[CANCER RESEARCH 40, 3222-3227, September 1980]0008-5472/80/0040-0000$02.00

Destruction of Extracellular Matrices Containing Glycoproteins, Elastin, andCollagen by Metastatic Human Tumor Celli

Peter A. Jones2 and Yves A. DeClerck

Departments of Pediatrics fP. A. J., V. A. D.j and Biochemistry (P. A. J.j. University of Southern California School of Medicine, and Department of Pediatrics,Division of Hematology-Oncology. Childrens Hospital of Los Angeles, Los Angeles, California 90027

ABSTRACT

Four human tumor cell lines were grown in direct contactwith the extracellular matrix proteins which had previouslybeen produced by cultured rat smooth muscle cells. The extracellular matrix contained glycoproteins, elastin, and collagen,and its digestion by the tumor cells was followed by the appearance of radioactive breakdown products in the supernatantmedium. All four tumor lines tested digested glycoproteinspresent in the matrix, whereas human fibroblasts were inactivein glycoprotein digestion. The human fibrosarcoma cell line(HT1080) demonstrated elastolytic and collagenolytic activityin addition to a plasmin-induced hydrolysis of glycoproteins.Removal of glycoproteins from the matrix was necessary forthe maximal digestion rate of elastin and collagen, and plasmingeneration by the tumor cell plasminogen activator thereforeplayed a pivotal role in the hydrolysis of all of the matrixcomponents. The elastolytic and collagenolytic activities werelocalized to the plasma membrane since no matrix digestionoccurred unless the tumor cells were grown in direct contactwith the connective tissue proteins. These activities were notinhibited by a wide spectrum of protease inhibitors. The degradation of elastin and collagen required active protein synthesis suggesting a relatively short half-life for the degradativeenzyme(s). These quantitative studies, in which tumor cellswere grown in contact with a complex extracellular matrixpossessing some of the characteristics of connective tissue,should have a bearing on tumor cell invasion.

INTRODUCTION

One of the most lethal properties of malignant cells is theirability to infiltrate normal tissue and to metastasize to distantsites. The normal connective tissues consist of cells embeddedin an extracellular matrix containing variable quantities andsubclasses of the connective tissue proteins including glycoproteins, collagen, elastin, and proteoglycans. There havetherefore been suggestions that tumor-associated proteasesmay aid in the invasive process by removal of these matrixproteins (6).

Several studies have concentrated on this aspect of tumorcell biology, and increased protease production has beenobserved with many transformed cells. Included in these arecollagenase activity (5, 14, 16), lysosomal enzymes (21 , 22),and plasminogen activator (11, 15, 26). However, since someof the extracellular matrix components (e.g., collagen andelastin) are not efficiently degraded at physiological pH by

@ This work was supported by Research Grant CD-i 8 from the AmericanCancer Society.

2 To whom requests for reprints should be addressed, at the Division of

Hematology-Oncology, Childrens Hospital of Los Angeles, 4650 Sunset Bouleyard, Los Angeles, Calif. 90027.

Received December 26, 1979; accepted May 23, 1980.

general proteases (8, 9), it might be anticipated that the totaldegradation of the matrix would require a battery of enzymesacting together. It is also likely that the extracellular matrixcomponents interact so as to modify their accessibility to anddegradation by proteolytic enzymes (1). The digestion of mixtures of matrix proteins by tumor cells should therefore bestudied in addition to work with purified substrates.

In this study, we describe the use of the extracellular matrixproduced by cultured rat smooth muscle cells (13) as such amixed substrate for human tumor cells. The substrate containsglycoproteins, elastin, and collagen and may be considered arepresentation of a connective tissue matrix which might beencountered by a tumor cell in the animal. All 4 tumor cell linestested were capable of hydrolyzing the glycoprotein components ofthe matrix, but only the HT1080 line (23) demonstratedmeasurable elastolytic and collagenolytic activity. We havetherefore attempted to define the relationships between theproteases produced by these cells and to determine theirrelevance to the total destruction of the extracellular matrix.

MATERIALS AND METHODS

Cell Culture. The humantumorcell linesHT1060 (23), TE85(1 8), RD (1 9), and LAN-i (25) were grown in Eagle's minimum

essential medium (Grand Island Biological Co., Santa Clara,Calif.) containing 10% fetal calf serum (Irvine Scientific, Irvine,Calif.), 2% tryptose phosphate broth (Difco Laboratories, Inc.,Detroit, Mich.), penicillin (100 units/mI), and streptomycin (100@g/ml).Human fibroblasts were obtained from a skin biopsy.

