ICANCER RESEARCH 54. 6027-6031, December 1, I994@

Advances in Brief

Cathepsin B Expression and Localization in Glioma Progression and Invasion'

Sandra A. Rempel, Mark L. Rosenblum,2 Tom Mikkelsen, Pei-Sha Van, Kristin D. Ellis, William A. Golembieski,Mansoureh Sameni, Jurij Rozhin, Grace Ziegler, and Bonnie F. Sloane

Henry Ford Midwest Neuro-Oncology Center and Department of Neurological Surgery (S. A. R., M. L. R., T. M., P-S. Y., K. D. E., W. A. G.j and Department of NeurologyfT. MI, Henry Ford Hospital, Detroit, Michigan 48202, and Department ofPharmacology, Wayne State University, Detroit, Michigan 48201 IM. S., J. R., G. Z, B. F. S.]

Abstract Materials and Methods

ThepoorprognosisofhumanmalignantgliomasIsduetotheirinvasionand recurrence,the molecularmechanismsof whichremainpoorlycharacterized. We have accumulated substantial evidence implicating the cys

telne protease cathepsin B in human glioma malignancy. Increases in

cathepsln B expression were observed throughout progression. In primary

brain tumor tissue, transcript abundance (Northern blot analysis) increased In low-grade astrocytoma to high-grade glioblastoma from 3- to6-fold, respectively, above normal brain levels. This increase correlatedwith Increases in protein abundance (from + to +++) as measured byimmunohistochemistry. Furthermore, in glioblastoma ceH lines increases

in transcriptabundance(rangingfrom3- to 12-fold)wereaccompaniedbyincreases in enzyme activity (44-133 nmol/min x mg protein). Altered

subcellular localization was observed both immunohistochemically and byIndirect Immunofluorescence confocal microscopy and was found to correlate with increased grade. In addition, this increase in cathepsin B

expression and altered subcellular localization correlated with histomorphologicalinvasionand clinicalevidenceof invasionas detected by magnetic resonanceimaging.Thesedata support the hypothesisthat cathepsinB playsa role in humangliomaprogressionand invasion.

Introduction

An association between cathepsin B and malignancy has beenreported for human colon (1—5),breast (6—8),prostate (9, 10), and

bladder (1 1) tumors. Changes in expression as measured by changesin mRNA, protein, and activity levels (1—11)as well as alterations inactivation and processing (12, 13)@and intracellular trafficking (8)have been reported. Recent studies on human bladder (1 1), prostate (9,10), and colon carcinoma (5) have demonstrated a heterogeneity instaining for cathepsin B protein, mRNA, and in cathepsin B activity,with the most intense staining and highest activity at the invasive,leading edge of the tumors. These data suggest that the regulation ofcathepsin B in tumors may be altered at more than one level and thatthese alterations may contribute to the invasive phenotype. Promptedby these observations we have examined the expression and localization of cathepsin B in glioma and related the results to the degree oftumor progression. Our data demonstrate changes in cathepsin Bexpression and subcellular localization that correlate with increasedgrade, local invasion, and clinical evidence of invasion thereby implicating cathepsin B in both glioma progression and invasion.

Received 8/19/94; accepted 10/19/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by USPHS Grants CA 36481 and CA 5658 to B. F.

S. andCA62432to M. L R.2 To whom requests for reprints should be addressed, at Neurosurgery Editorial Office,

Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202.3 M. Sameni, J. Rozhin, G. Ziegler, and B. F. Sloane. Transfection of human breast

epithelial cells with c-Ha-ras oncogene delays processing of cathepsin B, manuscript inpreparation.

Tissue Samples. Immediately after surgical removal, specimens were

taken for pathological diagnosis and fixation and embedding while the remaining tissue was snap frozen in liquid nitrogen. Glioma specimens were categorized by grade (II, III, and IV) according to the WHO grading system (14).Informed consent was obtained from all patients or the guardian of that patient.

