Cell type-dependent pathogenic functions of overexpressed human cathepsin B in murine breast cancer progression.

The cysteine protease cathepsin B (CTSB) is frequently overexpressed in human breast cancer and correlated with a poor prognosis. Genetic deficiency or pharmacological inhibition of CTSB attenuates tumor growth, invasion and metastasis in mouse models of human cancers. CTSB is expressed in both cancer cells and cells of the tumor stroma, in particular in tumor-associated macrophages (TAM). In order to evaluate the impact of tumor- or stromal cell-derived CTSB on Polyoma Middle T (PyMT)-induced breast cancer progression, we used in vivo and in vitro approaches to induce human CTSB overexpression in PyMT cancer cells or stromal cells alone or in combination. Orthotopic transplantation experiments revealed that CTSB overexpression in cancer cells rather than in the stroma affects PyMT tumor progression. In 3D cultures, primary PyMT tumor cells showed higher extracellular matrix proteolysis and enhanced collective cell invasion when CTSB was overexpressed and proteolytically active. Coculture of PyMT cells with bone marrow-derived macrophages induced a TAM-like macrophage phenotype in vitro, and the presence of such M2-polarized macrophages in 3D cultures enhanced sprouting of tumor spheroids. We employed a doxycycline (DOX)-inducible CTSB expression system to selectively overexpress human CTSB either in cancer cells or in macrophages in 3D cocultures. Tumor spheroid invasiveness was only enhanced when CTSB was overexpressed in cancer cells, whereas CTSB expression in macrophages alone did not further promote invasiveness of tumor spheroids. We conclude that CTSB overexpression in the PyMT mouse model promotes tumor progression not by a stromal effect, but by a direct, cancer cell-inherent mode of action: CTSB overexpression renders the PyMT cancers more invasive by increasing proteolytic extracellular matrix protein degradation fostering collective cell invasion into adjacent tissue.

Cathepsin K in the bone microenvironment: link between obesity and prostate cancer?

The skeleton is the most common site of metastasis in patients with advanced prostate cancer. Despite many advances in targeting skeletal metastases, the mechanisms behind the attraction of prostate cancer cells to the bone are not known. Osteoclast cathepsin K, due to its ability to effectively degrade bone matrix collagen I, has been implicated in colonization and growth of prostate tumours in the bone. Identification of new cathepsin K substrates in the bone microenvironment and the recent findings demonstrating its involvement in obesity and inflammation suggest additional roles for this enzyme in skeletal metastases of prostate cancer.

Plasma membrane association of cathepsin B in human prostate cancer: biochemical and immunogold electron microscopic analysis.

