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Keywords:

  • cathepsin B;
  • cysteine protease inhibitors;
  • lysosomal enzyme;
  • cellular assays

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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 µm at pH 7.0 and 37 °C. This is nearly the same as the Km of 430 µm 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.

Abbreviation
PAB

pericellular assay buffer

Cathepsin B (EC 3.4.22.1) has been suggested to play a role in growth and metastasis of many types of cancer. Although a strong role for cathepsin B has been established by invasion and metastasis studies in vitro, there is a relative paucity of studies demonstrating the efficacy of cathepsin inhibitors as anti-metastatic agents in vivo. Cathepsin B is immunolocalized at the invasive edges of human tumor biopsy specimens [1–3] and is differentially redistributed to the plasma membrane with some forms of the enzyme secreted by tumor cells [4]. Often it is the association of cathepsin B with the plasma membrane that correlates with metastatic potential of cells [5] and that occurs upon oncogenic transformation of cell lines [6]. Tumor cell lines with abundant pericellular (cell surface and secreted) cathepsin B would seem appropriate for use in models in which to test the efficacy of cathepsin B inhibitors against growth and metastasis in vivo. In the few studies which have examined the efficacy of cysteine protease inhibitors in vivo, different in vitro methods were employed to verify that the inhibitors reduced cathepsin B activity in the tumor cells. Leto et al. [7] cultured tumor cells in the presence and absence of E-64 and then prepared a crude postnuclear fraction of the cells for enzymatic assay finding that E-64 indeed reduced cathepsin B activity within the cells. Alternatively, both Redwood et al. [8] and Navab et al. [9] verified the ability of E-64 to block tumor cell invasion through an artificial basement membrane (Matrigel) prior to its use in vivo. Van Noorden et al. [10] developed a new synthetic fluorogenic substrate for cathepsin B allowing localization of the enzyme in living cells using confocal microscopy. Using this substrate in vitro, they confirmed that their cathepsin B inhibitor, Mu-Phe-homoPhe-fluoromethylketone, reduced cathepsin B activity in the rat colon cancer cell line subsequently used in an experimental model of liver metastasis [10]. Preparation of subcellular fractions and the Matrigel invasion assay are fairly labor intensive techniques and confocal microscopy requires a significant investment in equipment. Therefore, we sought to develop a more facile method to survey cell lines for cathepsin B activity and inhibition of this activity. We describe here an assay to measure pericellular cathepsin B using the fluorogenic substrate Z-Arg-Arg-AMC and a standard fluorescence plate reader. This is an adaptation of one previously described in which we measured pericellular cathepsin B activity generated by living cells grown on cover slips [4,11]. Enzymatic activity correlated with the presence of cathepsin B protein as detected by immunoblotting. Such an assay should be of use in choosing cell lines that express cathepsin B activity and for verifying the efficacy of inhibitors prior to testing in in vivo models.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

The vinyl sulfone cathepsin inhibitors Mu-Tyr(3,5 I2)-HphVSPh, Mu-Leu-HphVSPh and Mu-Np2-HphVS-2Np were synthesized as described [12]. The urokinase inhibitor B428 [13] was a generous gift of B. Littlefield, Eisai Research Institute (Andover, MA, USA). E-64 was purchased from Sigma (St Louis, MO, USA). Leupeptin, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC were obtained from Bachem (King of Prussia, PA, USA). CA-074 was purchased from Peptide Institute (Lexington, KY, USA). Purified human liver cathepsin B and polyclonal rabbit anti-human cathepsin B, H and L sera were purchased from Athens Research and Technology (Athens, GA, USA). NuPAGE SDS gels, electrophoresis reagents and the Western Breeze™ chemiluminescent immunodetection system were obtained from Novex (San Diego, CA, USA).

