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Cellular detachment by purified lysosomal cathepsin B
Europ. ~. Cancer Vol. 4, pp. 559-562. Pergamon Press 1968. Printed in Great Britain
Cellular Detachment by Purified Lysosomal Cathepsin B B. SYLV]~N The Cancer Research Division of Radiumhemmet, Karolinska Institute, Stockholm 60, Sweden
calcium- and magnesium-free Hank's solution (CMFH) at a carefully checked pH of 7.1 (no serum albumin added). Crystalline trypsin (Worthington) and salt-free lyophilized cathepsin B was used at concentrations mentioned below. This enzyme hydrolyses similar C-N bonds as trypsin does, and is activated [7, 8] by chelating agents like EDTA or lactic a c i d + added -SH compounds like cysteine or glutathione (GSH), but is inhibited by added heavy metal ions (Mn s÷ and Mg*+). This enzyme has been isolated by Snellman [9] from lysosomal concentrate obtained from calf liver and was supplied in a highly purifed state without admixture of cathepsin D, aminopeptidases or other enzymes. Proteinase activity was maxim u m at pH 5.8, falling to one third at p H 7. Cathepsin B could not be activated in the usual way by addition of 0.05 M E D T A + 0 . 0 0 4 M cysteine since the ceUswould deteriorate. Hence, the environmental conditions in tissue fluids, particularly of solid tumours, were mimicked; instead of EDTA a weak chelation was applied by addition of 0.2% lactic acid to C M F H and cysteine was replaced by 0.16% GSH. The acid pH shift was corrected by added NaOH until full stability was reached for 1 hr. Only a fraction of the digestive capacity of cathepsin B was thus utilised at a stable pH of 7" 1 as compared with the much higher proteinase activity of similar amounts of added trypsin.
CELLULAR detachment from glass and other substrata is produced by different proteinases such as trypsin [1], pronase(s) [2] and collagenase(s) [3]. Short time incubation in weak trypsin solutions (0.05-0.2%) has become an established method for cell separation although this involves some temporary damage to cell surfaces by removing part of the protein coating rich in N-acetyl neuraminic acid [4]. It is also known that dead cells become rapidly digested by trypsin treatment [5], and that cells first treated by a chelating agent or a non-ionic detergent become more vulnerable to a subsequent trypsin treatment [6]. These various proteinases either do not occur in normal mammalian tissues and tumours or else thay are effectively inhibited, like trypsin, by specific inhibitors in blood and tissue fluid. It is therefore of interest to demonstrate that a normally occurring cellular hydrolase like cathepsin B, once in a free and active state outside the cytoplasm, will exert a marked capacity to detach cells. This enzyme has now been found to exert a cell surface effect qualitatively very similar to that of trypsin. MATERIAL A N D M E T H O D S For the study of cellular detachment the C13 hamster fibroblasts (Stoker line) were grown as still cultures in Eagle's basal medium with 10% fetal calf serum added. All controls and short-time incubations were performed in
RESULTS C13 fibroblasts subcultured on coverslips for 24 hr in Petri dishes presented a rather dense
Submitted for publication 16 July 1968. Accepted 19 August 1968.
559
s6o
sylva
felt-like monolayer with long pseudopodia and slender cell bodies; all cells were firmly attached to the glass. Such cultures were just washed in C M F H and transferred to cavity slides for enzyme treatment applying about 0.25 ml enzyme solution to each monolayer. The results were very obvious and qualitatively registered under the phase-microscope with green light at magnifications up to 500 times. The results summarized in Table 1 were similar using either trypsin or activated cathepsin B. Inside 3-10 min all pseudopodia were withdrawn, the cells rounded up, became short, plump, strongly refractile and most cells left their attachment to the coverslips. Cellular aggregation due to stickiness was less marked with cathepsin B than following trypsin treatment. After 20-30 min treatment no further changes were noticed. It was not established whether this was due to deterioration of enzyme activity (both enzymes are rather instable). An interesting fact was that the cathepsin B effect was reversible at an enzyme concentration up to 1.5 mg/ml. When returned to their original growth m e d i u m the cells attached to glass again, became elongated and fully resumed their original growth pattern in about 5 hours' time. T h e actual percentage of cell death, as ascertained by the Lissamine green dyeexclusion test [10], was not increased after 30 min treatment with cathepsin B while trypsin treatment at a concentration of 1.5 mg/ml, caused both cell death and some cytolysis. Table 1.
