The Osteoclast-Associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1

Home , Cathepsin K

CANCER RESEARCH57. 5386-5390. December I, 19971 The Osteoclast-associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1

Amanda J. Littlewood-Evans,' Graeme Bilbe, Wayne B. Bowler, David Farley, Brenda Wlodarski, Toshio Kokubo, Tetsuya Inaoka, John Sloane, Dean B. Evans, and James A. Gallagher

Novartis Pharina AG, CH-4002 Base!. Switzerland (A. I. L-E., G. B., D. F., D. B. El; Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology 1W. B. B., B. W., J. A. G.J, and Department of Pathology If. S.), University of Liverpool, Liverpool 5.69 3BX, United Kingdom [W. B. B., B. W., J. S., J. A. G.]; and Takarazuka Research Institute, Novartis Pharma lid., Takarazuka 665, Japan IT. K., T. LI

ABSTRACT (ZR75â€”l,Hs578Tduct, BT474, ZR75â€”30,BT549, and T-47D), adenocarci noma (MDA-MB-231, SK-BR3, MDA-MB-468, and BT2O), and breast car Human cathepsin K is a novel cysteine protease previously reported cinoma[MVLNandMTLN,transfectedMCF-7-derivedclonesobtainedfrom to be restricted in its expression to osteoclasts. Immunolocalization of Dr. J. C. Nicolas (Unit de Recherches sur Ia Biochemie des Steroides Insern cathepsin K in breast tumor bone metastases revealed that the invading U58, Montpelier, France), and MCF-7]. The MCF-7 cells were obtained from breast cancer cells expressed this protease, albeit at a lower intensity two different sources, ATCC and Dr. Roland Schuele (Tumor Klinik, Freiburg, than in osteoclasts. In situ hybridization and immunolocalization stud Germany). These are indicated in the figure legend as clone 1 (ATCC) and ies were subsequently conducted to demonstrate cathepsin K mRNA clone 2 (Dr. Schuele). Other breast lines consisted of HBL-lOObreast epithe and protein expression in samples of primary breast carcinoma. Ex hal line isolated from the milk of a nursing mother, two different cultures of pression of cathepsin K mRNA was confirmed by reverse transcription MCF1OA(from two laboratories in Novartis Pharma AG) derived from mam PCR and Southern analysis in a number of human breast cancer cell mary tissue of a patient with fibrocystic breast disease, the human mammary lines and in primary human breast tumors and their metastases. As epithelial line HMEC4144 (Clonetics, San Diego, CA), and MTSVI.7neo, a this protease is known to degrade extracellular matrix, including bone nonmalignant human breast-derived cell line (6). RNA samples from various matrix proteins, it is possible that cathepsin K may contribute to the primary breast tumors and their metastases were obtained from the Cancer Invasive potential of breast cancer cells, Including those that metasta Tissue Bank Research Center (University of Liverpool). All primary carci size to bone. Thus, cathepsin K may be a potential target leading to the noma tissues were graded by the modified Bloom and Richardson system (7). design of novel drugs for cancer therapy. Immunohistochemistry. Blocks of tissue were fixed with formaldehyde for 24 h prior to paraffin embedding and were further subjected to fixation in INTRODUCTION methacarn. Paraffin sections (5 @.tm)weremounted onto Vectabond (Vector Laboratories, Petersborough, United Kingdom)-treated slides and dried over Cathepsins are a family of proteases that have been proposed to night in a 50Â°Covenbefore dewaxing with xylene. The immunohistochemistry be involved in the progression of a number of disease processes was performed as described previously (5). A specific chicken anticathepsin K including rheumatoid arthritis (1) and tumor invasion (2). Re antibody was used for the immunostaining. This antibody has been character cently, a novel cysteine protease, cathepsin K, was cloned from ized and validated for selectivity by Western blot analysis, which revealed that rabbit and human osteoclast-derived libraries (3, 4) and was found the antibody reacted only with cathepsin K and not with any other cathepsins to exhibit a variable amino acid homology to other members of the (5, 