The cells were passaged regularly using trypsin-EDTA (GrandIsland Biological Co.) and were added to radioactively labeledmatrices at 1O@cells/i 6-mm plastic well (Costar, Cambridge,Mass.) or 2 x 1o@cells/35-mm dish (Falcon Plastics, Oxnard,Calif.). The matrix-producing rat smooth muscle cells were theR22 strain (13) and were cultured in the same medium.

Productionand Analysisof Extracellular Matrix. The radioactive extracellular matrix elaborated by R22 cells was prepared as described previously (12, 13). Briefly, R22 smoothmuscle cells were seeded at 0.5 x iO@cells/16-mm well(Costar) or 10@cells/35-mm dish (Falcon). The cultures received ascorbic acid (50 @g/ml)on the following day and freshascorbic acid daily. L-[3,4-3H]Proline (New England Nuclear,Boston, Mass.) was added at a final concentration of 0.3 to 1@Ci/ml5 days after seeding, and the matrix was prepared 7

days later. The cultures were washed once with 0.9% NaCIsolution, and the cells were lysed in 0.25 M NH4OHfor 30 mmat room temperature. The matrix was then vigorously washedwith distilled water followed by 70% ethanol and allowed to dryat 37°.The dishes were sterilized with 70% ethanol andwashed with phosphate-buffered saline [NaCI (8 g/liter), KCI(0.2 g/liter), Na2HPO4(1.15 g/liter), and KH2PO4(0.2 g/Iiter,pH 7.2)] before the subsequent addition of test cells.

CANCERRESEARCHVOL. 403222

Research. on February 23, 2021. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

AB100

v)0

0iLlU)

Id

@ 50

Matrix Hydrolysis by Tumor Cells

The complete matrices contain glycoproteins, elastin, andcollagen (13), and glycoprotemn-depleted matrices were prepared from them by the addition of trypsin (Sigma type III,pretreated with elastin to adsorb possible elastase contaminants) at a concentration of 20 @tg/mlin 0. 1 M Tnis-HCI, pH7.6, containing 10 mr@iCaCI2. Incubation was for 5 hr at 37°after which time the residual matrix (containing elastin andcollagen) was washed with water and 70% ethanol. Retreatment of trypsin-pretreated matrices with trypsin resulted in thefurther solubihization of less than 3% of the original total radioactivity.

Matrices were analyzed for glycopnotein, elastin, and collagen content by sequential enzyme treatment with trypsin, elastase, and finally collagenase as described previously (13). Theenzymes were used at final concentrations of 10 j@g/mlin 0.1M Tnis-HCI, pH 7.6, containing 1 0 m@ CaCI2, and the amount

of radioactivity solubilized by each treatment determined in100-@lsamples after the addition of 5 ml of Biofluon (NewEngland Nuclear). The R22 cells produced a matrix in whichapproximately 37% of the prohine radioactivity was trypsinsensitive, 31% was elastase sensitive, and 31% was collagenase sensitive (2).

Digestion Studies. Test cells were seeded onto [3H]pnolinelabeled matrices in the required serum. Ahiquots(100 @l)of thesupennatant medium were taken at the indicated times, and theradioactivity was determined following the addition of 5 ml ofBiofluon. Medium was changed every 48 hr, and the resultsgiven in the text refer to the cumulative totals of solubilizedradioactivity. Background values for matrices incubated withmedium without cells (usually less than 3% of the total radioactivity present) have been subtracted from these values.

Protease Inhibitors. Soybeantrypsininhibitorwas obtainedfrom Miles Laboratories, Inc., Elkhart, Ind.; trasylol and €-aminocaproic acid were obtained from Sigma Chemical Co., St.Louis, Mo. The other inhibitors shown in Table 3 were kindlysupplied by Dr. Walter Troll, State University of New York,through the generosity of the United States-Japan CooperativeCancer Research Program.

Serum. Acid-treatedserumwas prepared as describedpreviously (10). Plasmmnogen-depletedserum was prepared by thepassage of fetal calf serum through a lysine-Sepharose column(10) and was verified to be zymogen free using ‘25I-Iabeledfibnin plates with urokinase as activator. Human plasminogenwas prepared by the method of Deutsch and Mertz (4).