BACKGROUND: Cathepsin B (CB), a cysteine protease, is usually found in perinuclear lysosomes of epithelial cells of normal organs and non-malignant tumors, but is associated with the plasma membranes of many solid organ malignant tumors. Plasma membrane localized CB facilitates degradation of extracellular matrix proteins and progression of tumor cells from one biological compartment to another. The activities of CB and its subcellular distribution have not been investigated in malignant prostate. Our objective was to examine the subcellular distribution of CB by determining the activities of CB in lysosome and plasma membrane/endosome subcellular fractions and its subcellular localization by immunogold electron microscopy. METHODS: Prostate tissue pieces obtained immediately after prostatectomy were homogenized and fractionated into subcellular components for determining biochemical activities of CB and cysteine protease inhibitors (CPIs). Distribution of CB was compared with that of prostate specific antigen (PSA, a serine protease), which is abundant in secretory vesicles and granules of normal prostate, benign prostatic hyperplasia (BPH) and malignant prostate cells. Localization of CB was investigated in resin embedded lysosomes and plasma membrane/endosome subcellular fractions and in prostate tissue sections by immunogold electron microscopy. RESULTS: We have demonstrated the specificity of CB activity in human prostate homogenates by using a variety of inhibitors in our assay. We did not find any difference in the specific activity of CB based on protein or DNA content in homogenates of malignant prostate (Gleason histologic scores 5-7) and BPH (no histological evidence of cancer) whether it was measured by chromogenic or fluorogenic peptide substrate assay techniques. We found significantly higher activities of CB in the plasma membrane/endosome fractions of malignant prostate than in BPH. In contrast, CPI activity was increased relative to CB activity in plasma membrane/endosome fraction of BPH versus prostate cancer. Our data indicated a shift in the balance of enzyme to inhibitor that would favor increased activities of CB in prostate cancer. The immunogold microscopic study showed specific localization of CB in plasma membrane. They also showed localization of CB in lysosomes that were often adjacent to luminal and/or basal surfaces of malignant cells in contrast to the usual perinuclear distribution of lysosomes in hyperplastic prostate glands. PSA was localized in secretory granules and vesicles, including the plasma membranes and secretory blebs in malignant prostate cells. Occasional PSA positive secretory vesicles or membrane profiles were seen in the plasma membrane/endosomal and lysosomal fractions. CONCLUSIONS: The increased activity of CB in plasma membrane/endosomal fractions is associated with malignant prostate and not with BPH or normal prostate. Morphologic distribution CB is associated with the plasma membranes or lysosomes adjacent to apical and basal cell surfaces. This distribution is characteristic feature prostate cancer cells, but not in BPH or normal prostate cells. Subcellular distribution of PSA occurs in secretory vesicles and granules of the cytoplasm, but not in lysosomes. Our biochemical and morphological data could be used to distinguish malignant prostates from non-malignant tumors.

Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosa.

C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection.

Ratio of cathepsin B to stefin A identifies heterogeneity within Gleason histologic scores for human prostate cancer.

BACKGROUND: Cathepsin B (CB), a lysosomal cysteine protease, is involved in degradation of extracellular matrix proteins and progression of tumor cells from one biological compartment to another in many solid organ cancers, including prostate cancer. Our objective was to identify patterns of distribution of CB and its endogenous cellular inhibitor stefin A in cryostat sections of frozen BPH and prostate cancer tissue samples and to define these patterns in relation to Gleason histologic scores, clinical stages, and serum total PSA levels. METHODS: We localized CB and stefin A in the same sections using polyclonal and monoclonal antibody immunoglobulin G (IgGs) against CB and stefin A using immunofluorescence and confocal microscopic techniques. Only cryostat sections of frozen prostates were used in localizations of CB and stefin A. RESULTS: Benign prostatic hyperplasia (BPH) showed similar localization patterns for CB and stefin A and a ratio of 1 was indicated by CB = stefin A. Confocal studies indicated that most CB and stefin A sites in BPH glandular cells overlapped as shown by the yellow fluorescence of their co-localization. We found considerable variability in individual localization of CB and stefin A within and between Gleason histologic scores for prostate cancers. This variability was also found in Gleason score 6 tumors that are otherwise considered similar histologically and morphologically. Negative control sections did not show localization of CB by FITC, stefin A by Cy3 or yellow fluorescence for co-localization. Our analysis of the ratio of CB to stefin A showed three patterns, namely CB = stefin A, CB > stefin A, and CB < stefin A, within each Gleason score evaluated by us. Confocal microscopy showed more sites of yellow fluorescence when the ratio was CB = stefin A than those found in CB > stefin A or CB < stefin A. Statistical analyses showed prostate cancer cases with ratios of CB > stefin A (P < 0.05) and CB < stefin A (P < 0.05) significantly different from normal prostate and BPH which had ratios of CB = stefin A. Regression analysis did not show any specific relationship between the ratio of CB to stefin A and Gleason scores, clinical stages, and serum total prostate specific antigen (PSA) levels in prostate cancers. Analysis of our data indicates that the homeostatic balance between the enzyme and inhibitor was altered even in Gleason histologic score 6 tumors that are usually considered histologically similar by glandular differentiation. CONCLUSIONS: We have shown that prostate cancer is a heterogeneous tumor within each Gleason histological score regardless of the progression indicated by lower to higher Gleason score tumors. The ratio of CB > stefin A would indicate a preponderance of enzyme that would favor degradation of extracellular matrix proteins and progression of tumor cells in biological compartments. These tumors are expected to be aggressive prostate cancers. In contrast, prostate tumors showing ratios of CB < stefin A and CB = stefin A are expected to be less aggressive prostate cancers. This is the first report to define heterogeneity within any Gleason score for prostate cancers by the ratios of CB to stefin A.

