Localization Of Cathepsin Research Articles

Cathepsins B, D, and G Are Expressed in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma.

AimWe have previously demonstrated the presence of two cancer stem cell (CSC) subpopulations within metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC) expressing components of the renin-angiotensin system (RAS), which promotes tumorigenesis. Cathepsins B, D and G are enzymes that constitute bypass loops for the RAS. This study investigated the expression and localization of cathepsins B, D, and G in relation to CSC subpopulations within mHNcSCC.MethodsImmunohistochemical staining was performed on mHNcSCC tissue samples from 20 patients to determine the expression and localization of cathepsins B, D, and G. Immunofluorescence staining was performed on two of these mHNcSCC tissue samples by co-staining of cathepsins B and D with OCT4 and SOX2, and cathepsin G with mast cell markers tryptase and chymase. Western blotting and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were performed on five mHNcSCC samples and four mHNcSCC-derived primary cell lines, to determine protein and transcript expression of these three cathepsins, respectively. Enzyme activity assays were performed on mHNcSCC tissue samples to determine whether these cathepsins were active.ResultsImmunohistochemical staining demonstrated the presence of cathepsins B, D and G in in all 20 mHNcSCC tissue samples. Immunofluorescence staining showed that cathepsins B and D were localized to the CSCs both within the tumor nests and peri-tumoral stroma (PTS) and cathepsin G was localized to the phenotypic mast cells within the PTS. Western blotting demonstrated protein expression of cathepsin B and D, and RT-qPCR demonstrated transcript expression of all three cathepsins. Enzyme activity assays showed that cathepsin B and D to be active.ConclusionThe presence of cathepsins B and D on the CSCs and cathepsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for mHNcSCC.

Glomerular Damage in Trichloroethylene-Sensitized Mice: Targeting Cathepsin L-Induced Hyperactive mTOR Signaling.

Trichloroethylene (TCE) is a serious health hazard for workers with daily exposure, causing occupational medicamentosa-like dermatitis due to TCE (OMDT) and glomerular damage. Recent studies suggest that mTORC1 signaling is activated in various glomerular disorders; however, the role of mTORC1 signaling in TCE-induced glomerular damage remains to be explored. In the present study, 6 OMDT patients were enrolled and a TCE-sensitized mouse model was established to investigate molecular mechanisms underlying the glomerular damage associated with OMDT. Glomerular damage was assessed by levels of urine nephrin, H&E staining, and renal function test. Ultrastructural change of podocyte was investigated by transmission electron microscopy. The podocyte-related molecules including nephrin, α-actinin-4, and integrin β1 were visualized by immunofluorescence. The activation of mTORC1 signaling was confirmed by Western blot. Glomerular apoptosis was examined by the TUNEL test and Western blotting. Expression and location of cathepsin L (CTSL) were assessed by RT-PCR and immunofluorescence. Our results showed that TCE sensitization caused damage to glomerular structural integrity and also increased the activation of mTORC1 signaling, which was accompanied by podocyte loss, hypertrophy, and glomerular apoptosis. Importantly, we also found that over-expressed CTSL was mainly located in podocyte and CTSL inhibition could partially block the activation of mTORC1 signaling. Thus, our findings suggested a novel mechanism whereby hyperactive mTOR signaling contributes to TCE sensitization–induced and immune-mediated glomerular damage via CTSL activation.

Expression of cathepsins B and D by cancer stem cells in head and neck metastatic malignant melanoma.

We have previously demonstrated cancer stem cell (CSC) subpopulations in head and neck metastatic malignant melanoma (HNmMM), and the expression of components of the renin-angiotensin system (RAS) by these CSCs. Cathepsins B, D and G are involved in carcinogenesis and constitute bypass loops of the RAS. This study investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. Immunohistochemical staining demonstrated expression of cathepsins B, D and G in HNmMM sections from all 20 patients. Western blotting confirmed the presence of cathepsins B and D proteins in all six HNmMM tissue samples and four HNmMM-derived primary cell lines. RT-qPCR showed transcript expression of cathepsins B, D and G in all six HNmMM tissue samples, and cathepsins B and D but not cathepsin G in all four HNmMM-derived primary cell lines. Enzymatic activity assays demonstrated cathepsins B and D were active in all six HNmMM tissue samples. Immunofluorescence staining performed on two of the HNmMM tissue samples demonstrated expression of cathepsins B and D by the CSCs, and cathepsin G by cells within the peritumoral stroma. Our novel findings suggest the possibility of targeting these CSCs by modulation of paracrine RAS signaling.

Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study.