Another question was whether cathepsin B migh digest damaged and dead cells as observed following trypsin treatment. This was tested on suspensions of ELD tumour cells in C M F H recently killed by repeated freezing and thawing. Several series of cells counts were made at frequent intervals following the addition of trypsin and activated cathepsin B at the same concentrations. Cathepsin B was equally effective as trypsin; dead cells permeable to the dye were digested at a similar very rapid rate inside 10 min by added cathepsin B, but in addition cells less damaged and not yet permeable to the dye also became digested by the more powerful trypsin activity but not by added cathepsin B. Both suspensions were rich in ghosts and cell remnants. The effect of extraneous cathepsin B was most likely increased by activation of the lysosomal proteinase in damaged cells, since the rate of cytolysis became markedly increased also in cases where only lactic acid-J-GSH were added. Hence, the percentage of living cells, as determined by the dye-exclusion test was overestimated, owing to digestion of the dead cells by cathepsin in 30 min. COMMElCrS The experiments demonstrate that a normal intracellular proteinase with an activity extending above p H 7 once released and activated will bring about both cellular detachment and rapid digestion of damaged cells. T h e digestive capacity of liver cathepsins during autolysis was studied by Sawant et al. [11]. Cellular
Cellular detachment at different concentrations of added Trypsin and activated Cathepsin B p H of incubation medium carefully maintained at 7.1
Test solutions Controls In C M F H only In C M F H + 0 . 2 % lactic acid added In C M F H + L . a c i d + 0 . 1 5 % GSH added
Crystalline trypsin added 0.05% in CMFI-I 0.1% and 0- 15% in C M F H Purified cathepsin B added In C M F H containing L. acid and GSH for activation+0.05% cathepsin B 0" 1% and 0.15% cathepsin B
Changes of C 13 fibroblasts No changes in shape for 60 min Some rounding up, no detachment Some retraction of pseudopodia, but detachment
no
Slow and partial detachment of cells Rapid pseudopodia retraction in a few minutes; most cells detached Partial detachment of cells and general pseudopodia retraction Rapid and complete pseudopodia retraction in 3-10 min. Almost all cells lost attachment to glass
Cellular Detachment by Purified Lysosomal Cathepsin B detachment was cursorily tested by Leon Weiss who used an impure and non-activated lysosomal concentrate [12]. Present results with pure cathepsin B thus open new and important avenues of approach into several fields of pathology notably with a view to the study of cell detachment associated with the invasiveness of tumours, the chemical mechanisms of their destructive activity, and possibly to metastasis formation (cf. references 13 and 14). The investigations are being continued in order
561
to obtain more quantitative in vitro data [15] and in order to study the topical distribution in tumours of cathepsin B with histochemical [8, 16] and biochemical micromethods [17]. Acimowledlgements--My thanks are due to Professor
E. J. Ambrose of the Chester Beatty Research Institute for the gift of C13 fibroblasts (originally derived from Professor Stoker). Samples of purified cathepsin B preparations were kindly supplied by Dr. O. Snellman of this laboratory. This investigation was supported by the Jubilee Fund and the Cancer Societyof Stockholm.
