calibratedunder the indicated sample and elution conditions forelution volume (Ve) of six different protein standards:bovine serum albumin (Koch-Light, Colnbrook, Bucks,UK), Mr67000; ovalbumin (Serva), Mr43 000; pep-sin (Sigma), Mr33 000; chymotrypsinogen A (Serva),MT25 700; soybean trypsin inhibitor (Serva), Mr20 100; andcytochrome c (Serva), Mr12500. The void (Vo) and totalbed (Vt) volumes of the column were estimated from theelution positions of thyroglobulin (Pharmacia), M,669 000,and adenosine 5'-phosphate, MT 347-2, respectively. For thedetermination of apparent Mr value a calibration curve wasconstructed by plotting the calculated /Qv values:

K . v = [ ( V e - V o ) / ( V t - V o ) ] ,

against the log Mr of protein standards.

Assay of cathepsin B activity and evaluation of someproperties of the enzyme

The activity of cathepsin B was measured with the substrateZ-Arg-Arg-NHMec using a newly developed endpoint fluor-escence assay working with flat-bottomed 96-well blackpolystyrene microtitre plates and an automated fluorescencereader AM140 (Dynatech Laboratories, Alexandria, VA,USA). The method, including the synthesis of cathepsin Bsubstrate Z-Arg-Arg-NHMec, will be described in detailelsewhere (E. Kfepela & J. Vicar, unpublished).

In the standard assay procedure the cell homogenatesamples, in lOOmM-sodium Mes buffer, pH6-0, containinglmM-DTT and 1 mM-Na2EDTA (MED buffer), werepreincubated (50 yh per well) in the microtitre plates for15 min at 37 ± 0-2 deg. C. The enzymic reaction was startedby addition of 0-2mM-Z-Arg-Arg-NHMec in MED buffer(50/il per well; the substrate solution was kept at 38°C),incubated for 10—20 min at 3 7 ± 0 2 d e g . C and terminatedby addition of lOOmM-sodium monochloracetate in 500mM-sodium acetate buffer, pH4-5 (100\A per well). In controls,the enzyme samples were added after the terminating buffer.The enzymically liberated NH2Mec was measured at 450 nmwith excitation at 365 nm. The instrument was calibratedwith a Fluorescence Reference Disc (Dynatech Labora-tories; Cat. no. 6320083001) having an RFI (relative fluor-escence intensity) corresponding to 600pmol NH2Mec. Theassay was performed under conditions where a linear re-lationship between the RFI and the concentration ofNH2Mec was obtained. The assay was proved to be linearin respect to the reaction time as well as to the amount ofenzyme (up to 10% of the substrate cleaved). Unlessindicated otherwise, the enzyme activity was expressed innkatmg"1 of DNA or in pkatmg"1 of total protein.

For the estimation of the pH-activity profile, the enzymicreaction was started by the addition of preactivated enzymesamples in lOmM-sodium acetate buffer, pH5-5, containing2mM-DTT and 1 mM-Na2EDTA (kept at 38°C), to thebuffered substrate solutions containing 0-2mM-Z-Arg-Arg-NHMec in 200mM-sodium Mes-1 mM-Na2EDTA buffer,pH 4-0—7-4. The reaction mixture (100 /xl per well) wasincubated for 10 min at 37 ±0-2 deg. C and terminated asdescribed for the standard assay. The controls included theenzyme samples added after terminating buffer and theenzyme reacted in the presence of 2f*M-E-64.

The pH stability of the enzyme in cell homogenates wasevaluated as follows. A 20^1 sample of the homogenate wasmixed with 180 \A of deionized and redistilled water and with200/il of 50mM-NaH2PO4-Na2HPO4 buffer, pH6-0, 6-5,7 0 or 7-4. The samples were preincubated at 37°C for up to60 min. Then 500 \i\ of 200 mM-sodium Mes buffer, pH 6 0 ,containing 2 mM-DTT and 2 mM-Na2EDTA was added, andthe preincubation samples were made up to 1 ml with water.In controls without preincubation, the phosphate bufferswere added as the last components to the enzyme samplemixtures. The enzyme activity in preincubation and controlsamples was assayed as described for the standard assay.