Gap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell called connexosomes or annular gap junctions. Here, we systematically investigated the fate of connexosomes in intact ovarian follicles. High-pressure frozen, serial-sectioned tissue was immunogold labeled for connexin 43 (Cx43, also known as GJA1). Within a volume corresponding to ∼35 cells, every labeled structure was categorized and had its surface area measured. Measurements support the concept that multiple connexosomes form from larger invaginated gap junctions. Subsequently, the inner and outer membranes separate, Cx43 immunogenicity is lost from the outer membrane, and the inner membrane appears to undergo fission. One pathway for processing involves lysosomes, based on localization of cathepsin B to some processed connexosomes. In summary, this study demonstrates new technology for high-resolution analyses of gap junction processing.This article has an associated First Person interview with the first author of the paper.

Immunoreactivity of cytotoxic T-lymphocyte antigen 2 alpha in mouse pancreatic islet cells.

Cells of the pancreatic islets produce several molecules including insulin (beta cells), glucagon (alpha cells), somatostatin (delta cells), pancreatic polypeptide (PP cells), ghrelin (epsilon cells), serotonin (enterochromaffin cells), gastrin (G cells) and small granules of unknown content secreted by the P/D1 cells. Secretion mechanism of some of these molecules is still poorly understood. However, Cathepsin L is shown to regulate insulin exocytosis in beta cells and activate the trypsinogen produced by the pancreatic serous acini cells into trypsin. The structure of the propeptide region of Cathepsin L is homologous to Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2 alpha) which is also shown to exhibit selective inhibitory activities against Cathepsin L. It was thought that if CTLA-2 alpha was expressed in the pancreas; then, it would be an important regulator of protease activation and insulin secretion. The purpose of this study was, therefore, to examine by immunohistochemistry the cellular localization and distribution pattern of CTLA-2 alpha in the pancreas. Results showed that strong immunoreactivity was specifically detected in the pancreatic islets (endocrine pancreas) but not in the exocrine pancreas and pancreatic stroma. Immunostaining was further performed to investigate more on localization of Cathepsin L in the pancreas. Strong immunoreactivity for Cathepsin L was detected in the pancreatic islets, serous cells and the pancreas duct system. These findings suggest that CTLA-2 alpha may be involved in the proteolytic processing and secretion of insulin through regulation of Cathepsin L and that the regulated inhibition of Cathepsin L may have therapeutic potential for type 1 diabetes.

Increased expression and altered localization of cathepsin Z are associated with progression to jaundice stage in primary biliary cholangitis

Our recent genome-wide association study found that the NELFCD/CTSZ locus was significantly associated with progression of primary biliary cholangitis (PBC) to jaundice stage in the Japanese population. In this study, we investigated the role of cathepsin Z in the etiology and pathology of PBC. Serum cathepsin Z levels were measured using enzyme-linked immunosorbent assay. The expression and localization of cathepsin Z in liver specimens were analyzed by western blotting and immunohistochemistry. In PBC patients, serum cathepsin Z levels were significantly increased with disease progression. In addition, its levels were positively correlated with alanine transaminase, aspartate transaminase and total bilirubin, and were negatively correlated with platelet count and albumin. Cathepsin Z expression was markedly increased in hepatocytes at later stages of PBC, and its localization was altered from the peri-bile canaliculus to the cytoplasm, where a fraction was no longer colocalized with endosomal/lysosomal vesicles. Similar altered expression of cathepsin Z was observed in end-stage of other cholestatic liver diseases including sepsis, obstructive jaundice, and Alagille syndrome. Our results indicate that altered expression and localization of cathepsin Z in hepatocytes are characteristic features of PBC and other cholestatic liver diseases, and are implicated in the progression of PBC.

Expression and Localization of Cathepsins B, D and G in Cancer Stem Cells in Liver Metastasis From Colon Adenocarcinoma.

AimWe have previously identified and characterized cancer stem cell (CSC) subpopulations in liver metastasis from colon adenocarcinoma (LMCA). In this study we investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs.Methods3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D and G was performed on 4μm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients. Immunofluorescence (IF) IHC staining was performed on three representative samples of LMCA from the original cohort of nine patients, to determine the localization of these cathepsins in relation to the CSC subpopulations. NanoString mRNA analysis and Western Blotting (WB) were used to examine the transcript and protein expression of these cathepsins, respectively. Enzyme activity assays were utilized to determine their functional activity. Data acquired from counting of cells staining positively of the cathepsins on the DAB IHC-stained slides and from Nanostring mRNA analysis were subjected to statistical analyses to determine significance.ResultsDAB IHC staining demonstrated expression of cathepsins B, D and G within LMCA. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4− cells within the tumor nests and the OCT4+ CSC subpopulation within the peritumoral stroma. NanoString mRNA analysis showed significantly greater transcript expression of cathepsin B and cathepsin D, compared to cathepsin G. WB confirmed expression of cathepsin B and cathepsin D proteins, while cathepsin G was below detectable levels. Enzyme activity assays showed functional activity of cathepsin B and cathepsin D.ConclusionOur study demonstrated novel finding of the expression of cathepsin B, cathepsin D, and possibly cathepsin G by the putative CSC subpopulations within LMCA.