Northern Blots. To determine whether increases in cathepsin B transcriptabundance correlated with malignant progression in gliomas, Northern blot

analyses were performed using total RNA from normal brain, astrocytoma andoligodendroglioma (grade II), anaplastic astrocytoma (grade III), and glioblastoma multiforme (grade IV) biopsy samples and glioblastoma cell lines. RNAwas extracted using the guanidium thiocyanate procedure (15), quantitated bymeasuring absorbance at 260 nm, electrophoresed through a 1.2% formaldehyde-agarose gel in the presence of ethidium bromide, and transferred toHybond N (16). Following prehybridization, the filter was hybridized to a32P-radiolabeledcathepsin B probe (2), stringency washed, and exposed toX-rayfilm. The abundanceof the 2.1-kilobasecathepsinB was determinedbyquantifying hybridization signals on X-ray film using densitometric analysis asreported previously (16). The abundance for each sample was corrected forvariations in sample loading by normalizing the values to the 18S rRNA signal.The percentage transcript abundance for each sample was expressed as apercentage of the maximum integral value (U87 = 100%). The fold increasein abundance was calculated as a fold increase above levels seen in normaltissue.The meanvalue is presentedfor the tumors.

Immunohistochemistry. Antisera were raised in rabbits (New ZealandWhitemales)againsta cathepsinB-derivedsyntheticpeptide(residues135—147)as describedpreviously(17). IgG fractionswere purifiedand stored at—20°C.The specificities of the IgGs for cathepsin B have been confirmed byslot blotting and immunoblotting using purified human liver and tumor cathepsin B as well as homogenates and/or extracts of human normal and tumortissues(17).The monospecificanti-cathepsinB IgGsrecognizeprocathepsinB3 andsingle- anddouble-chainformsof the matureenzyme in immunoblots(17). Immunohistochemical analyses (18, 19) were performed. Paraffin-embedded tissue sections were subjected to routine deparaffinization and rehydration. Sections were incubated for 10 mm with 3% hydrogen peroxide indistilled water to inactivate endogenous peroxidases. Slides were then placedin a Coplin jar with 10 mM sodium citrate buffer (pH 6.0) and boiled for 3—5

mmin a microwaveovenwitha capacityof 650—720W.Afterthebufferwaschanged, boiling was repeated for an additional 5 mm. Slides were permittedto cool to room temperature (20 min) followed by rinsing in PBS. Allsubsequent steps were performed at room temperature. Sections were thenincubated with 10% normal goat or horse serum for 30 mm to block nonspecific binding sites. The slides were incubated overnight with a 1:500 dilutionof primaryanti-cathepsinB antibodyin PBS. Afterthreewashes in PBS bufferthe slides were incubated for 30 mm with biotinylated secondary antibody(1:200 dilution in PBS) and then washed and incubated for 45 mm at roomtemperature with the Vectastain ABC kit (Vector Laboratories, Burlingame,CA). Finally,the sections were washedandreactedwith diaminobenzidinein0.1 MIris buffer (jH 7.6) with 0.03% hydrogen peroxide, followed by rinsingin tap water, counterstaining, and mounting. Slides were scored and blindlyreviewed by a neuropathologist. Staining intensity was graded as negative (—),weak (+), moderate (++), or strong (+++).

EnzymeActivityAssays. The activityof cathepsinB (Vmax)in cell linesand tissues was determined against Z—Arg—Arg—NHMecas we have described(20).ProteinandDNAcontentsweredeterminedusingthe bicinchoninicacidmethodof PierceChemicalCo. (Rockford,IL)withbovineserumalbuminas

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CATHEPSIN B IN GLIAL TUMORIGENESIS

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Fig. 1. Northem blot analysis of cathepsin B mRNAin normal and tumor brain tissues and glioblastoma celllines. Total RNA was extracted from normal brain,tumor tissues, and exponentially growing cells (15).TotalRNA(10 p@g/1ane)waselectrophoresedthrough1.2% formaldehyde-agarose gel, transferred to a nylonmembrane, and hybridized with a 32P-radiolabeledcathepsin B probe (16). Top, 2.1- and 4.0-kilobase iranscripts were detected in normal brain, low-gradeglioma(A), anaplasticastrocytoma (AA), glioblastoma multiforme(GBM),andcelllines.Bars,positionsof28Sand185 rRNA. Bottom, hybridization to 18S rRNA probe(16).Note:HF24is incorrectlylabeled.It is andwasevaluated as a glioblastoma. HF1 was reclassified as ananaplastic astrocytoma based on our analyses (see “Resuits and Discussion―).