Cathepsin B in breast epithelial cells 147

The effect of several proteinase inhibitors on enzymiccleavage of Z-Arg-Arg-NHMec was evaluated with 120 000 £cell homogenate supernatants. The enzyme samples in MEDbuffer, containing various proteinase inhibitors and thecontrols without added inhibitors, were preincubated for15min at 37±0-2deg. C. The enzyme reaction was thencompleted as described for the standard assay. The panelof tested inhibitors was the same as indicated for thecytochemistry of cathepsin B (with the exception of1,10-phenanthroline), and included additional sodiummonochloracetate (BDH Chemicals, Poole, UK), Z-Phe-AlaCHN2 and Z-Phe-PheCHN2 (Bachem Feinchemikalien,Bubendorf, Switzerland). The experiments evaluating theinfluence of D T T and Na2EDTA on the enzymic reactionwere carried out in the absence of one or both of thesecompounds.

The enzymic activity in fractions from gel chromatogra-phy was measured in 15 mM-sodium acetate buffer, pH 5'5,containing 0-3 mM-Z-Arg-Arg-NHMec, 1 mM-DTT, 1 mM-NazEDTA, 50mM-NaCl. The incubations were carried outat 37 ± 0-2 deg. C for 10-20 min.

Other methodsDNA was measured in cell homogenates by the fluorescencemithramycin binding assay (Groyer & Robel, 1980) usingmithramycin A (Sigma) and purified double-stranded calfthymus DNA as standards (kindly given by Dr E. Palec'ek,DrSc , from the Institute of Biophysics, CzechoslovakAcademy of Sciences, Czechoslovakia). Total protein wasdetermined by the method of Markwell et al. (1981) usingcrystalline bovine serum albumin (Koch-Light) as standard.

Fig. 1. Breast cancer cells BT-474 (A,B) and ZR-75-1 (C,D); primary culture of organoid-derived normal breast epithelialcells (E,F). Staining for cathepsin B activity (A,C,E) and inhibition of enzyme activity by IO/ZM-E-64 (B,D,F).Bar, 20 fim.

148 E. Krepela et al.

Results

Cytochemical staining for cathepsin B activity incultured cells

When stained with the substrate Z-Arg-Arg-2-NNapOMe in the presence of 5-nitrosalicylaldehyde,all studied cell cultures showed an accumulation ofbrightly yellow fluorescing microgranules. The micro-granules were distributed at various densities through-out the cytoplasm, in the perinuclear zone, in somecytoplasmic projections, and at the cell surface. Theresults of these experiments with breast cancer celllines BT-20, BT-474, ZR-75-1, MCF-7, with thetransformed HBL-100 cells, and with organoid-derived normal breast epithelial cells are shown in

Figs 1 and 2A,C,E. A substantially improved detec-tion of initial enzyme reaction centres, especially incells with high enzyme activity, was achieved bytreatment with E-64 subsequent to prior incubation ofcells with the substrate staining solution.

The control cell cultures stained for enzyme activityin the presence of 10/ZM-E-64 or Z-Phe-AlaCH2Fremained completely negative; these inhibitors wereeffective at a concentration of 1 ^M (Figs 1, 2B,D,F).Leupeptin and antipain also completely suppressedthe formation of the reaction product in the studiedcells. On the other hand, pepstatin A, PMSF and1,10-phenanthroline were without any obvious inhibi-tory effect at the cytochemical level. The couplingreagent, 5-nitrosalicylaldehyde, did stain proteins,

Fig. 2. Breast cancer cells BT-20 (A,B) and MCF-7 (C,D); transformed mammary epithelial cells HBL-100 (E,F).Staining for cathepsin B activity (A,C,E) and inhibition of enzyme activity by Z-Phe-AlaCH2F at 10/iM (B,F) or 1 ̂Bar, 20fim.

(D).

Cathepsin B in breast epithelial cells 149

400-

0 -

7-6

Fig. 3. pH-activity profiles of the enzyme cleaving thesubstrate Z-Arg-Arg-NHMec in homogenates of T-47D,PMC42 and MCF-7 cells. Inhibition of the enzymicreaction with E-64 at 2/^M is indicated.

particularly those associated with the nucleus, produc-ing a difuse non-specific weak yellow-green fluor-escence.