RESUME Ce travail ddcrit l'action in vitro de la cathepsine B hautement purifige, activie par l'acide lactique et le glutathion. L'enzyme active avait des effets trks semblables ~ ceux de la trypsine ; elle provoquait rapidement un ditachement des cellules des surfaces de verre et une digestion des cellules mortes. La signification de ces proprie'tgs est discutge en tenant compte du concept selon lequel le dgtachement des cellules tumorales serait un facteur essentiel dans la formation des mgtastases. SUMMARY In vitro studies are reported using highly purified cathepsin B which was activated by
added lactic acid+ glutathione. The active enzyme presented effects very similar to those of added trypsin; detachment of cells from glass surfaces as well as digestion of dead cells was rapidly achieved. The apparent implications are discussed with a view to the detachment of tumour cells forming an essential prerequisite to metastasis formation. ZUSAMMENFASSUNG
Hochgereinigtes Kathepsin B wurde durch Zugabe von Milchsdure und Glutathion aktiviert. Das aktive Enzym bewirkte in vitro Effekte, die denen yon Trypsin ~hnlich sind. Losliisung von Zellen vom Glase konnte ebenso wie die Verdauung toter ZeUen schnell erzielt werden. Die Bedeutung der Befunde wird im Hinblick auf die Losliisung von TumorzeUen diskutiert, die eine notwendige Voraussetzung der Metastasenbildung ist. BlgFERENCES 1. L. WEiss, The adhesion of cells. In International Review of Cytology (Edited by G. H. BOURNE and J. F. DANmLLI) Vol 9, p. 187. Academic Press, New York (1960). 2. R . B . L . GWATKINand J. L. THOMSON, A new method for dispersing the cells of mammalian tissues. Nature (Lond.) 201, 1242 (1964). 3. R . B . HowAm~, A. K. CHRISTENSEn,F. A. GraBS and I. A. PINCH, The enzymatic preparation of isolated intact parencymal cells from rat liver. J. cell Biol. 35, 675 (1967). 4. G. M. W. Cooiq D. H. HEARX) and G. V. F. SEAMnN,A sialomucopeptide liberated by trypsin from the human erythrocyte. Nature (Lond.) 1 ~ 1011 (1960). 5. C. DE Lucn, The use of ,trypsin for the determination of cellular viability. Exp. Cell Res. 40, 186 (1965). 6. A . G . MALENKOV,S. A. BOGATYm~VA,V, P. BOZHKOVA,E. A. MaDJAOVA and Ju. M. Vnsmmv, Reversible alterations of the surface of ascites tumour cells induced by a surface active substance. Exp. Cell Res. 411, 397 (1967). 7. B. SYLVAN and O. SN~.LLMAN,Haptoglobin acting as a natural inhibitor of cathepsin B activity. Nature (Lond.) 216~ 1033 (1967). 8. B. SYLVgN and O. SNELLMAN,Studies on the histochemical "leucine aminopeptidase" reaction. V. Cathepsin B as a potential effector of LNA hydrolysis. Histochemie, 12, 240 (1968).
562
B. Sylvan 9. 10. I 1. 12. 13.
O. SN~.LLM~, Separation of cathepsin B and D from calf liver and the role of haptoglobin as a cathepsin B inhibitor. (In preparation.) B. HOLMBSRO,On the permeability to Lissamine green and other dyes in the course ofceU injury and cell death. Exp. Cell Res. ~p 406 (1961). P . L . SAWANT, I. D. D~.SAXand A. L. TAPP~L, Digestive capacity of purified lysosomes. Biochim. biophys. Acta (Amst.) 85, 93 (1964). L. W~.rss, Studies on eellular adhesion in tissue culture--VIII. Some effects of antisera on cell detachment. Exp. Cell Res. 37~ 540 (1965). B. SYLV~N, The host-tumor interzone and tumor invasion. In Biological
Interactions in Normal and Neoplastic Growth. Henry Ford Hos[Ktal International Symposium, p. 635. Little, Brown & Co., Boston (1962). 14.
B. SYLVtN, Biochemical factors accompanying growth and invasion.
In
Endogenous Factors Influencing Host-Tumor Balance (Edited by R. W. WISSLER, T. L. DAO and JR. S. WOOD) p. 267. University of Chicago Press (1967). G.C. EASTY,D. M. EaSTY and E. J. AMBROSE,Studies of cellular adhesiveness. Exp. Cell Res. 19, 539 (1960). 16. B. SYLVANand I. BOIs-SvENSSON, Studies on the histochemical "leucine aminopeptidase" reaction--IV. Chemical and histochemical characterization of the intracellular and stromal LNA reactions in solid tumor transplants. Histochemie ~ 135 (1964). 17. B. SYLVAN, Lysosomal enzyme activity in the interstitial fluid of solid mouse tumour transplants. Europ. aT. Cancer 4~ 463. 15.