Imaging proteolysis by living human glioma cells.

Degradation of basement membrane is an essential step for tumor invasion. In order to study degradation in real time as well as localize the site of proteolysis, we have established an assay with living human cancer cells in which we image cleavage of quenched-fluorescent basement membrane type IV collagen (DQ-collagen IV). Accumulation of fluorescent products is imaged with a confocal microscope and localized by optically sectioning both the cells and the matrix on which they are growing. For the studies described here, we seeded U87 human glioma cells as either monolayers or spheroids on a 3-dimensional gelatin matrix in which DQ-collagen IV had been embedded. As early as 24 hours after plating as monolayers, U87 cells were present throughout the 3-dimensional matrix. Cells at all levels had accumulated fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Similar observations were made for U87 spheroids and the individual cells migrating from the spheroids into the gelatin matrix. Both the migrating cells and those within the spheroid contained fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Thus, glioma cells like breast cancer cells are able to degrade type IV collagen intracellularly, suggesting that this is an important pathway for matrix degradation.

Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases.

It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general natural inhibitor of cysteine proteinases, (82.8+/-1.6% inhibition of cell invasion), followed by the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). CLIK-148, a specific inhibitor of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.

Expression of cathepsins B and S in the progression of prostate carcinoma.

Cathepsins B and S (CatB, CatS) are lysosomal cysteine proteases which, among other functions, appear to play a role in cancer progression in different tumor models due to their matrix-degrading properties. To investigate their possible involvement in the development of prostate carcinoma, we immunohistochemically analyzed CatB and CatS in 38 primary human prostatic adenocarcinomas, as well as concomitant high-grade prostatic intra-epithelial neoplasia, nodular hyperplasia and normal tissue. CatB expression was observed in 28 (74%) and CatS in 32 (84%) carcinomas, being concomitant in 24 cases (63%). High-grade intra-epithelial neoplasia expressed CatB in 20/23 cases (87%), and a similar result was obtained for CatS, with expression of both coinciding in 18 cases (78%). In non-neoplastic tissue, strong expression of both proteases was observed in macrophages, inflamed glands and transitional metaplasia, whereas atrophic glands and basal cells of normal glands displayed intense CatB positivity. We conclude that CatB and CatS are often expressed together in neoplastic prostatic cells from pre-invasive to invasive and clinically detectable stages, suggesting a putative role in local invasion, though other functions cannot be ruled out.

Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix.

Transfection of Rat1 fibroblasts with an activated form of rac1 (V12rac1) stimulated cell migration in vitro compared to transfection of Rat1 fibroblasts with vector only or with dominant negative rac1 (N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal assay to evaluate the ability of the Rat1 transfectants to degrade a quenched fluorescent protein substrate (DQ-green bovine serum albumin) embedded in a three-dimensional gelatin matrix. Cleavage of the substrate results in fluorescence, thus enabling one to image extracellular and intracellular proteolysis by living cells. The Rat1 transfectants accumulated degraded substrate intracellularly. V12rac1 increased accumulation of the fluorescent product in vesicles that also labeled with the lysosomal marker LysoTracker. Treatment of the V12rac1-transfected cells with membrane-permeable inhibitors of lysosomal cysteine proteases and a membrane-permeable selective inhibitor of the cysteine protease cathepsin B significantly reduced intracellular accumulation of degraded substrate, indicating that degradation occurred intracellularly. V12rac1 stimulated uptake of dextran 70 (a marker of macropinocytosis) and polystyrene beads (markers of phagocytosis) into vesicles that also labeled for cathepsin B. Thus, stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 may be responsible for the increased internalization and subsequent degradation of extracellular proteins.