Biochemical characterization of the studied enzymein cultured cells

In different cell lines and normal breast epithelial cellsthe optimal enzyme activity in the whole cell hom-ogenates was found between pH6-2 and 6-8 (Fig. 3and Table 1). As indicated in Fig. 3, a cysteineproteinase specific inhibitor, E-64 (Barrett et al.1981), completely inhibited the enzymic reaction inthe studied pH range. As shown in Fig. 4, the pHstability of the enzyme in the cell homogenate was afunction of pH and time of preincubation. Theenzyme was rapidly inactivated above pH7-0 (Fig. 4and Table 1).

It was found that Zwittergent® 3-12 detergent at3 mM slightly enhanced the enzyme activity in cellhomogenates (by about 12%). The yields of enzymeactivity in 120 000 # cell homogenate supernatantsrepresented 81 •4-96-8 % of the sum of enzyme activi-ties measured in supernatants and pellets of differentcell lines and normal breast epithelial cells. The rate ofinactivation of the enzyme at pH7-4 measured in120 000 £ cell homogenate pellets (data not shown) wassimilar to that in whole cell homogenates (Table 1).

The gel chromatography of the enzyme from differ-ent cell lines and normal breast epithelial cells on acolumn of Sephadex G-75 resulted in a single sharppeak of activity. The experiment with T-47D cells ispresented in Fig. 5. The apparent MT values of the

Table 1. Some properties of the enzyme cleaving the substrate Z-Arg-Arg-NHMec and its activity in culturedhuman breast epithelial cells

Cells

BT-20CAMA-1MDA-MB-231ZR-7S-1BT-474MCF-7HBL-100PMC42T-47DNBEC

n"

43

33544333

pHoptimumb

6-2-6-66-2-6-46-4-6-66-2-6-86-2-6-46-2-6-46-2-6-46-4-6-66-2-6-86-6-6-8

22-641-349-962-051052-046-362-745-225-1

pH-stabiliry (%)

pH7-0

(18-0-33-6)(32-0-55-8)(30-8-56-8)(55-4-66-7)(36-0-75-9)(45-8-63-6)(24-9-60-5)(44-5-73-8)(30-8-64-5)(8-2-34-4)

6-27-0

16-013-413-721-17-5

20-610-55-4

b.c.f

pH7-4

(3-9-8-6)(2-9-14-4)(9-8-22-9)(10-7-16-0)(9-8-24-9)(15-4-25-4)(3-5-13-0)(10-2-26-8)(5-0-18-2)(2-7-10-2)

ApparentMr

2630026300263002640026 3002630029800272002920027800

Enzyme

(nkatmg"1 of DNA)

1-11 (0-74-1-61)1-21 (1-05-1-41)2-55 (1-32-3-43)3-04 (1-83-4-43)3-13 (2-28-4-63)3-28 (2-21-4-33)4-03 (3-25-5-26)5-29 (2-38-7-32)

13-25 (11-14-14-58)17-52 (0-92-36-39)

activityb'

(pkatmg"

22-959-4

121-2119-2114-9135-3261-0258-8333-0596-6

o.f

"' of total protein)

(8-5-42-0)(56-9-69-3)(55-4-186-0)(80-7-177-0)(80-7-168-8)(117-5-154-4)(169-1-314-9)(210-0-337-6)(299-0-351-2)(70-6-1343-6)

*n = number of independent experiments.bEnzyme activity measured in cell homogenates (see Materials and methods).c % of enzyme activity remaining after preincubation at 37°C for 1 h (see Materials and methods).d Determined by gel chromatography on a column of Sephadex G75 (see Materials and methods)."Enzyme activity measured by the standard assay method (see Materials and methods). The intra-assay coefficient of variation did not

exceed 5%.fData are expressed as mean and range.'NBEC, the organoid-derived normal breast epithelial cells in primary culture.

150 E. Kfepela et al.

100-

20 40Preincubation time (min)

60

Fig. 4. Stability of the enzyme cleaving the substrate Z-Arg-Arg-NHMec in homogenate of PMC42 cells as afunction of pH and time of preincubation.

enzyme from the studied cells lie in the range from26300 to 29800 (Table 1).