Cell lines

EJ human bladder carcinoma cells were a generous gift of S. Aaronson, Mt. Sinai School of Medicine (New York, NY, USA). The MatLyLu-PYN rat prostate carcinoma and Mat B III rat adenocarcinoma lines were obtained from S. Rabbani, McGill University, Montreal, Canada. The Lewis lung carcinoma line was obtained from the National Cancer Institute, Bethesda, MD, USA. The U87 human glioma, BT-20 human breast carcinoma, BT-549 human breast carcinoma, B16a mouse amelanotic melanoma, the CaCo-2 human colon carcinoma line, the PC-3 human prostate carcinoma line, the HT-1080 human fibrosarcoma line, the HCT-116 human colon carcinoma line and the MiaPaCa human pancreatic carcinoma line were obtained from ATCC, Rockville, MD, USA. Cells were maintained in 75-cm2 flasks in serum containing growth medium as specified by ATCC in a humidified atmosphere at 37 °C and 5% CO2 and passaged 1–2 times per week as needed.

Enzymatic assay for cathepsin B

A 1 : 3000 dilution working stock of human liver cathepsin B corresponding to 225 ng·mL−1 of enzyme with a specific activity of 588 U·mg−1 was prepared in pericellular assay buffer (PAB; Hank's balanced salt solution lacking sodium bicarbonate and containing 0.6 mm CaCl2, 0.6 mm MgCl2, 2 mm l-cysteine, 25 mm Pipes) adjusted to pH 7.0 [11]. The enzymatic reaction (0.2 mL volume) was run in a 96 well plate (Dynex™ microfluor) at a final 1 : 30 000 dilution (22.5 ng·mL−1; 0.9 mm) of enzyme utilizing 60 µm Z-Arg-Arg-AMC substrate. Inhibitors and their dilutions were prepared in dimethylsulfoxide while the untreated reactions were run with an equal volume of dimethylsulfoxide containing vehicle (0.1% v/v, final). Velocity (relative fluorescence units per min) was determined on an Fmax fluorescent plate reader and associated softpro software (Molecular Devices, Sunnyvale, CA, USA) set in kinetic mode with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The reaction was run for 10 min at 37 °C with readings taken every 30 s.

Cellular assay for cathepsin B activity

Monolayer cultures. Cells were plated in 96 well culture plates (Corning Costar) and grown to 80% confluence (i.e. between 10 000–20 000 cells per well). The growth medium was then aspirated and the monolayers washed with 0.1 mL Dulbecco's NaCl/Pi. The cells were then incubated with 0.2 mL of PAB at 37 °C for 30 min after which time the buffer was replaced with fresh PAB containing 100 µm Z-Arg-Arg-AMC in the presence or absence of inhibitors and 0.1% Triton X-100 to liberate total cellular cathepsin B activity. Progress of fluorescent product formation was recorded using a Labsystems Fluoroskan II microplate reader on kinetic mode at 37 °C with an 355-nm excitation filter and a 460-nm emission filter. Data analysis was performed with the aid of deltasoft 3 ELISA analysis software for MacIntosh from Biometallics, Inc. (Princeton, NJ, USA).

Cell suspensions. The monolayers of cell cultures (90–95% confluent) were decanted of growth medium and rinsed with Ca2+ and Mg2+-free D-NaCl/Pi, pH 7.4 (GIBCO, Grand Island, NY, USA). The cells were nonenzymatically liberated from the flasks in Versene (GIBCO) and were resuspended at a concentration of 1 × 106 cells·mL−1 in PAB with 2.0 mm l-cysteine, pH 7.0. The enzymatic reaction for cellular cathepsin B was run in a Dynex™ microfluor 96 well plate in a 0.2-mL final volume using either 20 000 or 150 000 cells per well as noted in the figure legends. The Km of U87 cell associated cathepsin B was determined using various concentrations of Z-Arg-Arg-AMC. For all other cell lines, a final concentration of 200 µm Z-Arg-Arg-AMC was used to assess activity. The reactions were run for 10 min at 37 °C with readings taken every 30 s. For the cell assays in the presence of cysteine protease inhibitors, the concentration of dimethylsulfoxide was limited to 0.1%. In some experiments, 0.1% Triton X-100 was added to liberate total cellular cathepsin B activity. Data was analyzed using the enzymekinetics software package (Trinity Software, Fort Pierce, FL).