8). cathepsin family, ranging from 17% with cathepsin D to 48% in Situ Hybridization. An EcoRlJSall cDNA fragment corresponding to amino acid homology with cathepsin S. the first 455 bp from the ATGof the humancathepsin K gene was cloned We have previously demonstrated the osteoclast-specific expres into Bluescript KS+ and checked in GenBank to ensure that there was no sion of cathepsin K using immunolocalization and in situ hybridiza homology to other sequences that would result in cross-hybridization of the tion techniques in various normal and bone disease conditions and probes. Sense and antisense RNA probes for in situ hybridization were labeled with the SP6, T7, T3 in vitro transcription kit with DIG-labeled also in multinucleated cells in giant cell tumor of bone (5). However, UTP (Boehringer Mannheim) according to the manufacturer's instructions. in sections of human bone in which skeletal breast cancer metastases The RNA probes were stored at â€”20Â°C. were present, we have also observed a positive immunoreactivity, Freshly excised tissue from surgical procedures was submerged in pre albeit oflower intensity, in the breast cancer cells that had invaded the chilled n-hexane, transferred into sterile 50-mi falcon tubes, and stored at bone tissue. This unexpected observation has lead us to investigate â€”70Â°Cuntil use. A cryostat was cooled to â€”35Â°Cand 10 micron sections were further the possible association of the expression of cathepsin K in cut and mounted onto 3-aminopropyltriethoxysilane-coated slides. The in situ other human breast cancer tissues and cells. In the current study, we hybridization procedure was performed as described by Komminoth (9). For therefore analyzed the expression of cathepsin K using immunocyto the detection of the DIG-labeled probes, the protocol of the manufacturer's kit chemistry, in situ hybridization, and RT-PCR2 in surgically removed was used (Boehringer Mannheim). The reaction was stopped in 10 mMTris, 1 primary breast tumors, bone metastases, and human breast cancer cell mM EDTA, pH 8.1. Slides were dipped briefly in water, counterstained with lines cultured in vitro. 1% (w/v) light green in 1% acetic acid, mounted, and photographed. RT-PCR Analysis. Total RNA was reverse transcribedaccordingto the manufacturer's instructions (Promega). For the primary breast tumor samples MATERIALSAND METHODS and their metastasis, 2.5 .tg of total RNA were used for cDNA synthesis in a 50-pi reaction volume. Two primers for cathepsin K were designed and used Materials. Breastcancer cell lines (purchasedfrom ATCC unless other to amplify an expected product of 617 bp (sense, 5'-TFC CCG CAG TAA wise stated) were classified into subtypes: infiltrating ductal carcinoma TGA CAC C; antisense, 5'-lTF CCC CAG iTT TCT CCC C). In a [email protected] reaction volume, the following components were added: 10 @.dofPCR buffer Received 7/10/97; accepted 10/1/97. (lOX stock, Boehringer Mannheim), S @tlofDMSO, 2 pi ofdNTP mmxture (10 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 with mM stock), 0.5 i@gof each primer, 0.5 @tlof Taq polymerase (Boehringer 18 U.S.C. Section 1734 solely to indicate this fact. Mannheim), water to 97 pJ, and 3 @dofreverse-transcribed RNA. The PCR 1 To whom requests for reprints should be addressed. at Friedrich Miescher-Institute was performed so that the amplification was in the log phase. The conditions R1066.4. 16, Maulbeer Strasse, Basel. CH4002, Switzerland. Phone: 061-6977876; Fax: of denaturation, annealing, and extension used were 94Â°Cfor 3 mm followed 061-6973976; E-mail: liulaml @fmi.ch. 2 The abbreviations used are: RI-PCR, reverse transcription PCR: ATCC, American by 35 cycles of 94Â°CforI mm, 48Â°Cfor1.5 mm, and 72Â°Cfor2.5 mm. At Type Culture Collection; DIG, digoxygenin. the end of the cycling phase, a further extension period of 10 mm at 72Â°Cwas 5386

CAThEPSIN K IN BREAST CARCINOMA @ A@' %:@ B @@@ â€œ*s.â€¢ @â€˜ -a, @. I â€œâ€¢

%4@@@,;;lT;s@1 @. ;â€˜.-â€˜â€˜s@tai11:ss(.,...@ J.;@ P@

@@@ @@â€˜,â€˜:â€¢ V â€˜â€¢@t'@rAl@@@:? a 1:5.