Fluorescent microplate assay for cancer cell-associated cathepsin B.

Cathepsin B and in particular cell-surface and secreted cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for cathepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Intact human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a Km of 460 microM at pH 7.0 and 37 degrees C. This is nearly the same as the Km of 430 microM obtained with purified cathepsin B assayed under the same conditions. The pericellular (i.e. both cell-surface and released) cathepsin B activity was inhibited by the cysteine protease inhibitors E-64, leupeptin, Mu-Np2-HphVS-2Np, Mu-Leu-HpHVSPh and the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver cathepsin B. Other human cancer cell lines with measurable pericellular cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlated with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.

Observing proteases in living cells.

The lysosomal cysteine protease cathepsin B has been implicated in tumor progression and metastasis in part due to its altered trafficking. In order to analyze the trafficking of cathepsin B in living cells, we utilized enhanced green fluorescent protein (EGFP) fused to various cathepsin B constructs for transfecting two cell lines: an invasive human breast adenocarcinoma cell line (BT20) and a cathepsin B deficient mouse embryonic fibroblast cell line (MEF T -/-). The cells were transiently transfected with four cathepsin B-EGFP fusion constructs: full-length preprocathepsin B-EGFP, cathepsin B preregion-EGFP, cathepsin B prepro region-EGFP, and cathepsin B prepro region-EGFP with a mutation of the glycosylation site in the pro region. The full length construct showed vesicular distribution throughout the cells in both cell lines. In both BT20 and MEF T -/- cells, preregion-EGFP was localized in a ring tightly associated with the cell nucleus, suggesting distribution to the endoplasmic reticulum. The distribution of the prepro region-EGFP construct was similar except that it also included some patchy areas adjacent to the nucleus. This suggested that the cathepsin B prepro region-EGFP might have entered the Golgi. Distribution of the mutated cathepsin B prepro region-EGFP was similar to that of wild-type prepro region-EGFP in the MEF T -/-. In the invasive BT20 cells, however, the mutated prepro region-EGFP showed a vesicular distribution throughout the cytoplasm and in cell processes. This distribution is similar to that of endogenous cathepsin B in these cells. Our results suggest that: 1) tumor cells have an alternative mechanism for trafficking of cathepsin B which is independent of the mannose-6-phosphate receptor pathway, and 2) the pro region of cathepsin B may contain the sorting sequence necessary for its trafficking via this pathway.

Human procathepsin B interacts with the annexin II tetramer on the surface of tumor cells.

To study potential roles of plasma membrane-associated extracellular cathepsin B in tumor cell invasion and metastasis, we used the yeast two-hybrid system to screen for proteins that interact with human procathepsin B. The annexin II light chain (p11), one of the two subunits of the annexin II tetramer, was one of the proteins identified. We have confirmed that recombinant human procathepsin B interacts with p11 as well as with the annexin II tetramer in vitro. Furthermore, procathepsin B could interact with the annexin II tetramer in vivo as demonstrated by coimmunoprecipitation. Cathepsin B and the annexin II tetramer were shown by immunofluorescent staining to colocalize on the surface of human breast carcinoma and glioma cells. Taken together, our results indicate that the annexin II tetramer can serve as a binding protein for procathepsin B on the surface of tumor cells, an interaction that may facilitate tumor invasion and metastasis.

Transcription of human cathepsin B is mediated by Sp1 and Ets family factors in glioma.