Preparation of cell lysates, electrophoresis and immunoblotting. Cells in T-75 cm2 flasks were decanted of growth medium and rinsed twice with D-NaCl/Pi. A 1.5-mL aliquot of extraction buffer (50 mm Tris/HCl, 1% Triton X-100, 1% Na deoxycholate, 150 mm NaCl, 5 mm Na4-EDTA, 0.1% SDS, pH 7.4) with protease inhibitor cocktail (Complete™; Boehringer-Mannheim, Germany) was added to each flask and the contents scraped and subjected to three freeze/thaw cycles at −80 °C/37 °C. The crude lysate was then collected into microcentrifuge tubes and passed through a tuberculin syringe needle three times prior to centrifugation at 12 000 g for 10 min at 4 °C in a Beckman microcentrifuge. The clarified supernatants were collected into screw capped cryovials and stored (−20 °C) for subsequent electrophoresis and immunoblotting. The protein content of cell lysates was determined using the method of Bensadoun and Weinstein [14] with bovine serum albumin as a standard. Samples (5 µg protein) of cell lysates in NuPAGE sample buffer (Novex) were subjected to electrophoresis using NuPAGE Mes-SDS running buffer (Novex) under reducing conditions on 4–12% NuPAGE Bis-Tris gels (Novex). The separated proteins were transferred onto nitrocellulose filters using NuPAGE transfer buffer with 20% methanol (v/v). The filters were blocked, blotted and developed using the Novex Western Breeze™ chemiluminescent immunodetection system with rabbit anti-(human cathepsin B) IgG (1 : 4000 dilution), rabbit anti-(human cathepsin H) IgG (1 : 8000 dilution) or rabbit anti-(human cathepsin L) IgG (1 : 8000 dilution) according to the manufacturer's protocol. The anti-(cathepsin B) IgG does not cross react with cathepsins D, H and L while the anti-(cathepsin H) IgG does not cross react with cathepsins B, L and D and the anti-(cathepsin L) IgG does not cross react with cathepsins B, D and H at the respective dilutions used for immunoblotting.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Intact U87, CaCo-2 and BT-20 cells possess the ability to hydrolyze the cathepsin B selective substrate Z-Arg-Arg-AMC when added directly to cell monolayers. As seen in Fig. 1, both pericellular and total cathepsin B activity can be measured in monolayer cultures of cells. Triton X-100 treatment of these cells resulted in many fold more Z-Arg-Arg-AMC hydrolyzable activity which could be almost completely abolished with 10 µm of the cathepsin B highly selective inhibitor CA-074 (Fig. 1). CA-074 is amply documented as an active site irreversible inhibitor of cathepsin B [11–15]. These studies show that our cellular assay is specific for cathepsin B. However, the number of cells that can be assayed is limited to the number that can grow as a monolayer in an individual well of a 96 well plate and could limit the detection of enzymatic activity in cells that secrete low levels of the enzyme. Cathepsin B activity may be undetectable in monolayers of cells such as BT-20 which have low pericellular levels of the enzyme. Other compounds, namely the vinyl sulfone irreversible broad spectrum cysteine protease inhibitors, were then tested to show their efficacy in these cell lines.

image

Figure 1. Cathepsin B activity of cell lines in monolayer culture. Cells were plated in 96-well plates and grown to 80% confluence (10 000–20 000 cells per well) and then assayed for cathepsin B activity in the presence and absence of 0.1% Triton X-100 and 10 µm CA-074 and 100 µm Z-Arg-Arg-AMC as described in Materials and methods. Velocity measurements are expressed as the mean ± SEM of the maximum rate of fluorescence unit increase per min where n = 8.