@ 5â€¢ â€¢ â€˜ â€¢%@ 40 @ A ., tt. â€˜.. @.@: # , (5*1@. @a D .S

A@.

E L@' F S 4'

I â€˜a

â€˜A (@â€˜

@ ., ,

@ A .â€˜a5'@'

â€˜S @ .

@ G :@ ;. : .. H

@ , .. â€¢,@@:â€˜;@. @ : â€˜@

@% @ .â€¢;. -a@t#4 5' ; â€”. I . â€¢ _ @@@@@ . : -. :@ :,@

Fig. 1. Immunoreactivity of cathepsin K in breast cancer cells. A, immunolocalization of cathepsin K in a skeletal metastasis of breast tumor. Positive staining can be seen in osteoclasts (medium arrows), adjacent to bone matrix (small arrow), and in breast cancer cells (large arrow), present in the marrow space. B, invasive carcinoma of no special type (grade 3) showing strong cytoplasmic positivity. The stromal cells are negative. C, intravascular, probably intralymphatic, tumor showing strong cytoplasmic positivity (arrow). The adjacent arteriole shows positivity of the smooth muscle coat. D, nonneoplastic terminal duct lobular units showing microcystic change. There is cytoplasmic positivity that is restricted to the myoepithelial cells. E, a normal terminal duct lobular unit showing no positivity. F, ductal carcinoma in situ of high nuclear grade and comedo growth pattern showing strong cytoplasmicpositivity.Thereisweakstainingofthestromalmyofibroblasts.G,sametumorasshowninB,exhibitingnegativityforcathepsinKinthisparticulararea.Thereis vascular invasion in this field, and the intravascular tumor is also negative. H. invasive carcinoma of no special type (grade 2) showing strong cytoplasmic positivity. The stroma is negative except for the pericytes of small blood vessels.

5387

CAThEPSIN K IN BREAST CARCINOMA

A B A

@ . S @ , @: @A @ @I. I-@; . .,.@ @ A . @_.,%,

@ @â€˜I,. â€œa

.* @ â€˜ @1 â€˜ Fig. 2. Localization of cathepsin K mRNA in an invasive ductal carcinoma (grade 2). All sections were counterstained with 1% (w/v) light green in 1% (vlv) acetic acid. A, in situ hybridizationwitha DIG-labeledcathepsinKantisenseRNAprobe.TheepithelialcellsareexpressingcathepsinKas visualizedbythepurplecoloration(arrow).B,serialcathepsin K sense RNA probe hybridized section. This shows no background hybridization of the sense probe.

performed, the PCR reactions were electrophoresed on a I% (wlv) agarose gel, NaCI. The membrane was hybridized at 42Â°Covernight with the full-length and the bands were visualized with ethidium bromide. cathepsin K cDNA probe labeled with [32P]dATP (Amersham). Southern Analysis. The amplified DNA products were transferred over night onto a Hybond membrane (Amersham Corp.) in 0.4 MNaOH and 0.6 M RESULTS