Cathepsin B expression is increased at both the mRNA and protein levels in a wide variety of tumors. The mechanisms responsible for this regulation are not well elucidated. We have isolated a 2.2-kb cathepsin B genomic fragment that contains the 5'-flanking region of the cathepsin B gene. Using reporter gene analysis in human glioblastoma U87MG cells, we have mapped a 228-bp fragment (-172 to +56) having high promoter activity. This promoter region has a high G+C content; contains potential Spl, Ets, and USF binding motifs; and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site. Cotransfection experiments demonstrated that Spl and Ets1 could trans-activate cathepsin B transcription, whereas Ets2 could not. Electrophoretic mobility shift assays and supershift assays revealed that three of the four putative Sp1 sites in this promoter region form a specific complex containing the Sp1 transcription factor. Mutating all four of the Spl binding sites individually markedly reduced the promoter activity of transfected reporter genes in U87 cells. Cotransfection of this cathepsin B promoter construct with Spl family expression vectors in Schneider's Drosophila line 2 (SL2) cells demonstrated that Spl and Sp3, but not Sp4, activated cathepsin B transcription. Taken together, these results suggest that Sp1, Sp3, and Ets1 are important factors in cathepsin B transcription. The regulation of cathepsin B transcription by Sp1- and Sp1-related factors is mediated through multiple GC boxes.

Cell surface complex of cathepsin B/annexin II tetramer in malignant progression.

The cysteine protease cathepsin B is upregulated in a variety of tumors, particularly at the invasive edges. Cathepsin B can degrade extracellular matrix proteins, such as collagen IV and laminin, and can activate the precursor form of urokinase plasminogen activator (uPA), perhaps thereby initiating an extracellular proteolytic cascade. Recently, we demonstrated that procathepsin B interacts with the annexin II heterotetramer (AIIt) on the surface of tumor cells. AIIt had previously been shown to interact with the serine proteases: plasminogen/plasmin and tissue-type plasminogen activator (tPA). The AIIt binding site for cathepsin B differs from that for either plasminogen/plasmin or tPA. AIIt also interacts with extracellular matrix proteins, e.g., collagen I and tenascin-C, forming a structural link between the tumor cell surface and the extracellular matrix. Interestingly, cathepsin B, plasminogen/plasmin, t-PA and tenascin-C have all been linked to tumor development. We speculate that colocalization through AIIt of proteases and their substrates on the tumor cell surface may facilitate: (1) activation of precursor forms of proteases and initiation of proteolytic cascades; and (2) selective degradation of extracellular matrix proteins. The recruitment of proteases to specific regions on the cell surface, regions where potential substrates are also bound, could well function as a 'proteolytic center' to enhance tumor cell detachment, invasion and motility.

Unraveling the role of proteases in cancer.

Investigators have been studying the expression and activity of proteases in the final steps of tumor progression, invasion and metastasis, for the past 30 years. Recent studies, however, indicate that proteases are involved earlier in progression, e.g., in tumor growth both at the primary and metastatic sites. Extracellular proteases may co-operatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. In this review, we use cathepsin B as an example to examine the involvement of proteases in tumor progression and metastasis. We discuss the effect of interactions among tumor cells, stromal cells, and the extracellular matrix on the regulation of protease expression. Further elucidation of the role of proteases in cancer will allow us to design more effective inhibitors and novel protease-based drugs for clinical use.

Imaging proteolysis by living human breast cancer cells.

Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549) through the use of quenched-fluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin B-selective cysteine protease inhibitor, intracellular fluorescence was decreased approximately 90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence approximately 50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1) a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2) the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.

Cathepsin B and glioma invasion.

Increased expression of cathepsin B has been reported in a number of human and animal tumors. This has also been observed in human gliomas where increases in cathepsin B mRNA, protein, activity and secretion parallel malignant progression. In the present study, we showed that cathepsin B was directly involved in glioma cell invasion. Activity of cathepsin B was an order of magnitude higher in glioma tissue than in matched normal brain. Inhibitors of cysteine proteases reduced invasion of glioma cells in two in vitro models: invasion through Matrigel and infiltration of a glioma spheroid into a normal brain aggregate. Glioma spheroids expressed higher levels of cathepsin B than did monolayers and the ability of subclones differing in cathepsin B activity to infiltrate normal brain aggregates paralleled their cathepsin B activity. We confirmed that intracellular staining for cathepsin B occurs at the cell periphery and in cell processes and observed extracellular staining on the cell surface. In addition, we demonstrated that intracellular cathepsin B located at the cell periphery and in processes was active. The cell surface cathepsin B colocalized with areas of degradation of an extracellular matrix component. We hypothesize that the increased expression of active cathepsin B in gliomas leads to increases in invasion in vitro and in vivo and have developed a xenotransplant model in which this hypothesis can be tested.