HF66.3).Thusthefoldincreasesshownin Table1mayunderestimatethe actual differences in transcript abundance between matched normal brain and gliomas. Nevertheless, our data suggest that the abundance of cathepsin B transcripts increases during malignant progression of gliomas. The fold increase in transcript abundance variedamong the cell lines ranging from a 3- to 12-fold increase comparedwith levels seen in normal brain (Fig. 1; Table 1).

An exception was tumor HF1. This low-grade astrocytoma had a6-fold increase in transcript levels, i.e., comparable to that seen for thehigh-grade glioblastomas (Table 1). On review of the clinical file ofthe patient it was determined that this tumor was a recurrent tumor atthe site of two previously diagnosed and resected anaplastic astrocytomas. Furthermore, a section taken from the tumor, which wasimmediately adjacent to the Northern blot analysis sample, had aKi-67 (18) proliferative index of 12%, and morphological featureswere consistent with the diagnosis of a glioblastoma with the exception of frank necrosis. This sample therefore has been reclassified torepresent at least an anaplastic astrocytoma and was included in thisgrade for data analysis. Thus the sample used for the initial histomorphological examination was not representative of the most malignantpart of the tumor, a sample of which was analyzed in our Northernblot. These results emphasize the problem of sample heterogeneitywithin glioma tumors. Importantly, our data demonstrate the potentialusefulness of cathepsin B as a diagnostic marker.

Immunohistochemical analysis (19) was performed using a polyclonal anti-cathepsin B antibody which detects both mature and precursor forms of cathepsin B (8, 17). To ensure that cathepsin Bstaining occurred in tumor cells and not in infiltrating macrophages,adjacent sections for all samples were stained with a macrophagespecific monoclonal antibody KP-1 (data not shown). Sections oftumors of various grades were stained and compared with the expression pattern observed in normal brain. Staining of sections of normalbrain showed significant expression of cathepsin B in neuronal cellbodies and processes. However, no positive staining of astrocytes oroligodendrocytes was observed (Fig. 2A). Scattered positive stainingwas noted in microglia in both white and gray matter. Positivecathepsin B staining in low-grade glioma was found to be weak (+),agranular, and perinuclear (Fig. 2B). Cathepsin B expression wasintermediate (+ +) in anaplastic astrocytomas (Fig. 2C). In glioblastomas it was strong (+ + +), and the number of cells staining positivefor cathepsin B ranged from 10 to 90% in different areas within the

standard and the fluorometric procedure of Downs and Wilfinger (21),respectively.

Confocal Microscopy. Intracellular cathepsin B was localized using apreviously described modification (8) of the general immunocytochemicalmethodologies described by Willingham (22). Antisera were raised in rabbits(New Zealand White males) against the mature double-chain form of humanliver cathepsin B (17). The specificities of the IgGs were the same as thecathepsin B antipeptide IgGs. Cells grown on glass coverslips were fixed withmethanol(—2O°C)for 5 mm. After being washed with PBS, cells were blockedwith 2 mg/ml bovine serum albumin in PBS. All subsequent antibody and washsolutions contained 0.1% saponin. Cells were incubated with primary antibodies (rabbit anti-human liver cathepsin B IgG plus, in the double-labelingstudies, mouse anti-@-tubulinIgG1) for 2 h and washed. In controls, preimmune serum was substituted for the primary antibody. After blocking withnormal donkey serum (5% in PBS-0.1% saponin) cells were incubated for 1 h

with fluorescein- or Texas red-conjugated affinity-purified donkey anti-rabbitIgG and fluorescein- or Texas red-conjugated affinity-purified donkey anti

mouse IgG at 20 @Wml.Cells were then washed, mounted with Slow Fade

antifade reagent, and observed on a Zeiss LSM 310 microscope.MRI4 Scans. Scans were blindly reviewed by a neurologist and classified

as either focal with no evidence of regional invasion, locally invasive, ordiffuse with widespread invasion. In addition, each scan was determined as to

whether the tumor was enhancing or nonenhancing with respect to gadoliniumcontrasting agent.