Cellular Detachment by Purified Lysosomal Cathepsin B B. SYLV]~N The Cancer Research Division of Radiumhemmet, Karolinska Institute, Stockholm 60, Sweden
calcium- and magnesium-free Hank's solution (CMFH) at a carefully checked pH of 7.1 (no serum albumin added). Crystalline trypsin (Worthington) and salt-free lyophilized cathepsin B was used at concentrations mentioned below. This enzyme hydrolyses similar C-N bonds as trypsin does, and is activated [7, 8] by chelating agents like EDTA or lactic a c i d + added -SH compounds like cysteine or glutathione (GSH), but is inhibited by added heavy metal ions (Mn s÷ and Mg*+). This enzyme has been isolated by Snellman [9] from lysosomal concentrate obtained from calf liver and was supplied in a highly purifed state without admixture of cathepsin D, aminopeptidases or other enzymes. Proteinase activity was maxim u m at pH 5.8, falling to one third at p H 7. Cathepsin B could not be activated in the usual way by addition of 0.05 M E D T A + 0 . 0 0 4 M cysteine since the ceUswould deteriorate. Hence, the environmental conditions in tissue fluids, particularly of solid tumours, were mimicked; instead of EDTA a weak chelation was applied by addition of 0.2% lactic acid to C M F H and cysteine was replaced by 0.16% GSH. The acid pH shift was corrected by added NaOH until full stability was reached for 1 hr. Only a fraction of the digestive capacity of cathepsin B was thus utilised at a stable pH of 7" 1 as compared with the much higher proteinase activity of similar amounts of added trypsin.
CELLULAR detachment from glass and other substrata is produced by different proteinases such as trypsin [1], pronase(s) [2] and collagenase(s) [3]. Short time incubation in weak trypsin solutions (0.05-0.2%) has become an established method for cell separation although this involves some temporary damage to cell surfaces by removing part of the protein coating rich in N-acetyl neuraminic acid [4]. It is also known that dead cells become rapidly digested by trypsin treatment [5], and that cells first treated by a chelating agent or a non-ionic detergent become more vulnerable to a subsequent trypsin treatment [6]. These various proteinases either do not occur in normal mammalian tissues and tumours or else thay are effectively inhibited, like trypsin, by specific inhibitors in blood and tissue fluid. It is therefore of interest to demonstrate that a normally occurring cellular hydrolase like cathepsin B, once in a free and active state outside the cytoplasm, will exert a marked capacity to detach cells. This enzyme has now been found to exert a cell surface effect qualitatively very similar to that of trypsin. MATERIAL A N D M E T H O D S For the study of cellular detachment the C13 hamster fibroblasts (Stoker line) were grown as still cultures in Eagle's basal medium with 10% fetal calf serum added. All controls and short-time incubations were performed in
RESULTS C13 fibroblasts subcultured on coverslips for 24 hr in Petri dishes presented a rather dense
Submitted for publication 16 July 1968. Accepted 19 August 1968.
559
s6o
sylva
felt-like monolayer with long pseudopodia and slender cell bodies; all cells were firmly attached to the glass. Such cultures were just washed in C M F H and transferred to cavity slides for enzyme treatment applying about 0.25 ml enzyme solution to each monolayer. The results were very obvious and qualitatively registered under the phase-microscope with green light at magnifications up to 500 times. The results summarized in Table 1 were similar using either trypsin or activated cathepsin B. Inside 3-10 min all pseudopodia were withdrawn, the cells rounded up, became short, plump, strongly refractile and most cells left their attachment to the coverslips. Cellular aggregation due to stickiness was less marked with cathepsin B than following trypsin treatment. After 20-30 min treatment no further changes were noticed. It was not established whether this was due to deterioration of enzyme activity (both enzymes are rather instable). An interesting fact was that the cathepsin B effect was reversible at an enzyme concentration up to 1.5 mg/ml. When returned to their original growth m e d i u m the cells attached to glass again, became elongated and fully resumed their original growth pattern in about 5 hours' time. T h e actual percentage of cell death, as ascertained by the Lissamine green dyeexclusion test [10], was not increased after 30 min treatment with cathepsin B while trypsin treatment at a concentration of 1.5 mg/ml, caused both cell death and some cytolysis. Table 1.