RESULTS

The kinetics of hydrolysis of complete and trypsin-pretreatedmatrices by human fibrosarcoma cells (HT1080) are shown inChart 1. Considerable hydrolysis of the complete matrix occurred when the fibrosarcoma cells were plated in acid-treatedfetal calf serum. The rate of matrix hydrolysis was less whenthe HT1080 cells were seeded in the presence of fetal calfserum. Since acid treatment destroys protease inhibitors present in serum (17), the results suggest that these inhibitors slowthe rate of matrix degradation by the fibrosarcoma cells. Therate of degradation of trypsmn-pretreated (i.e. , glycoprotemndepleted) matrices was greater than that of the complete matrixand was once again stimulated by the use of acid-treatedserum. Reutilization of the [3H]pnohinelabel by the tumor cellsdid not appear to be significant, since the total amount of

2 4 6 8 0 2 4 6 8 0DAYS AFTER PLATING

Chart i . Degradation of complete or trypsin-pretreated extracellular matricesby HTi 080 cells. HTi 080 cells (10@/16-mm well) were plated onto complete (A)or trypsin-pretreated (B) extracellular matrices which had previously been daborated by rat smooth muscle cells in the presence of (3Hjproline. Medium waschanged every 2 days, and the progress of matrix degradation was followed bythe appearance of radioactivity in the culture medium in the presence of 10%fetal calf serum (•)or 10% acid-treated fetal calf serum (0). Results given arethe mean values for duplicate dishes; similar results were obtained in severalseparate experiments.

radioactivity detected in the supernatant medium correspondedapproximately to the amount missing from the residual matrix(see below).

The residual matrices at the end of the experiment shown inChart 1 were analyzed by sequential enzyme digestion todetermine which proteins had been digested from the complexsubstrates. When HT1080 cells were grown on complete matrices in the presence of fetal calf serum, mainly glycoproteinswere digested (Table 1). However, when the cells were incubated under identical conditions on trypsin-pretreated matrices, considerable elastin and collagen hydrolysis occurred.Elastin and collagen were also digested from the completenative matrix when the cells were incubated in the presence ofacid-treated fetal calf serum. Almost complete removal of elastin and collagen was achieved if the HT1080 cells were incubated on a glycoprotein-depleted matrix in the presence ofacid-treated fetal calf serum. Since these matrices containedapproximately 100 @zgof elastin and 30 @tgof collagen per 35-mm dish (2), the tumor cells solubihizedapproximately 80 @gofelastin and 10 j.tg of collagen during the 10-day incubationperiod.

The kinetics of hydrolysis of complete and trypsin-pretreatedmatrices by other human tumor cells and human fibroblastswere also studied (Chart 2). Whereas all 3 lines of human tumorcells digested some of the complete matrix, no release ofradioactivity was seen when human fibroblasts were grown onsuch matrices (Chart 2A). In contrast, none of the cells causedextensive hydrolysis of trypsin-pretneated matrices (Chart 2B).We have not attempted to normalize these results for cellnumber because of difficulties in interpretation of such longterm experiments caused by (a) substrate depletion, (b) cellturnover, (c) the importance of cell-matrix contact (see below),and (d) differences in cell size and protein content. However,there were no significant differences between the attachmentof fibroblasts ontumor cells to either matrix type, as determinedby cell counts performed 24 hr after plating (not shown).

The residual matrices at the end of the experiment shown inChart 2 were analyzed by sequential enzyme hydrolysis (Table2). Human fibroblasts released little radioactivity from completematrices but released 9% of the collagen from trypsin-pre

SEPTEMBER 1980 3223

Research. on February 23, 2021. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

Digestion of complete and trypsin-pretreated extracellular matrices by human fibrosarcoma cellsHT1080 cells were grown on the complete matrix labeled with [3Hjproline in the indicated serum for 10

days. The fibrosarcoma cells were removed by NH4OHhydrolysis, and the residual matrix was analyzed bysequential enzyme digestion with trypsin. elastase. and collagenase. The amount of each component whichhad been removed from the matrices by the tumor cells was then calculated by comparison to duplicatematrices incubated for 10 days without cells. Under the same conditions, HT1080 cells were plated ontrypsin-pretreated matrices. Further treatment of these matrices with trypsin resulted in the release of lessthan 3% of the original total radioactivity. Results are given as the mean values for duplicate dishes andwere similar to those obtained in another separateexperiment.Complete

matrix Trypsin-pretreatedmatrix%

of gly- % of % of col- % of % of colSerum supple- coprotein elastin lagen di- % of glycoprotein elastin lagen di

ment digested digested gested digested digestedgestedFetal

calf serum 25 0 2 None present 2633Acid-treated

fetal 51 20 17 None present 80 73calf serum

AB500...o

0―

@@..-:;--;:

collagenolytic activity of the human fibrosarcoma cells therefore required active protein synthesis for the maximal hydrolysis rate to be achieved.