Results and Discussion

Northern blot hybridization with a 32P-radiolabeled cathepsin Bprobe revealed a major 2.1-kilobase transcript and a minor 4.0-kilobase transcript (Fig. 1), as has been observed previously in othernormal and tumor tissues (2, 8, 20). Normalized densitometric analysis was performed using 185 rRNA levels (16) as the standardizinginternal control. The percentage of transcript abundance was normalized to U87 RNA levels as 100%. The abundance of the 2.1- kilobasecathepsin B transcripts in glioblastomas was approximately twice thatseen in a low-grade glioma and anaplastic astrocytomas which in turnhad increases in transcript abundance relative to the normal brainspecimen (Table 1). The fold increases in individual tumors werecalculated relative to the random normal brain sample and not to thematched normal brain, with one exception. In the case of the glioblastoma sample HF66.19 there was an 11-fold increase in transcriptabundance when compared to its matched normal brain tissue (sample

4 The abbreviation used is: MRI, magnetic resonance imaging.

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Table 1 Human gliomas and cell lines: cathepsin B transcript abundance and enzyme activityThe fold increase in transcript abundance for the samples presented in Fig. 1 is presented as the mean ±SD for n = 1 for low grade glioma

n = 6 for glioblastoma multiforme, where n is the number of specimens.,n=9 foranaplasticastrocytoma,andFold

increase FoldincreaseActivityTissue(2.1-kilobase transcript) Cell lines (2.1-kilobase transcript)(nmollmin X mgprotein)Normal

U251MGn344Low-grade3 U251MGp1195gliomaAnaplastic

2 ±2 U8712133astrocytomaGlioblastoma

6 ±4 HF66ND―351bmultiforme

CATHEPSIN B IN GLIAL TUMORIGENESIS

a ND, not determined.b A 14-fold increase in cathepsin B activity was observed in tumor vs accompanying normal brain sample of patient HF66 (data not shown).

tumors (Fig. 2, D and E). The intensity of staining in these cells was

greatly increased (Fig. 2, D and E). We have now performed immunohistochemical analyses on a large number of tumors (16 low-gradetumors, 33 anaplastic tumors, and 33 glioblastomas). These analysesindicate that the results reported here are representative of cathepsin Bexpression for the various grades of glioma.5 Increased levels ofcathepsin B protein accompany increases in transcript abundance, andboth increase during glioma progression.

Whereas most glioblastomas have staining patterns as indicated(Fig. 2, D and E), several tumors also displayed a pattern in whichthe occasional capillary was surrounded by either a single layer oftumor cells that expressed cathepsin B (as in Fig. 2F) or endothehal cells in tumor vessels that expressed cathepsin B (data notshown).5 Our observations suggest that cathepsin B expression inthese tumor and endothelial cells may play a role in angiogenesisin glioblastomas (23).

If cathepsin B plays a role in glioma invasion, one would expecthigh expression at the invading edge of the tumor. In a high-powermagnification (Fig. 2H) of the interface between normal gray matterand tumor (Fig. 2G), positively staining cells were observed in thegray matter. Therefore, expression in the peripheral invading edge ofthe tumor places cathepsin B at the site of local invasion. Furthermore,cathepsin B-expressing tumor cells surrounded vessels in areas ofinfiltrated brain (Fig. 2F) which represent the histological invasionpattern known as secondary structures of Scherer (24).

To determine whether increases in the enzyme detected correlatedimmunohistochemically with increased function (since the antibodiesdetect both pro and mature forms), we assayed cathepsin B activity(20) in frozen sections of normal and tumor tissues and glioblastomacell lines (Table 1). Cathepsin B activity was found to be increased14-fold in glioblastoma versus normal for the matched normal/tumorpair HF66 (Table 1 legend) consistent with an 11-fold increase intranscript abundance and elevated protein levels (Fig. 2, A and D).Enzyme activity was also elevated 8-fold in glioblastoma versusnormal for an additional matched pair HF14O (data not shown).Increasing enzyme activity in the cell lines correlated with increasingtranscript abundance (Table 1).