Differentiating agents regulate cathepsin B gene expression in HL-60 cells.

We utilized HL-60 cells as a model system to examine the regulation of ctsb gene expression by differentiating agents. Inducers of monocytic differentiation [phorbol ester (PMA), calcitriol (D3), and sodium butyrate (NaB)] and inducers of granulocytic differentiation [all-trans retinoic acid (RA) and 9-cis retinoic acid (9-cis RA)] increase ctsb mRNA levels in a dose-dependent manner as determined by Northern blot hybridization. D3 and retinoids exert additive effects, suggesting that these agents act in part through distinct pathways. Actinomycin D decay experiments indicate that D3, NaB, RA, and 9-cis RA do not alter mRNA stability. In contrast, PMA markedly increases the half-life of ctsb mRNA. In transient transfection assays, PMA and NaB both stimulate transcription of the luciferase reporter gene placed under the control of ctsb promoter fragments. Thus, inducers of HL-60 cell differentiation can regulate the expression of the ctsb gene at both transcriptional and posttranscriptional levels.

Exocytosis of active cathepsin B enzyme activity at pH 7.0, inhibition and molecular mass.

Lysosomal cathepsin B has been implicated in parasitic, inflammatory and neoplastic diseases. Most of these pathologies suggest a role for cathepsin B outside the cells, although the origin of extracellular active enzyme is not well defined. The activity of extracellular cathepsin B is difficult to assess because of the presence of inhibitors and inactivation of the enzyme by oxidizing agents. Therefore, we have developed a continuous assay for measurement of cathepsin B activity produced pericellularly by living cells. The kinetic rate of Z-Arg-Arg-NHMec conversion was monitored and the assay optimized for enzyme stability, cell viability and sensitivity. To validate the assay, we determined that human liver cathepsin B was stable and active under the conditions of the assay and its activity could be inhibited by the selective epoxide derivative CA-074. Via this assay, we were able to demonstrate that active cathepsin B was secreted pericellularly by viable cells. Both preneoplastic and malignant cells secreted active cathepsin B. Pretreatment of cells with the membrane-permeant proinhibitor CA-074Me completely abolished pericellular and total cathepsin B activity whereas pretreatment with the active drug CA-074 had no effect. Immunoprecipitation and immunoblotting experiments suggested that the active enzyme species was 31-kDa single-chain cathepsin B. Exocytosis of cathepsin B was not related to secretion of proenzyme or secretion from mature lysosomes. Our results suggest an alternative pathway for exocytosis of active cathepsin B.

Molecular regulation, membrane association and secretion of tumor cathepsin B.

Upregulation, membrane association and secretion of cathepsin B have been shown to occur in many types of tumors and to correlate positively with their invasive and metastatic capabilities. To further understand changes in cathepsin B activity and localization, we have been examining its regulation at many levels including transcription and trafficking. Our studies indicate that there may be three promoter regions in the cathepsin B gene. Of these, continued examination of the promoter upstream of exon 1 has indicated possible control by several regulatory factors including E-box and Sp-1 binding elements. Upregulation of cathepsin B at this level may account for some of the secretion of cathepsin B found in tumors. We have also gathered evidence that endo- and exocytosis of cathepsin B may be regulated by ras and ras-related proteins in addition to previously described trafficking systems. There is also evidence that several populations of lysosomes may exist and that trafficking to different populations may determine whether cathepsin B is secreted from the tumor cell or remains intracellular. Our results indicate that membrane association and secretion of cathepsin B is not a random process in the tumor cell, but rather part of a tightly controlled system.