The effects of protease inhibitors on the digestion of complete and trypsin-pretreated matrices by living tumor cells areshown in Table 3. Inhibitors of plasmin and other serine proteases including soybean trypsin inhibitor, trasylol, €-aminocaproic acid, and leupeptin all inhibited the digestion of thecomplete matrix but had little effect on the degradation of theglycoprotein-depleted matrix. The result suggests that residualtrypsin on trypsin-pretreated matrices did not function as anactivator of latent tumor enzymes since the inhibitors wouldhave prevented further trypsin activity. The digestion of theglycoprotein-depleted matrix was increased by Colcemid, suggesting that Colcemid stimulated protease activity as has beenfound for macrophages (7). The only compound tested whichsignificantly inhibited elastin and collagen degradation wasdimethyl sulfoxide.

The next series of experiments was designed to determineboth the localization of the tumor proteases and the part playedby plasminogen activator in matrix digestion. Complete andglycoprotein-depleted matrices were therefore prepared on 25-mm plastic discs (Lux Scientific), and the HT1080 cells werecultured in different sera either in direct contact with thematrices or with the discs floating (matrix side down) in 35-mmdishes containing HT1080 cells. Removal of plasminogen fromacid-treated fetal calf serum decreased the rate of hydrolysisof the complete matrix by HT1080 cells grown in direct contactwith the proteins (Chart 4A). The rate of digestion was, however, stimulated when human plasminogen was readded to thezymogen-depleted serum. The presence of plasminogen therefore increased the hydrolysis rate as was found previously forstimulated mouse peritoneal macrophages (12).

Plasminogen was absolutely required for digestion if thematrix was not in direct contact with the tumor cells (Chart 4B).No digestion of the floating matrix occurred when the cellswere cultured in plasminogen-depleted serum. Digestion of thefloating matrix was, however, seen with acid-treated fetal calfserum or with plasminogen-reconstituted serum. Since glycoprotein components of the matrix are plasmin sensitive,3 the

3Z. Werb,M. Banda,andP. A. Jones.Degradationof connectivetissuematrices containing elastin, glycoproteins and collagen by macrophages inculture. I. Influence of matrix composition on rates of degradation by purifiedproteinases, manuscript in preparation.

P. A. Jones and V. A. DeC!erck

Table 1

0.U

Q0LU

LU_1LU

>-

2 4 6 8 0 2 4 6 8DAYS AFTER PLATING

Chart 2. Degradation of complete or trypsin-pretreated extracellular matricesby human fibroblasts and tumor cells. Human fibroblasts (•)or the human tumorlines TE85 (A). RD(0), or LAN-i (0) were plated (1O@/16-mm well) onto complete(A) or trypsin-pretreated (B) extracellular matrices labeled with [3HJprolinein thepresence of 10% acid-treated fetal calf serum. Medium was changed every 2days, and the release of radioactivity into the supernatant was determined at theindicated times after seeding. The results given are the meanvalues for duplicatedishes.

treated matrices. The other human tumor cells tested allshowed hydrolysis of the glycoprotein components of the complete matrices, but little or no elastolytic or collagenolyticactivity was seen with these cells, in marked contrast to thesituation with the human fibrosarcoma cells (HT1080). Furtherexperiments were therefore conducted with this line to investigate the nature of the tumor cell-mediated hydrolysis.

Chart 3 shows the effects of various metabolic inhibitors onthe digestion of complete or trypsin-pretreated matrices by theHT1080 cells. 1-f3-D-Arabinofuranosylcytosine, which inhibited[3H]thymidine incorporation by 98% at a concentration of 5,@M (not shown), had little effect on the rate of digestion of

either matrix type by the HT1080 cells, showing that activeDNA synthesis was not required for hydrolysis. Actinomycin D,which inhibited [3H]uridine incorporation by 80% (not shown),stimulated the rate of hydrolysis of both complete and trypsinpretreated matrices. The effect was seen despite the considerable cytotoxicity of the drug. Cycloheximide, which inhibited14C-Iabeled amino acid incorporation by 79% (not shown),reduced the extent of digestion of the complete matrix byHT1080 cells by 50% and was even more effective in inhibitingthe digestion of trypsin-pretreated matrices. The elastolytic and

3224 CANCERRESEARCHVOL. 40

Research. on February 23, 2021. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

duplicate dishes.Complete

matrixTrypsin-pretreatedmatrix%of%of%of%of%ofglyco

elastincelia elastincoliaproteindi gen di % of glycoproteindi gen di

CelltypedigestedgestedgesteddigestedgestedgestedHuman

fibroblasts300Nonepresent09TE853206Nonepresent06RD6506Nonepresent06LAN-i1435None

present00

Effect of protease inhibitors, Colcemid, and dimethyl sulfoxide on thedigestionofthe extracellular matrix by HT1O8OcellsHT1