Expression and Localization of Cathepsins B, D, and G in Dupuytren's Disease.

The pathogenesis of Dupuytren's disease (DD) remains unclear. An embryonic stem cell (ESC)-like population in the endothelium of the microvessels around tissues that expresses components of the renin-angiotensin system (RAS) has been reported. This study investigated if this primitive population expresses cathepsins B, D, and G, that contribute to RAS bypass loops. 3,3-Diaminobenzidine immunohistochemical (IHC) staining for cathepsins B, D, and G was performed on sections of formalin-fixed paraffin-embedded DD cords (n = 10) and nodules (n = 10). Immunofluorescence IHC staining was utilized to demonstrate co-expression of these cathepsins with ESC markers. Protein and gene expression of these cathepsins was investigated in snap-frozen DD cords (n = 3) and nodules (n = 3) by Western blotting and NanoString analysis, respectively. Enzymatic activity of these cathepsins was investigated by enzymatic activity assays. 3,3-Diaminobenzidine IHC staining demonstrated expression of cathepsins B, D, and G in DD cords and nodules. Gene expression of cathepsins B, D, and G was confirmed by NanoString analysis. Western blotting confirmed expression of cathepsins B and D, but not cathepsin G. Immunofluorescent IHC staining demonstrated high abundance of cathepsins B and D on the OCT4+/angiotensin converting enzyme+ endothelium and the smooth muscle layer of the microvessels. Cathepsin G was localized to trypase+ cells within the stroma in DD cords and nodules with limited expression on the microvessels. Enzyme activity assays demonstrated functional activity of cathepsins B and D. Cathepsins B, D, and G were expressed in the DD tissues, with cathepsins B and D localized to the primitive population in the endothelium of the microvessels, whereas cathepsin G was localized to phenotypic mast cells, suggesting the presence of bypass loops for the RAS.

Expression and Localization of Cathepsins B, D, and G in Two Cancer Stem Cell Subpopulations in Moderately Differentiated Oral Tongue Squamous Cell Carcinoma.

We have previously demonstrated the putative presence of two cancer stem cell (CSC) subpopulations within moderately differentiated oral tongue squamous cell carcinoma (MDOTSCC), which express components of the renin-angiotensin system (RAS). In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to these CSC subpopulations within MDOTSCC. 3,3-Diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining was performed on MDOTSCC samples to determine the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations. NanoString mRNA analysis and colorimetric in situ hybridization (CISH) were used to study their transcripts expression. Enzyme activity assays were performed to determine the activity of these cathepsins in MDOTSCC. IHC staining demonstrated expression of cathepsins B, D, and G in MDOTSCC. Cathepsins B and D were localized to CSCs within the tumor nests, while cathepsin B was localized to the CSCs within the peri-tumoral stroma, and cathepsin G was localized to the tryptase+ phenotypic mast cells within the peri-tumoral stroma. NanoString and CISH mRNA analyses confirmed transcription activation of cathepsins B, D, and G. Enzyme activity assays confirmed active cathepsins B and D, but not cathepsin G. The presence of cathepsins B and D on the CSCs and cathspsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for MDOTSCC.

Apolipoprotein E4 Elicits Lysosomal Cathepsin D Release, Decreased Thioredoxin-1 Levels, and Apoptosis.