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As an alternative, we used Versene to liberate intact cells from monolayer culture and counted and dispensed the dissociated cells into the assay plates. This enabled us to use more cells than can be naturally accommodated if grown as a monolayer (50 000 or 150 000 cells as specified in the figure legends) in order to approximate the level of enzymatic activity of the purified enzyme used. The cellular assays were then run under reaction conditions identical to those used with the purified human liver enzyme. In dissociated U87 cells, the Km for Z-Arg-Arg-AMC was determined to be 460 µm for this pericellular enzymatic activity at pH 7.0 and 37 °C (Fig. 2A). This is close to the 430 µmKm obtained with purified human liver cathepsin B at pH 7.0 and 37 °C (Fig. 2B). We were thus able to measure pericellular cathepsin B in both Versene dispersed cells and intact monolayer cultures.

image

Figure 2. Cathepsin B enzymatic activity. (A) Cathepsin B enzymatic activity of U87 cells. Cells (20 000 per well) were incubated (37 °C) with Z-Arg-Arg-AMC substrate for 10 min in PAB, pH 7.0 with 2 mm l-cysteine and fluorescence readings taken every 30 s as described in Materials and methods. Graph inset depicts the Lineweaver–Burk plot of the transformed data indicating a Km of 460 µm. Velocity measurements are in relative fluorescence units·min−1. (B) Enzymatic activity of purified human liver cathepsin B. Human liver cathepsin B (13.5 ng·mL−1) was incubated (37 °C) with Z-Arg-Arg-AMC substrate for 10 min in PAB, pH 7.0 with 2 mm l-cysteine and fluorescence readings taken every 30 s as described in Materials and methods. Graph inset depicts the Lineweaver–Burk plot of the transformed data indicating a Km of 430 µm. Velocity measurements are in relative fluorescence units·min−1.

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Moreover, this pericellular (cell surface and secreted) cathepsin B activity was inhibited by several types of cysteine protease inhibitors with potency similar to that observed against purified human liver cathepsin B (Fig. 3 and Table 1). We used several types of broad spectrum cysteine protease inhibitors of the vinyl sulfone class which are irreversible mechanism based inhibitors [12]. The IC50 value for Mu-Tyr(3,5 I2)-HphVSPh (which is a selective vinyl sulfone inhibitor designed to be most active against cathepsin B-like activity) was < 100 nm on both pericellular and purified cathepsin B enzymes. The vinyl sulfone inhibitor Mu-Leu-HphVSPh (cathepsin L-like inhibitor) and the vinyl sulfone inhibitor Mu-Np2-HphVS-2Np (cathepsin S-like inhibitor) were less potent against pericellular and purified cathepsin B enzymes with IC50 values in the low µm range. Both E-64, an irreversible epoxide broad spectrum cysteine protease inhibitor and leupeptin, a reversible aldehyde broad spectrum cysteine protease inhibitor, were effective in inhibiting pericellular and purified cathepsin B with IC50 values in the 100–200 nm and 50 nm range, respectively. B428, an inhibitor of the serine protease urokinase [13], was unable to inhibit the activity of either pericellular cathepsin B or purified cathepsin B at concentrations of 100 µm (Fig. 3 and Table 1).

image

Figure 3. Effect of cysteine protease inhibitors on pericellular cathepsin B activity. Purified human liver cathepsin B (A) and U87 cells (B) were incubated with and without leupeptin, E-64, B428, Mu-Tyr(3,5 I2)-HphVSPh, Mu-Leu-HphVSPh or MuNp2-HphVS-2Np as indicated and Z-Arg-Arg-AMC for 10 min at 37 °C in PAB (pH = 7.0) with 2 mm l-cysteine as described in Materials and methods. In the presence of vehicle only, the control velocity for purified enzyme was 32 ± 1 relative fluorescent units·min−1 and the control velocity for pericellular enzyme was 48 ± 1 relative fluorescent units·min−1. Results shown are the mean ± SEM of 2 separate experiments where n = 3. Points without bars had standard errors too small to depict.