Immunolocalization of Cathepsin K in Metastatic and Primary â€”@ A us.) 10 Breast Cancer Tissues. Fig. IA demonstrates the immunohisto @ ,, 0 @ .5 chemical localization of cathepsin K in breast cancer cells of a zz.! bone metastasis in the bone marrow cavity. Although the intensity @ @-.@OO of the staining was lower than that observed in the osteoclasts, the protein was clearly present within the tumor cells. This observation led us to extend our study, investigating the expression of cathep sin K in primary breast tumors (Fig. 1, Bâ€”H).In sections of all five primary carcinomas and the one metastatic carcinoma studied, moderate to strong positivity was seen in the cytoplasm of between 20 and 90% of tumor cells. Staining was heterogeneous, however, being present in some parts of the tumor (Fig. 1, B and H) but not in others (Fig. 1G). Both positive and negative cathepsin K im munostaining was observed in intravascular tumor (Fig. 1, C and G). Insufficient cases were studied by this method to determine S â€¢â€¢@ ISSIN whether there was any relationship between cathepsin K immu nopositivity and any other pathological characteristics, such as B tumor grade, estrogen receptor status, or metastatic potential. Im << munoreactivity was also localized in the cytoplasm of smooth @ ,@0 ,@0 Q (@> @ La. LI. = W muscle cells forming the walls of stromal blood vessels (Fig. 1C) @ 0 0 m I- and in myofibroblasts (Fig. iF). Four cases of ductal carcinoma in situ accompanying the invasive carcinoma or occurring in the absence of invasive disease showed similar heterogeneous positiv ity (Fig. iF). In sections of two normal breast tissue samples, immunostaining was either negative or only weakly positive, lo 617 bp â€” calized either in the epithelial or myoepithelial cells (Fig. 1, D and E) of a limited number of lobules or ducts. Sections of a normal male breast were completely negative (data not shown). No stain ing was observed with the omission of the primary antibody, indicating the specificity of the immunolocalization reaction (data 617bp not shown). In Situ Localization in Primary Breast Carcinomas. In situ hybridization of cathepsin K mRNA showed a histological pattern of Fig. 3. RT-PCR of human breast cancer cell lines (A) and noncancerous breast lines expression similar to that observed for the protein as assessed by (B). A, top panel, ethidium bromide-stained 1% (wlv) agarose gels. A I-kb DNA ladder immunohistochemistry. Hybridization with the antisense RNA probe marker (Life Technologies, Inc.) was used. The 6l7-bp product is evident in various breast cancer lines. B, two sources of MCF-7 were used: ATCC and Dr. R. Schuele (see revealed an intense signal within the tumor cells of an invasive ductal â€œMaterialsandMethodsâ€•).Bottom panel, Southern blot of the agarose gel probed with the carcinoma (Fig. 2A). No signal was observed in the serial section full-length cathepsin K probe. B, top panel. agarose gel showing no PCR products in incubated with the sense RNA control (Fig. 2B). noncancerous breast lines. Two different laboratories in Novartis Pharma AG provided the MCF-lOA cell. Bottom panel. Southern analysis showing no hybridization with the RT-PCRAnalysis of Human Breast Cancer Cell Lines. RT cathepsin K probe. PCR analysis using specific cathepsin K primers revealed the pres 5388