Another question was whether cathepsin B migh digest damaged and dead cells as observed following trypsin treatment. This was tested on suspensions of ELD tumour cells in C M F H recently killed by repeated freezing and thawing. Several series of cells counts were made at frequent intervals following the addition of trypsin and activated cathepsin B at the same concentrations. Cathepsin B was equally effective as trypsin; dead cells permeable to the dye were digested at a similar very rapid rate inside 10 min by added cathepsin B, but in addition cells less damaged and not yet permeable to the dye also became digested by the more powerful trypsin activity but not by added cathepsin B. Both suspensions were rich in ghosts and cell remnants. The effect of extraneous cathepsin B was most likely increased by activation of the lysosomal proteinase in damaged cells, since the rate of cytolysis became markedly increased also in cases where only lactic acid-J-GSH were added. Hence, the percentage of living cells, as determined by the dye-exclusion test was overestimated, owing to digestion of the dead cells by cathepsin in 30 min. COMMElCrS The experiments demonstrate that a normal intracellular proteinase with an activity extending above p H 7 once released and activated will bring about both cellular detachment and rapid digestion of damaged cells. T h e digestive capacity of liver cathepsins during autolysis was studied by Sawant et al. [11]. Cellular
Cellular detachment at different concentrations of added Trypsin and activated Cathepsin B p H of incubation medium carefully maintained at 7.1
Test solutions Controls In C M F H only In C M F H + 0 . 2 % lactic acid added In C M F H + L . a c i d + 0 . 1 5 % GSH added
Crystalline trypsin added 0.05% in CMFI-I 0.1% and 0- 15% in C M F H Purified cathepsin B added In C M F H containing L. acid and GSH for activation+0.05% cathepsin B 0" 1% and 0.15% cathepsin B
Changes of C 13 fibroblasts No changes in shape for 60 min Some rounding up, no detachment Some retraction of pseudopodia, but detachment
no
Slow and partial detachment of cells Rapid pseudopodia retraction in a few minutes; most cells detached Partial detachment of cells and general pseudopodia retraction Rapid and complete pseudopodia retraction in 3-10 min. Almost all cells lost attachment to glass
Cellular Detachment by Purified Lysosomal Cathepsin B detachment was cursorily tested by Leon Weiss who used an impure and non-activated lysosomal concentrate [12]. Present results with pure cathepsin B thus open new and important avenues of approach into several fields of pathology notably with a view to the study of cell detachment associated with the invasiveness of tumours, the chemical mechanisms of their destructive activity, and possibly to metastasis formation (cf. references 13 and 14). The investigations are being continued in order
561
to obtain more quantitative in vitro data [15] and in order to study the topical distribution in tumours of cathepsin B with histochemical [8, 16] and biochemical micromethods [17]. Acimowledlgements--My thanks are due to Professor
E. J. Ambrose of the Chester Beatty Research Institute for the gift of C13 fibroblasts (originally derived from Professor Stoker). Samples of purified cathepsin B preparations were kindly supplied by Dr. O. Snellman of this laboratory. This investigation was supported by the Jubilee Fund and the Cancer Societyof Stockholm.