The major genetic risk factor for Alzheimer’s disease (AD), apolipoprotein E4 (ApoE4), has been suggested to have detrimental effects on neurons, including direct toxicity via apoptosis. Thioredoxin-1 (Trx1) is an endogenous antioxidant protein important for redox regulation and participates in the regulation of apoptosis through the inhibition of apoptosis signal-regulating kinase-1 (Ask-1). In this study, we have investigated the effects of ApoE on Trx1 in the brain. Our results showed that the protein levels of Trx1 were reduced in the hippocampus of ApoE4 targeted replacement (TR) mice compared to ApoE3 TR mice. The reduction was also seen in vitro after treatment of both human primary cortical neurons and neuroblastoma cells with human recombinant ApoE4 (rApoE4). Furthermore, ApoE4 caused a disruption of lysosomal integrity and a shift in the localization of Cathepsin D, an enzyme known to degrade Trx1. ApoE4 treatment induced in addition apoptosis through translocation of Death-domain associated protein-6 (Daxx) from the nucleus to the cytosol, suggesting an activation of the Ask-1 pathway. This toxicity was prevented by overexpression of Trx1 and other endogenous Ask-1 inhibitors. Our data suggests that down-regulation of Trx1 is involved in the toxicity caused by ApoE4. An activated ASK-1 pathway might indeed make cells more vulnerable to other insults such as amyloid-β, which could partially explain the mechanism behind the strongest genetic risk factor for AD.

Localization of cathepsin D in mouse reproductive tissues and its acquisition onto sperm surface during epididymal sperm maturation

Sperm maturation in the epididymis involves multiple complex events, that include the adsorption of epididymal secretory proteins, re-organization and removal of sperm surface ligands. In this study, we investigated the existence and distribution of cathepsin D (CAT-D) transcripts and proteins in mouse reproductive tissues and proposed a transfer mechanism of CAT-D to the sperm surface. CAT-D transcripts were highly expressed in cultured Sertoli cells, but not in germ cells. The transcriptional level was relatively higher in the caput epididymis (CP) than in the cauda epididymis (CD). At the translational level, CAT-D was detected in testicular somatic cells and in the principal and basal cells in the CP. The expression of CAT-D was fairly specific to the clear cells in the CD. All forms of CAT-D were detected in ultracentrifuged epididymosomes. In conjunction with the expression levels in epididymal epithelium and epididymosomes, CAT-D expression level on the sperm surface was relatively high in CP sperm, but gradually declined toward the CD. Overall, our results indicated that CAT-D was not inherent to sperm themselves, but rather of epididymal origin and was presumably transported to the sperm surface via epididymosomes.

Cathepsin H functions as an aminopeptidase in secretory vesicles for production of enkephalin and galanin peptide neurotransmitters.

Peptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N- and/or C-terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin-like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N-terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR-ME and KK-ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and immunofluorescence deconvolution microscopy. Purified human cathepsin H sequentially removes N-terminal basic residues to generate ME, with peptide products characterized by nano-LC-MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N-terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production.

Immunohistochemical localization of cathepsin D and a possible role of melanocytes during tail resorption in tadpoles of a tropical toad

Programmed cell death during anuran tail resorption is primarily brought about by apoptosis. Cathepsin D, a lysosomal aspartyl protease, is involved in the death of tail tissues. Thus, anuran tail resorption presents an ideal model to study cathepsin-mediated cell death during vertebrate development. Present study describes the trend of specific activity of cathepsin D in the tail of different developmental stages and immunohistochemical localization of cathepsin D in the tail tissues of the common Asian toad, Duttaphrynus melanostictus. Cathepsin D was involved in programmed cell death in epidermis, muscle, spinal cord, and blood cells in the resorbing tail. Interestingly, it was also involved in the pre-resorbing tail before visible tail resorption which indicates initiation of cell death even before actually the tail resorbs. Melanocytes were found to be one of the causative agents in degrading tail tissues and were associated with the degradation of muscle, epidermis and spinal cord of the resorbing tail.

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.

Localization of Cathepsins D and B at the Maternal-Fetal Interface and the Invasiveness of the Trophoblast during the Postimplantation Period in the Mouse

A survey of existing data suggests that trophoblast cells produce factors involved in extracellular matrix degradation. In this study, we correlated the expression of cathepsins D and B in the murine ectoplacental cone with the ultrastructural progress of decidual invasion by trophoblast cells. Both proteases were immunolocalized at implantation sites in lysosome-endosome-like compartments of trophoblast giant cells. Cathepsin D, but not cathepsin B, was also detected ultrastructurally in extracellular compartments surrounded by processes of the invading trophoblast containing extracellular matrix components and endometrial cell debris. The expression of cathepsins D and B by trophoblast cells was confirmed by RT-PCR in ectoplacental cones isolated from implantation chambers at gestation day 7.5. Our data addressed a positive relationship between the expression and presence of cathepsin D at the extracellular compartment of the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period, suggesting a participation of invading trophoblast cells in the cathepsin D release. Such findings indicate that mouse trophoblast cells might exhibit a proteolytic ability to partake in the decidual invasion process at the maternal-fetal interface.

Quantitative determination and localization of cathepsin D and its inhibitors.