RT-PCRSampleTable IDetails ofprimary breast carcinomas usedfor demonstrated with cathepsins D (1 1), B, and L (12). In the present description1no. Tumortissue study, none of the breast epithelial cell lines derived from noncancer ducts] carcinoma, grade 3 ous breast tissue expressed cathepsin K as assessed by RT-PCR 2 Infiltratinglobularcarcinoma,grade2 followed by subsequent Southern analysis. However, weak immu 3 Infiltrating ductal carcinoma, grade 3 nopositivity was seen in histological sections of normal ducts and 4 Infiltrating ductal carcinoma, grade 3 5 Infiltrating ductal carcinoma, grade 3 lobules, principally in the myoepithelial cells. The reasons for this 6Infiltrating Infiltrating ductal carcinoma, grade 2 apparent discrepancy are not yet clear, but myoepithelial differentia tion, stromal contact, and the proximity of carcinoma are possible factors based on current observations. carcinomasSampleTable 2 Details of metastatic breast Our cathepsin K in situ hybridization and immunolocalization data detailsIno.Tumor tissue parallel other studies that have investigated the expression of cathep of infiltrating ductal carcinoma, grade 2, sin D in breast cancer tissue (13). Cathepsin D is a ubiquitously removedfromauxilIarylymphnode expressed acidic protease that is overproduced by breast cancer cells 2 Metastasis of infiltrating ductal carcinoma. grade 3, removed from auxilIary lymph node and has been associated with the proliferative and invasive potential 3 Metastasis of infiltrating ducts] carcinoma, grade 3. of breast carcinomas ( 14). Interestingly, this protease was present in a removed from auxillary lymph node high proportion of, but not all, breast tumors (15). Others have 4Metastasis Metastasis of infiltrating ductal carcinoma, grade 3. removedfromauxilIarylymphnode demonstrated that the occasional cathepsin D immunostaining ob served in the stromal cells correlated with the increased likelihood of tumor metastasis and decreased survival time of the patient (16, 17). ence of cathepsin K mRNA in 12 of 14 hormone-dependent and Both cathepsin K and cathepsin D are active under acidic condi hormone-independent human breast cancer cell lines cultured in vitro tions and have been shown to be capable of degrading extracellular as visualized by a PCR product of the expected 6l7-bp size (Fig. 3A). matrix proteins ( 15, 18), providing a possible mechanism by which The primers were specifically designed to span an intron3 to eliminate breast cancer cells can metastasize to secondary sites. Indeed, bone, the possibility of genomic DNA contaminants in the RNA prepara which is a frequent site for breast tumor metastasis, is rich in os tions. The agarose gel was Southern blotted and probed with a specific teonectin and collagen, which are both substrates for the degradative cathepsin K cDNA probe to confirm the identity of the 6l7-bp PCR action of cathepsin K. The identification of cathepsin K in primary band (Fig. 3A). Furthermore, several of the 6l7-bp fragments from breast cancer cells and in cells that are metastatic to the bone indicates different lines were subcloned and sequenced to confirm unequivo that this protease may play a contributory role in tumor cell invasion. cally their identity as cathepsin K transcripts. When the RT-PCR The mechanisms by which breast cancer metastases induce local analysis was performed on noncancerous human breast lines MCF bone destruction are unknown. Animal models have been used to lOA, HBL100, HMEC4144, and MTSV1.7neo, we were unable to demonstrate that tumor cells induce osteolysis by enhancing the detect any amplifiable PCR products or hybridization signal following process of osteoclast-mediated bone resorption (19). It is likely that Southernanalysis(Fig. 3B). tumor cells release soluble factors, such as cytokines and growth RT-PCRof Tumor Samples.RT-PCRamplificationofcDNAs factors, which regulate bone cell metabolism within the vicinity of the derived from five of six primary breast carcinomas and all four metastasis (20â€”22). There is also evidence that tumor cell lines, axillary lymph node metastases studied (Tables 1 and 2) generated a including MCF-7, and tumor-associated macrophages can directly 617-bp amplifiable PCR product, the specificity of which was con resorb bone (23â€”26).In the present study, we demonstrated by RT firmed by Southern analysis and subsequent hybridization with the PCR analysis that MCF-7 cells express cathepsin K, thus providing an full-length cathepsin K cDNA probe (Fig. 4). indication that breast tumor cells may be able to directly resorb bone via the release of this proteolytic enzyme. Future studies using neu DISCUSSION tralizing antibodies, antisense oligonucleotides, or inhibitors of ca thepsin K should help to clarify this potential association. Thus, This is the first report demonstrating the expression of cathepsin K in human breast cancer tissue and cell lines. Previously, this protein has only been identified in osteoclasts (5, 10), the cells responsible for bone resorption.Drake et a!. (10) failed to detect cathepsinK in a panel of normal human tissues including lung, kidney, heart, spleen, :@ Primarybreast Breast and liver. In addition, they observed little or no expression of cathep sin K in cDNA libraries derived from ovarian, prostatic, and pancre atic cancers. In the present study, cathepsin K immunopositivity was seen in normal breast tissue, but this was weak in intensity and limited to epithelial and myoepithelial cells. It thus seems likely that the @ â€” 617bp detection of cathepsin K in normal tissues may be determined by the origin of the tissue and the sensitivity of the detection method used. In the current study, we localized cathepsin K in primary and metastatic breast tumors by the techniques of in situ hybridization and immunolocalization. Furthermore, we amplified cathepsin K by RT