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Table 1. Effect of cysteine protease inhibitors on cathepsin B activity. For Cathepsin B: purified human liver cathepsin B (1 : 3000), 10-min kinetic assay in PAB with 2 mm l-cysteine, pH 7.0. Final [dimethylsulfoxide] was 0.1%; Z-Arg-Arg-AMC was 60 µm. For U87 cells: 20 000 cells per well, 10-min kinetic assay in PAB with 2 mm l-cysteine, pH 7.0. Final [dimethylsulfoxide] was 0.1%; Z-Arg-Arg-AMC was 525 µm. Thumbnail image of

In a survey of other human cancer cell lines, we found that there was measurable pericellular cathepsin B activity in MiaPaCa, HT-1080, HCT-116 and PC-3 cell lines using the Z-Arg-Arg-AMC peptide substrate (Fig. 4A). This activity was abolished by the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh at a 0.3-µm concentration (Fig. 4A). When we detected pericellular cathepsin B activity, we also detected cathepsin B protein in cell lysates using anti-(cathepsin B) IgG (Fig. 5). Both active forms (31 kDa single chain and 25 kDa heavy chain of double chain cathepsin B) are depicted in the immunoblots. In BT-549 and EJ cells, we were unable to detect cathepsin B (Fig. 4A) or significant immunoreactivity (Fig. 5) under these conditions whereas in BT-20 cells we detected marginal levels of activity and protein (Figs 4A and 5). Therefore, there was catalytically active pericellular cathepsin B in a diverse range of human cancers (glioblastoma, colon, pancreatic, fibrosarcoma, prostate and breast). No immunodetectable bands were present in any of these lysates using either anti-(cathepsin H) IgG or anti-(cathepsin L) IgG (data not shown).

image

Figure 4. Assessment of cathepsin B pericellular enzymatic activity of cancer cell lines in culture. (A) Human cell lines; (B) rat and murine cell lines. Cells (150 000 per well) were incubated with 200 µm Z-Arg-Arg-AMC substrate for 10 min with fluorescence readings taken every 30 s as described in Materials and methods. Velocity measurements are in relative fluorescence units per min. Results shown are the mean ± SEM of two separate experiments where n = 4. Bars without standard error markings were too small to depict.

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image

Figure 5. Immunoblot analysis of human cancer cell lysates using a 1 : 4000 dilution of anti-(human liver cathepsin B) serum. Each lane had 5 µg of cell lysate loaded as indicated. The lane marked Cat B indicates purified human liver cathepsin B protein (1.5 ng) run as a standard. Arrows indicate the migration of the 31-kDa and 26-kDa molecular mass markers.

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Examination of several murine and rat cancer cell lines revealed that measurable cathepsin B enzymatic activity was associated with B16a amelanotic melanoma cells, Lewis lung carcinoma cells, Mat B III rat adenocarcinoma cells and MatLyLu rat prostate cancer cells (Fig. 4B). This activity was inhibited by the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh (Fig. 4B). There was not sufficient cross reactivity of the anti-(human cathepsin B) serum with murine and rat cathepsin B to detect cathepsin B protein in lysates of these nonhuman cell lines.

Triton X-100 treatment of the cells prior to assay liberated an additional fivefold to sevenfold Z-Arg-Arg-AMC hydrolyzable activity and this activity was > 90% inhibited by 10 µm Mu-Tyr(3,5 I2)-HphVSPh (Fig. 6A). When the less selective substrate Z-Phe-Arg-AMC was used, we found a twofold to threefold overall increase in activity liberated by Triton X-100 treatment (Fig. 6B). The cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh was slightly less effective (75–90%) in abolishing Z-Phe-Arg-AMC hydrolyzable activity. It should be noted that there are other species of cysteine proteases within the cells which can hydrolyze Z-Phe-Arg-AMC (e.g. cathepsin L) and thus can contribute to the proteolytic activity measured by this substrate [16]. Therefore, the use of the dibasic substrate is preferred over the monobasic substrate. Our results suggest that the pericellular cathepsin B activity we observed in the absence of detergent was secreted or membrane-associated cathepsin B. Studies performed in the presence of detergent show that the majority of cathepsin B activity was intracellular. The active pericellular enzyme is the 31 kDa species [11].