The activity of the enzyme in homogenates ofexamined cell lines showed great variability (Table 1).An unexpectedly high variability in enzyme activitywas observed in growth-stimulated normal breastepithelial cells in primary culture (Table 1).

The enzyme from different cell lines and normalbreast epithelial cells was fully active in the presence ofboth D T T and Na2EDTA. The most reduced enzymeactivity, ranging from 1 to 29 % of the activity ofcontrol incubations, was found when both D T T andNa2EDTA were omitted from assayed enzymesamples. After preincubation with various proteinaseinhibitors (in the presence of D T T and Na2EDTA),the enzyme reaction was almost completely inhibitedby E-64, Z-Phe-AlaCH2F and leupeptin (Table 2).Strong inhibitory effects were exhibited also byZ-Phe-AlaCHN2, antipain and sodium monochlor-acetate. On the other hand, the compounds Z-Phe-PheCHN2, PMSF and pepstatin A produced onlyslight inhibition of the enzyme reaction (Table 2).

Discussion

It has been described previously that N-terminallyprotected diarginyl substrates differing in the C-terminal leaving group, or tripeptide derivatives withan alanine residue in the position P3 are sensitivesubstrates for cysteine proteinases, cathepsins B, I, Kand M (McDonald & Ellis, 1976; Mort et al. 1980;Singh & Kalnitsky, 1980; Barrett & Kirschke, 1981;

120- -0-01

- 0

80Elution volume (ml)

120 160

Fig. 5. T-47D cells. Sephadex G-7S gel chromatography of the enzyme cleaving the substrate Z-Arg-Arg-NHMec in120 000£ cell homogenate supernatant. The elution positions of different protein standards are indicated.

Catkepsin B in breast epithelial cells 151

Pontremoli et al. 1982; Kalnitsky et al. 1983;Kirschke et al. 1984; Liao & Lenney, 1984; Smith,1984), and for serine proteinases, trypsin (McDonald& Ellis, 1976) and plasma kallikrein (Smith, 1984). Inthis study we have demonstrated that human breastepithelial cells, regardless of their origin from cancer-ous or normal breast tissue, contain a proteinaseactivity cleaving Z-Arg-Arg-arylamide substrates. Inorder to determine the mechanistic type of the pro-teinase(s) involved in the cytochemical reaction weused various class-specific proteinase inhibitors. Theformation of the microgranular fluorescent reactionproduct in breast epithelial cells was not suppressed byPMSF, pepstatin A and 1,10-phenanthroline, i.e. byinhibitors of serine, aspartic and metallo proteinases,respectively (Barrett & McDonald, 1980). The com-plete inhibition of the hydrolysis of Z-Arg-Arg-2-NNapOMe in the presence of a specific inhibitor ofcysteine proteinases, E-64 (Barrett et al. 1981, 1982),provided evidence that the enzyme reacting in studiedcells belongs to the class of cysteine proteinases. Thiswas confirmed biochemically; the enzyme was acti-vated by D T T and was strongly inhibited by Z-Phe-AlaCHN2, another specific inhibitor of cysteine pro-teinases (Green & Shaw, 1981).

The biochemical studies of the cysteine proteinasein breast epithelial cells provided evidence about itsnature. As compared with cathepsin I (I here denotesIowa), which is very stable at alkaline pH and resistantto inhibition by leupeptin (Singh & Kalnitsky, 1980;Kalnitsky et al. 1983), the activity of the enzyme frombreast epithelial cells was rapidly lost under similarconditions. In contrast to cathepsin K, which has anative Mr value of about 650000 and is inhibitedonly moderately by Z-Phe-AlaCHN2 at 1 /UM (Liao &Lenney, 1984), the proteinase from breast epithelial

cells had an apparent native MT in the range of 26 300to 29 800 and was inhibited strongly by 1 /IM-Z-Phe-AlaCHN2- Unlike cathepsin M, which is stable atalkaline pH (Pontremoli et al. 1982, 1983), the cys-teine proteinase from studied cells was rapidly inacti-vated at pH7-4. Taken together, the biochemicalcharacteristics of the cysteine proteinase in breastepithelial cells are closely related to the properties ofcathepsin B with respect to Mr value (Ritonja et al.1985), pH optimum, pH stability, substrate specificityand activation by DTT and Na2EDTA (Barrett,1973; Mort et al. 1980; Barrett & Kirschke, 1981),and sensitivity to inhibitors including E-64 and Z-Phe-AlaCHN2 (Barrett et al. 1982), Z-Phe-AlaCH2F(Rasnick, 1985), leupeptin (Knight, 1980) and anti-pain (Evans & Etherington, 1978).