@ PCR in breast cancer cell lines and in primary and metastatic breast â€”â€” @â€” â€”617bp tumors, indicating that the gene for this protease is expressed in these tissues. It would be of interest to perform a quantitative study to Fig.4. RT-PCRof breasttumorsamples.Toppanel,ethidiumbromide-stained1% correlate cathepsin K levels with tumor invasiveness, as others have (w/v) agarose gel. A I-kb DNA ladder(Life Technologies, Inc.) was used as a marker. The 6l7-bp RT-PCR product was present in the positive control tissue, osteoclastoma, and in five of six primary and four of four metastatic breast tumors. Bottompanel, Southern blot 3 Unpublished observations. of the agarose gel probed with the full-length cathepsin K probe. 5389

cathepsin K may provide a novel target for new therapeutic ap Lee-Rykacewski, E., Coleman, L., Rieman, D., Barthlow, R., Hastings, G., and proaches and should be assessed further as an alternative marker for Gowen,M.CathepsinK,but not CathepsinsB,L or S. is abundantlyexpressedin human osteoclasts. J. Biol. Chem., 271: 12511â€”12516,1996. metastatic potential of breast cancer and outcome of disease. I I. Johnson, M. D., Tom, J. A., Lippman, M. E., and Dickson, R. B. The role of cathepsin D in the invasiveness of human breast cancer cells. Cancer Res., 53: 873â€”877, 1993. ACKNOWLEDGMENTS 12. Scaddan, P. B., and Dufresne, M. J. Characterisation of cysteine proteases and their endogenous inhibitors in MCF-7 and adriamycin-resistant MCF-7 human breast We thank Dr. John Carron, Mathias Senften, and Brenda Wlodarski for their cancer cells. Invasion Metastasis, 13: 301â€”313, 1993. help in preparing the figures. Thanks to Christine Weber for help in preparing 13. Escot, C., Zhao, Y., and Rochefort, H. Cellular localization by in situ hybridisation of cDNA samples. cathepsin D, stromelysin 3, and urokinase plasminogen activator RNAs in breast cancer. Breast Cancer Res. Treat., 38: 217â€”226,1996. 14. Rochefort, H. Oestrogens, proteases and breast cancer. From cell lines to clinical REFERENCES applications. Eur. J. Cancer. 30A: 1583â€”1586,1994. 15. Rochefort, H. Cathepsin D in breast cancer. Breast Cancer Res. Treat., 16: 3â€”13, I. Gay. S., Gay, R. E., and Koopman, W. J. Molecular and cellular mechanisms of joint 1990. destruction in rheumatoid arthritis: two cellular mechanisms explain joint destruction? 16. Tetu, B., Brisson, J., Cote, C., Brisson, S., Potuin, D., and Roberge, N. Prognostic Ann. Rheum. Dis., 52: 539â€”547,1993. significance of cathepsin D expression in node positive breast carcinoma: an immu 2. vanderStappen,J.W.J., Paraskeva,C.,Williams,A.C.,Hague,A.,andMaciewicz, nohistochemical study. Int. J. Cancer, 55: 429â€”435, 1993. R. A. Relationship between the secretion of cysteine proteinases and their inhibitors 17. Joensuu, H., Toikkanen, S., and Isola, J. Stromal cell cathepsin D expression and long and malignant potential. Biochem. Soc. Trans., 19: 362S, 1991. term survival in breast cancer. Br. J. Cancer, 7/: 155â€”159,1995. 