Because the immunohistochemical staining suggested alterations incellular distribution of cathepsin B in glioblastomas compared withlower grade tumors and because alterations in the intracellular trafficking of cathepsin B have been implicated in tumongenesis of othertumor types (8), we have delineated the subcellular distribution ofcathepsin B using immunofluorescent staining (8) and confocal microscopy (Zeiss LSM 310) analysis. Sections were double stained fortubulin and cathepsin B using secondary antibodies conjugated toeither Texas red or fluorescein. Controls were incubated with

5 T. Mikkelsen, P-S. Yan, K-L Ho, M. Sameni, B. F. Sloane, and M. L Rosenblum.

Immunolocalization of cathepsin B in human glioma: implications for tumor invasion andangiogenesis, manuscript in preparation.

preimmune serum rather than primary antibody. In U251MGn cells,the staining for cathepsin B was localized in the perinuclear region(Fig. 3A). In contrast, in those tumor cells that have higher cathepsinB activity, staining for cathepsin B was distributedthroughoutthecells in both cytoplasmic processes and pennuclear regions (Fig. 3,B—D).Only a weak background fluorescence was observed in thepresence of preimmune IgO and secondary antibodies (data notshown). Cathepsin B is normally localized in lysosomes in the perinuclear regions of cells (8) as observed in the U251MGn cells.However, a redistribution of lysosomes toward the cell periphery

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Fig.2. Immunohistochemicalstainingof cathepsinB in normalbrainand gliomas.A-D, expression of cathepsin B throughoutglioma progression.A, normal brain HF66;x 80. B, astrocytoma HF792603; X 200. C, anaplastic astrocytoma HF39; X 200. D,glioblastoma multiforme HF66; X 200. E and F, patterns of cathepsin B expression inhigh-grade glioblastomas. E, glioblastoma multiforme HF31; X 200. F, glioblastomamultiforme HF5; X 200. Arrow, cathepsin B positively staining tumor cells. G and H,expression of cathepsin B at invading edge. G, glioblastoma multiforme HF31 invadingedge; X 100. White matter to the left, gray matter to the right. H, glioblastoma multiformeHF31; magnification of gray matter at invading edge; X 200.

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analysis of gadolinium-enhanced T1- and T2-weighted MRI scans isused to indicate regions of tumor growth and infiltration. Low-gradeand anaplastic astrocytomas most often present as focal and nonenhancing lesions, whereas malignant glioblastomas present as localand/or diffuse and enhancing lesions (Fig. 4). Often, however, MRIscans are difficult to interpret following the use of radiation therapy

which, along with chemotherapy, is the standard method of postsurgical treatment.Because MRIscans cannotbe relied upon solely thisnecessitates the use of other diagnostic indicators. We have correlatedthe staining intensity and transcript abundance of cathepsin B withclinical features of invasion as determined by MRI (Table 2). Elevatedcathepsin B transcript and protein abundance correlated with local ordiffuse and enhancing images which in turn correlated with increased

grade of tumor, indicating that enhanced cathepsin B expressioncorrelated with clinical evidence of invasion. These data suggest thatenhanced levels of cathepsin B might be predictive of invasion in vivo.

GradeSampleTranscriptabundance

(%)Staining intensityClinicalinvasion by

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HF26HF17HF47HF22HF!HF76HF31HF3HF5OHF667.3

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and nonenhancingFocal and nonenhancingFocalandnonenhancingFocal and nonenhancingFocal and enhancingDiffuse and enhancingDiffuse and enhancingLocal and enhancingDiffuse and enhancingLocal and enhancingDiffuse andenhancinga

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CATUEPSIN B IN GLIAL TUMORIGENESIS

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Fig. 3. Subcellular localization of cathepsin B. Immunocytochemical colocalization ofintracellular cathepsin B and @-tubulinin U251MGn (A), U251MGp (B), U87 (C), andHF66 (D) glioblastoma cell lines. Localization of cathepsin B was perinuclear in the lessinvasive cell line U2SIMGn, whereas cathepsin B staining was present throughout thecytoplasm, including the cell processes in the more invasive cell lines U25l MOp, U87,and HF66. Tubulin staining is green in B and C and red in A and D; cathepsin B stainingis red in B and C and green inA and D. The staining was repeated 3 times with comparableresults. X 1000.

appears to be common in cells that participate in degradative orinvasive processes. These alterations in cathepsin B subcellular distribution predict an increasing mobilization of cathepsin B throughoutglioma progression and suggest that altered trafficking of cathepsin Bin glioblastomas contributes to the malignant invasive phenotype.