A literature survey was performed of the methods of quantitative assessment of the activity and concentration of cathepsin D and its inhibitors. Usefulness of non-modified and modified proteins and synthetic peptides as measurement substrates was evaluated. The survey includes also chemical and immunochemical methods used to determine the distribution of cathepsin D and its inhibitors in cells and tissues.

Quantitative determination and localization of cathepsin D and its inhibitors.

Quantitative determination and localization of cathepsin D and its inhibitors.

Localization of cathepsin x in the mouse retina

Abstract Purpose Cathepsin X is a recently identified member of the papain‐like cysteine protease family that is expressed by many cell types. The in vivo functions of Cathepsin X are unknown. Hence, the aims of this study are to localize cathepsin X expression in the mouse retina and to compare the retinal morphology and ERG function of cathepsin X knock‐out mice (Ctsx‐/‐) to congenic wild type C57Bl/6 mice (Ctsx+/+). Methods Left eyes of adult mice were prepared for paraffin‐ and right eyes for cryo‐sections. Histology was accomplished by Hämalaun‐Eosin and PAS staining after depigmentation. Expression of cathepsin X and the proliferation marker Ki67 were investigated by immunohistochemistry. Retinal function was tested by ERG. Results Cathepsin X expression in WT mice accumulates along the external limiting membrane. Additionally, a netlike pattern of cathepsin X was found around the neuronal cells in the inner nuclear and ganglion cell layer. There is no significant difference between cathepsin X knock‐out and WT mice in retinal morphology, cell proliferation or ERG readings. Conclusion We found prominent expression of cathepsin X in WT mice retina. Normal retinal morphology and ERG function in cathepsin X knock‐out mice, however, suggest that other cathepsins might compensate for the loss of retinal cathepsin X.

Cathepsin D expression is decreased in Alzheimer's disease fibroblasts

Cathepsin D (CTSD), a protease detectable in different cell types whose primary function is to degrade proteins by bulk proteolysis in lysosomes, has been suggested to be involved in Alzheimer's disease (AD). In fact, there is increasing evidence that disturbance of the normal balance and localization of cathepsins may contribute to neurodegeneration in AD [Nakanishi H. Neuronal and microglial cathepsins in aging and age-related diseases. Aging Res Rev 2003; 2(4):367–81]. Here, we provide evidence of an altered balance of CTSD in skin fibroblasts from patients affected either by sporadic or familial forms of AD. In particular, we demonstrate that CTSD is down regulated at both transcriptional and translational level and its processing is altered in AD fibroblasts. The oncogene Ras is involved in the regulation of CTSD, as high expression level of the constitutively active form of Ras in normal or AD fibroblasts induces CTSD down-regulation. p38 MAPK signalling pathway also appears to down-modulate CTSD level. Overall results reinforce the hypothesis that a lysosomal impairment may be involved in AD pathogenesis and can be detected not only in the CNS but also at a peripheral level.

Matrigel influences morphology and cathepsin B distribution of prostate cancer PC3 cells

Increases in expression and activity of matrix-degrading enzymes such as the cysteine proteinases cathepsins B and L, and abnormal levels of their inhibitors, the cystatins, are associated with tumor cell invasion and metastasis. Environmental conditions have been shown to be causative factors in the development of a metastatic/invasive phenotype. We hypothesized that cell-matrix interactions affect the expression and activity of cathepsins B and L and their inhibitors in the prostate cancer cell lines, PC3 and DU145. To test this possibility, PC3 and DU145 were plated on uncoated surfaces or on surfaces coated with the reconstituted basement membrane, Matrigel. The cells were analyzed for cathepsins B and L immunolocalization, protein expression and activity 48 h after plating. Our data demonstrated that cathepsins B and L displayed a distinct punctate distribution with little co-localization; individual cells displayed a predominant staining for one or the other enzyme. Cathepsin B had a perinuclear distribution in PC3 grown on uncoated surfaces but a more peripheral staining in PC3 plated on Matrigel. Localization of cathepsin L remained predominantly perinuclear regardless of the plating surface. In addition to the translocation of cathepsin B from a perinuclear distribution to the cell periphery, growth of PC3 on Matrigel shifted cathepsin B activity from the cell extract to the media. There were no significant changes in cathepsins B and L immunolocalization or activity in DU145 with regard to plating surfaces. Likewise, the activity of endogenous cysteine proteinase inhibitors (CPIs) and protein expression of cystatin C remained unchanged in both cell lines. In conclusion, the interaction of PC3 prostate cancer cells with extracellular matrix components affects the distribution of cathepsin B protein and activity.