3. Tezuka, K., Tezuka, Y., Macjima, A., Sato, T., Nemoto, K., Kamioka, H., Hakeda, Y., 18. Bossard, M. J., Tomaszek, T. A., Thomson, S. K., Amegadzie, B. Y., Hanning, C. R., and Kumegawa, M. Molecular cloning of a possible cysteine proteinase predomi Jones, C., Kurdyla, J. T., McNulty, D. E., Drake, F. H., Gowen, M., and Levy, M. A. nantly expressed in osteoclasts. J. Biol. Chem., 269: 1106â€”1109,1994. Proteolytic activity of human osteoclast cathepsin K. J. Biol. Chem.. 27/: 125 17â€” 4. Inaoka, T., Bilbe, G., Ishibashi, 0., Tezuka, K., Kumegawa, M., and Kokubo, T. 12524,1996. Molecular cloning of human cDNA for cathepsin K: novel cysteine proteinase 19. Clohisy, D. R., Palkert, D., Ramnaraine, M. L., Pekurovsky, I., and Oursler, M. J. predominantly expressed in bone. Biochem. Biophys. Res. Commun., 206: 89â€”96, Human breast cancer induces osteoclast activation and increases the number of 1995. 5. Littlewood-Evans,A., Kokubo,T., Ishibashi,0., Inaoka, B., Wlodarski,B., osteoclasts at sites of tumor osteolysis. J. Orthop. Res., 14: 396â€”402, 1996. 20. Orr, F. W., Kostenuik, P., Sanchez-Sweatman, 0. H., and Singh, G. Mechanisms Gallagher, J. A., and Bilbe, G. Localization of cathepsin K in human osteoclasts by involved in the metastasis of cancer to bone. Breast Cancer Rca. Treat., 25: 151â€”163, in situ hybridization and immunohistochemistry. Bone, 20: 81â€”86,1997. 6. Bartek, J., Bartkova, J., Kyprianou, N., Lalani, E. N., Staskova, Z., Shearer, M., 1993. Chang, S., and Taylor-Papadimitriou, J. Efficient immortalization of luminal epithe 21. Mundy, G. R. Mechanisms of osteolytic bone destruction. Bone, 12 (Suppl. 1): hal cells from human mammary gland by introduction of simian virus 40 large tumor Slâ€”S6,1991. antigen with a recombinant retrovirus. Proc. NatI. Acad. Sci. USA, 88: 3520â€”3524. 22. Rubens,R. D. Improvingtreatmentforadvancedbreastcancer.CancerSurv.,/8: 1991. 199â€”209,1993. 7. Elston, C. W., and Ellis, I. 0. Pathological prognostic factors in breast cancer. I. The 23. Rossi,J. F. Bonetissueandcancer.Pathol.Biol.,38: 69â€”79,1990. value of histological grade in breast cancer: experience from a large study with long 24. Athanasou, N. A., and Quinn, J. M. Human tumour-associated macrophages are term follow up. Histopathology, 19: 403â€”410, 1991. capable of bone resorption. Br. J. Cancer, 65: 523â€”526,1992. 8. Inui, T., Ishibashi, 0., lnaoka, T., Origane. Y., Kumegawa. M.. Kokubo, T., and 25. Clohisy, D. R., Ogilvie. C. M., Carpenter, R. J., and Ramnaraine, M. L. Localized, Yamamura, T. Cathepsin K antisense oligonucleotide inhibits osteoclastic bone tumor associated osteolysis involves the recruitment and activation of osteoclasts. resorption. J. Biol. Chem., 272: 8109â€”81 12. 1997. J. Oi'thop. Res., 14: 2â€”6,1996. 9. Komminoth, P. Digoxigenin as an alternative probe labelling for in situ hybridization. 26. Eilon, G., and Mundy, G. R. Association of increased cyclic adenosine 3':S'- Diagn. Mol. Pathol., I: 142â€”150.1992. monophosphate content in cultured human breast cancer cells and release of hydro 10. Drake, F. H., Dodds, R. A., James, I. E., Connor, J. R., Debouck, C., Richardson, S., lyric enzymes and bone-resorbing activity. Cancer Res., 43: 5792â€”5794, 1983.

5390

Amanda J. Littlewood-Evans, Graeme Bilbe, Wayne B. Bowler, et al.

Cancer Res 1997;57:5386-5390.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/57/23/5386

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/57/23/5386. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.