RESUME Ce travail ddcrit l'action in vitro de la cathepsine B hautement purifige, activie par l'acide lactique et le glutathion. L'enzyme active avait des effets trks semblables ~ ceux de la trypsine ; elle provoquait rapidement un ditachement des cellules des surfaces de verre et une digestion des cellules mortes. La signification de ces proprie'tgs est discutge en tenant compte du concept selon lequel le dgtachement des cellules tumorales serait un facteur essentiel dans la formation des mgtastases. SUMMARY In vitro studies are reported using highly purified cathepsin B which was activated by
added lactic acid+ glutathione. The active enzyme presented effects very similar to those of added trypsin; detachment of cells from glass surfaces as well as digestion of dead cells was rapidly achieved. The apparent implications are discussed with a view to the detachment of tumour cells forming an essential prerequisite to metastasis formation. ZUSAMMENFASSUNG
Hochgereinigtes Kathepsin B wurde durch Zugabe von Milchsdure und Glutathion aktiviert. Das aktive Enzym bewirkte in vitro Effekte, die denen yon Trypsin ~hnlich sind. Losliisung von Zellen vom Glase konnte ebenso wie die Verdauung toter ZeUen schnell erzielt werden. Die Bedeutung der Befunde wird im Hinblick auf die Losliisung von TumorzeUen diskutiert, die eine notwendige Voraussetzung der Metastasenbildung ist. BlgFERENCES 1. L. WEiss, The adhesion of cells. In International Review of Cytology (Edited by G. H. BOURNE and J. F. DANmLLI) Vol 9, p. 187. Academic Press, New York (1960). 2. R . B . L . GWATKINand J. L. THOMSON, A new method for dispersing the cells of mammalian tissues. Nature (Lond.) 201, 1242 (1964). 3. R . B . HowAm~, A. K. CHRISTENSEn,F. A. GraBS and I. A. PINCH, The enzymatic preparation of isolated intact parencymal cells from rat liver. J. cell Biol. 35, 675 (1967). 4. G. M. W. Cooiq D. H. HEARX) and G. V. F. SEAMnN,A sialomucopeptide liberated by trypsin from the human erythrocyte. Nature (Lond.) 1 ~ 1011 (1960). 5. C. DE Lucn, The use of ,trypsin for the determination of cellular viability. Exp. Cell Res. 40, 186 (1965). 6. A . G . MALENKOV,S. A. BOGATYm~VA,V, P. BOZHKOVA,E. A. MaDJAOVA and Ju. M. Vnsmmv, Reversible alterations of the surface of ascites tumour cells induced by a surface active substance. Exp. Cell Res. 411, 397 (1967). 7. B. SYLVAN and O. SN~.LLMAN,Haptoglobin acting as a natural inhibitor of cathepsin B activity. Nature (Lond.) 216~ 1033 (1967). 8. B. SYLVgN and O. SNELLMAN,Studies on the histochemical "leucine aminopeptidase" reaction. V. Cathepsin B as a potential effector of LNA hydrolysis. Histochemie, 12, 240 (1968).
562
B. Sylvan 9. 10. I 1. 12. 13.
O. SN~.LLM~, Separation of cathepsin B and D from calf liver and the role of haptoglobin as a cathepsin B inhibitor. (In preparation.) B. HOLMBSRO,On the permeability to Lissamine green and other dyes in the course ofceU injury and cell death. Exp. Cell Res. ~p 406 (1961). P . L . SAWANT, I. D. D~.SAXand A. L. TAPP~L, Digestive capacity of purified lysosomes. Biochim. biophys. Acta (Amst.) 85, 93 (1964). L. W~.rss, Studies on eellular adhesion in tissue culture--VIII. Some effects of antisera on cell detachment. Exp. Cell Res. 37~ 540 (1965). B. SYLV~N, The host-tumor interzone and tumor invasion. In Biological
Interactions in Normal and Neoplastic Growth. Henry Ford Hos[Ktal International Symposium, p. 635. Little, Brown & Co., Boston (1962). 14.
B. SYLVtN, Biochemical factors accompanying growth and invasion.
In
Endogenous Factors Influencing Host-Tumor Balance (Edited by R. W. WISSLER, T. L. DAO and JR. S. WOOD) p. 267. University of Chicago Press (1967). G.C. EASTY,D. M. EaSTY and E. J. AMBROSE,Studies of cellular adhesiveness. Exp. Cell Res. 19, 539 (1960). 16. B. SYLVANand I. BOIs-SvENSSON, Studies on the histochemical "leucine aminopeptidase" reaction--IV. Chemical and histochemical characterization of the intracellular and stromal LNA reactions in solid tumor transplants. Histochemie ~ 135 (1964). 17. B. SYLVAN, Lysosomal enzyme activity in the interstitial fluid of solid mouse tumour transplants. Europ. aT. Cancer 4~ 463. 15.