image

Figure 6. Triton X-100 releasable cathepsin B activity of cell lines. Cells (150 000 per well) were incubated in the presence or absence of 0.1% Triton X-100 and 10 µm Mu-Tyr(3,5 I2)-HphVSPh as indicated and 200 µm Z-Arg-Arg-AMC (A) or 100 µm Z-Phe-Arg-AMC (B) substrate for 10 min with fluorescence readings taken every 30s as described in Materials and methods. Velocity measurements are in relative fluorescence units per min. Results shown are the mean ± SEM of two separate experiments where n = 4. Bars without standard error markings were too small to depict.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have demonstrated a facile method by which to survey cell lines for pericellular and total cathepsin B activity. There is good agreement between the enzymatic activity observed with artificial peptide substrates for cathepsin B in intact cells and the presence of cathepsin B protein in these cells by immunoblotting with specific antiserum. Our study has shown that pericellular cathepsin B has virtually the same kinetic profile and response to several classes of cysteine protease inhibitors as the purified human liver enzyme. This method can distinguish the relative amounts of catalytically active pericellular cathepsin B for a given number of cells from cell lines obtained from different tissues and different species. The relative secretion of cathepsin B vs. other cysteine proteases (such as cathepsin L and others) can also be determined using the Z-Phe-Arg-AMC substrate. Information gathered with this method would therefore be potentially useful in selecting cell lines appropriate for use in in vivo tumor models in order to assess the efficacy of cathepsin B inhibitors against tumor growth and metastasis.

While the major band that was observed for cathepsin B in the whole cell lysates of the cell lines was 25–26 kDa, corresponding to the fully processed double chain (25–26 + 5 kDa) enzyme form, the U87 and HT-1080 cells had an additional 31-kDa single-chain species of the enzyme that was prominent on the immunoblot (Fig. 5). This 31-kDa cathepsin B has previously been shown to be the active pericellular form of the enzyme [11]. Upon longer exposure of the immunoblot to film, the MiaPaCa cells also had a trace amount of the 31-kDa band (data not shown). Both single chain and double chain forms of human cathepsin B are active [17]. The highest levels of Z-Arg-Arg-AMC pericellular hydrolyzable activity, observed in the absence of Triton X-100, were from the U87, HT-1080 and MiaPaCa cells, respectively. This is consistent with our prior study suggesting that the 31-kDa single-chain cathepsin B is the active pericellular species of the enzyme [11]. The fact that the Km determinations are so close between the commercially purified and cellular forms of cathepsin B indicates that Z-Arg-Arg-AMC is a suitable substrate for both forms of this enzyme; the activity of the cellular and purified forms of the enzyme is identical against Z-Arg-Arg-AMC.

It is not surprising that the development of inhibitors of cathepsin B has gained broader interest given the potential functional link between cathepsin B and tumor invasion and metastasis. The fluorescent microplate assay described in this paper will be useful in screening for cathepsin B inhibitors able to block pericellular cathepsin B activity without affecting the intracellular enzyme. Such compounds may have the advantage of being less cytotoxic in vivo than compounds that penetrate inside cells and block total cathepsin B.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported in part by NIH grant CA 36481 to B. F. S.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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Footnotes
  1. Enzyme: cathepsin B (EC 3.4.22.1).

    Dedication: this paper is dedicated to Ellen R. Otis in honor of her retirement from Abbott Laboratories after nearly 30 years of devoted, efficient service and for beginning some of the preliminary studies which led to this paper.