Cathepsin B, as would be predicted for a lysosomal protease, has abroad substrate specificity and has been shown to be capable ofdegrading in vitro the extracellular matrix components proteoglycans(25), laminin (26, 27), fibronectin (27), and type IV collagen (27).Whether cathepsin B can degrade laminin, fibronectin, and type IVcollagen in vivo remains unanswered; however, use of cathepsinB-specific inhibitors has established a role for this enzyme in thedegradation of cartilage proteoglycans in vivo (28). This is of interestbecause the extracellular matrix of brain consists predominantly ofproteoglycans (29). Questions remain about the stability of cathepsinB outside the lysosome, yet cathepsinB retainsactivity for extendedtime periods (12 h) at neutral pH in the presence of laminin,fibronectin, and type IV collagen (27) suggesting that this enzymemight be active extracellularly. Since cathepsin B can degrade proteoglycans it may play a direct role in the degradation of brain matrix,or it may activate other proteases capable of degrading brain matrix.Recent studies by Kobayashi et a!. (30, 31) have established thatcathepsin B can activate receptor-bound prourokinase; in the case ofovarian tumors this activation by cathepsin B leads to a proteolyticcascade responsible for invasion of the ovarian tumor cells throughMatrigel. Whether this is the mechanism by which cathepsin Bfacilitates invasion of gliomas is not yet known; however, recent workhas shown that expression of urokinase is also up-regulated in gliomas(32, 33).

The results for the tumor cells in vitro (Table 1) suggest thatincreased cathepsin B levels seen in the primary tumors should alsocorrelate with in vivo evidence of enhanced invasion. Clinically, the

Table 2 Correlation of cathepsin B transcript abundance, staining intensity, and MRIinvasion patterns

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CATHEPSIN B IN GLIAL TUMORIGENESIS

Day, N. Alterations in processing and trafficking of cathepsin B during malignantprogression. In: N. Katunuma, K. Suzuki, J. Travis, and H. Fritz (eds.), BiologicalFunctions of Proteases and Inhibitors, pp. 131—147.Tokyo: Japan Scientific SocietiesPress,1994.

14. Kleihues, P., Burger, P. C., and Scheithauer, B. W. Histological Typing of Tumoursof the Central Nervous System, Ed. 2. Berlin: Springer-Verlag, 1993.

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16. Rempel, S. A., and Johnston, R. N. Steroid-induced cell proliferation is associatedwith increased c-myc proto-oncogene transcript abundance. Development (Camb.),104: 87—95,1988.

17. Mom, K., Day, N. A., Sameni, M., Hasnain, S., Hirama, T., and Sloane, B. F. Humantumour cathepsin B: comparison with normal human liver cathepsin B. Biochem.J., 285:427—434,1992.

18. Key, 0., Baker, M. H. 0., Duchrow, M., Schluter, C., and Gerdes, J. New 10-67equivalent murine monoclonal antibodies MIB1—3prepared against recombinant partsof 10-67 antigen. Anal. Cell. Pathol., 43: 181—186,1992.

19. Hsu, S. M., Raine, L., and Fanger, H. Use of avidin-biotin-peroxidase complex ABCin immunoperoxidase techniques: a comparison between ABC and unlabeledantibody PAP procedures. J. Histochem., 29: 557—580,1981.

20. Rozhin, J., Gomez, A. P., Ziegler, 0. H., Nelson, K. K., Chang, Y. S., Fong, D.,Onoda, J. M., Honn, K. V., and Sloane, B. F. Cathepsin B to cysteine proteinaseinhibitor balance in metastatic cell subpopulations isolated from murine tumors.Cancer Res., 50: 6278—6284,1990.

21. Downs, T. R., and Wilfinger, W. W. Fluorometric quantitation of DNA in cells andtissues. Anal. Biochem., 131: 538—547,1983.