The rapid inactivation of cathepsin B at pH 7-4 anditsMr value argue for its proteolytically mature state atsites of its distribution in studied breast epithelialcells. The granular pattern of distribution of cathepsinB initial reaction centres in breast epithelial cells isconsistent with the occurrence of the enzyme inlysosomes and secretory granules, as described forother cells (Smith & Van Frank, 1975; Bird et al.1977; Mort et al. 1981; Graf & Strauli, 1983;Docherty £< a/. 1984; Taugner et al. 1985). However,we regularly found the fluorescent microgranularreaction product at the extreme cell periphery. Thissuggests that in viable breast cancer cells, as well as intransformed mammary epithelial HBL-100 cells andgrowth-stimulated normal breast epithelial cells, theproteinase cathepsin B could be associated (at least invitro and at certain times) with specialized domains ofthe plasma membrane. It is clear, however, that theassociation of the active cathepsin B with the cellsurface should be precisely clarified at the electron

Table 2. Effect of various proteinase inhibitors on the enzyme cleaving the substrate Z-Arg-Arg-NHMec in 120000%homogenate supematants of cultured human breast epithelial cells

Added compound

NoneE-64Z-Phe-AlaCH2FZ-Phe-AlaCHN2

Z-Phe-PhcCHN2

LeupeptinAntipainSodium

monochloracetatePepstatin Ab'd

PMSP='d

Concentrationin preincubation

(mM)

0-0010-0010-0010-0010-0010-0011-0

0 11-0

BT-20

100016

7806

33

9181

CAMA-1

100113

9209

30

9679

MDA-MB-231

100015

960

1236

9380

% of enzyme activity

ZR-75-1

100015

922

1136

9178

BT-474

100126

861

1235

9583

MCF-7

10002

1094

11240

9086

HBL-100

100002

921

1232

9986

PMC42

100104

932

1433

9989

T-47D

100015

892

1339

9784

NBEC

100014

7939

31

9995

* NBEC, the organoid-derived normal breast epithelial cells in primary culture.b In the presence of 4% ethanol.c In the presence of 2% ethanol.d Ethanol at these concentrations did not influence the enzyme activity.

152 E. Kfepela et al.

microscopic level, and such studies are in progress inour laboratory. Previous biochemical studies of sub-cellular distribution of cathepsin B in human ectocer-vical cancer cells (Pietras & Roberts, 1981), and mousetumour cells and tissues (Cavanaugh et al. 1983;Sloane et al. 1986), indicated the presence of activecathepsin B in the plasma membrane.

The activity of cathepsin B in malignant and trans-formed breast epithelial cell lines, as well as in growth-stimulated normal breast epithelial cells obtained frommammary glands of different individuals, was highlyvariable. We have made no attempt in this study tocompare quantitatively the levels of enzyme activityamong normal, transformed and malignant breastepithelial cells. The quantitative comparison of cath-epsin B activity expressed by malignant and benignhuman breast epithelial cells requires investigation inrespect of cells obtained from the same patient at thesame time. In addition, the influence of in vitroconditions on the enzyme activity should be examinedat different numbers of passages. The present studyprovides qualitative evidence that malignant, as well astransformed and normal breast epithelial cells, expressactive cathepsin B and compartmentalize it intospecific subcellular sites within the cytoplasm and atthe cell surface.

It has been postulated that cathepsin B is a multi-targeted protein in cells in which it is expressed (SanSegundo et al. 1986). It will be important now tostudy whether cathepsin B, in respect of its targetedforms, plays a role in the invasive and destructiveactivities of breast cancer cells upon their interactionwith extracellular matrices.

We thank Mrs Tereza Kamenickd and Miss HanaFafflkovS for excellent technical assistance, and Dr JanKovaffk and Dr Jan Zaloudfk for their kind encouragementduring this study.

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{Received 22 July 1986-Accepted 16 October 1986)

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