22. Willingham, M. C. Immunocytochemical methods: useful and informative tools forscreening hybndomas and evaluating antigen expression. Focus (Idaho), 12: 62—67,1990.

23. Brem, S., Zagzag, D., Tsanaclis, A. M. C., Gately, S., Elkouby, M-P., and Brien, S. E.Inhibition of angiogenesis and tumor growth in the brain. Am. J. Pathol., 137:1121—1142,1990.

24. Scherer, H. D. Cerebral astrocytomas and their derivatives. Am. J. Cancer, 60:159—198,1940.

25. Morrison, R. I. 0., Barrett, A. J., and Dingle, J. T. Cathepsins Bi and D action onhuman cartilage proteoglycans. Biochim. Biophys. Acta, 302: 411—419,1973.

26. Lah, T. T., Buck, M. R., Honn, K. V., Crissman, J. D., Rao, N. C., Liotta L A., andSloane, B. F. Degradation of laminin by human tumor cathepsin B. Clin. Exp.Metastasis, 7: 461—468,1989.

27. Buck, M. R., Karustis, D. 0., Day, N. A., Honn, K. V., and Sloane, B. F. Degradationof extracellular matrix proteins by human cathepsin B from normal and tumourtissues. Biochem. J., 282: 273—278,1992.

28. Buttle, D. J., and Saklatvala, J. Lysosomal cysteine endopeptidases mediate interleukin 1-stimulated cartilage degradation. Biochem. J., 287: 657—661, 1992.

29. Rutka, J. T., Apodaca, 0., Stem, R., and Rosenblum, M. The extracellular matrix ofthe central and peripheral nervous systems: structure and function. J. Neurosurg., 69:155—170,1988.

30. Kobayashi, H., Schmitt, M., Goretzki, L., Chucholowski, N., Calvete, J., Kramer, M.,Gunzler, W. A., Janicke, F., and Graeff, H. Cathepsin B efficiently activates thesoluble and the tumor cell receptor-bound form of the proenzyme urokinase-typeplasminogen activator (pm-uPA). J. Biol. Chem., 266: 5147—5152,1991.

31. Kobayashi, H., Moniwa, N., Sugimua, M, Shinohara. H., Ohi, H., and Terao, T.Effects of membrane-associated cathepsin B on the activation of receptor-boundurokinase and subsequent invasion of reconstituted basement membranes. Biochim.Biophys.Acts, 1178:55—62,1993.

32. Mohanam, S., Sawaya, R., McCutcheon, I., Ali-Osman, F., Boyd, D., and Rao, J. S.Modulation of in vitro invasion of human glioblastoma cells by urokinase-typeplasminogen activator receptor antibody. Cancer Res., 53: 4143—4147,1993.

33. Yamamoto, M., Sawaya, R., Mohanam, S., Bindal, A. K., Bruner, J. M., Oka, K, Rao,V. H., Tomonaga, M., Nicolson, 0. L., and Rao, J. S. Expression and localization ofurokinase-type plasminogen activator in human astrocytomas in vivo. Cancer Res.,54: 3656—3661, 1994.

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Several lines of evidence (Northern blot analysis, immunohistochemical staining, and activity assays) presented in this report supportthe hypothesis that cathepsin B is a novel marker in glioma progression. Furthermore, alterations in subcellular localization and the expression of cathepsin B at the invading tumor edge are suggestive ofa role for this enzyme in local glioma invasion. In conjunction withMRI scans and traditional histomorphological grading, cathepsin Bmay serve as an important diagnostic marker for human gliomas.Furthermore, our preliminary studies with a series of cathepsin Binhibitors show significant inhibition of this enzyme in vitro andsuggest that this enzyme may eventually prove to be an importanttherapeutic target in glioma patients.

Acknowledgments

We thank Dr. Khang-Loon Ho for performing the Ki-67 analysis and thehistomorphological review for specimen HF!.

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1994;54:6027-6031. Cancer Res   Sandra A. Rempel, Mark L. Rosenblum, Tom Mikkelsen, et al.   and InvasionCathepsin B Expression and Localization in Glioma Progression

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