080 cells were plated onto complete matrices in medium containing10%fetalcalf serum (10@cells/ 16-mm well). Six days after seeding, the mediumwasreplaced

with medium containing 10% acid-treated fetal calf serum andtheindicatedInhibitors. The total radioactivity released after a further 48 hr incuba

tieri was then determined and expressed as a percentage of theradioactivityreleasedby HT1080 cells growin9 without added inhibitors. Under thesameconditions,

HT1080 cells were plated on trypsin-pretreated matrices. Allresultsarethe mean values for duplicate dishes in 2 separate experiments.Digestion

Digestionofcom- of trypsin

plete ma-pretreatedtrix(% of matrix(%inhibitor

Concentration control) ofcontrol)Soybean

trypsin inhibitor 100 @g/ml 44106Trasylol2 units/mI 40107a-Amlnocaprolc

acid 500 @g/ml 78126Leupeptin10@ig/ml 5894Antlpain

1 ig/ml 92100Elastatlnol10 @g/ml 9496PepstatinlOpg/ml 9398Phosphoramidan

1 @g/ml 91107Ammonlumacetate 4 mM 108134Colcemid

1 @g/ml 112185Dimethylsulfoxide 10 mg/mI 90 35

Matrix Hydro!ysis by Tumor Ce!!s

Table 2Digestion of complete and trypsin-pretreated extracellular matrices by human fibroblasts and tumor cells

The indicated cell types (see ‘‘Materialsand Methods'@ for description) were plated on complete ortrypsin-pretreated matrices in medium containing 10% acid-treated fetal calf serum, and the residualmatrices were analyzed (after 10 days of incubation) as outlined in Table 1. Results are the mean values for

g

0 100

I

0.U

I,)0

0

4LU-J

24 48 24 48

HOURSAFTERPLA11NG

Chart 3. Eftect of metabolic inhibitors on the digestion of complete or trypsinpretreated matrices by HT1080 cells. HT1080 cells were plated onto complete(A) or trypsin-pretreated (B) matrices in medium containing 1 0% fetal calf serum

at 10@ceils/16.rnm well. Six days after seeding, the medium was replaced withmedium containing 10% acid-treated fetal calf serum and actinomycin D (0.1 @g/ml) (t@),1-@8-o.arablnofuranosyIcytosine(5 tiM) f.D),cycloheximide (1 @g/ml)(0),or noinhibiter(•).Thereleaseof radioactivityfromthelabeledmatriceswasthen determined at the indicated times. Results given are the mean values forduplicate treatments; similar results were obtained in a separate experiment.

2 4 6 2 4 6DAYS AFTER PLATING

Chart 4. Effect of plasminogen on the digestion of complete extracellularmatrices in contact or separated from HT1080 cells. HT1080 cells were grownin contact with the matrix (A) or with the matrix floating above the cells (B) inmedium containin9 10% acid-treated fetal calf serum (•),plasminogen-depletedacid-treated serum (0), or plasminogen-reconstituted acid-treated serum (a).Medium was changed every 2 days, and the release of radioactivity from thematrix was then determined at the indicated times. Results given are the meanvalues for duplicate samples; similar results were obtained in a separate experiment.

Table 3

and secreted plasminogen activator, previous experience (11)has shown that some of the HT1080 activator is secreted orshed into the medium.

The localization of the HT1080 elastolytic and collagenolyticactivities was investigated in the experiments shown in Chart5. The presence or absence of plasminogen had no effect onthe hydrolysis rate when the HT1080 cells were cultured indirect contact with a trypsin-pretreated matrix (Chart 5A). Thisresult was in contrast to the situation with the complete matrix(Chart 4A) and demonstrated that plasmin generation had nodirect effect on elastin and collagen breakdown. Chart SBshows that no elastin and collagen hydrolysis occurred whenthe trypsin-pretreated matrix was not in direct contact with thecells. The enzyme activities were therefore probably localizedto the tumor cell surface or were rapidly inactivated aftersecretion. Several other experiments (not shown) failed todemonstrate significant elastolytic or collagenolytic activities inthe conditioned medium of HT1080 cells even after a 10-foldconcentration.

DISCUSSION

Most previous studies investigating tumor-associated proteases have utilized substrates of purified connective tissueproteins (5, 14, 16, 20). While such studies are essential to

results suggest that the plasmin generated by the HT1080tumor cell plasminogen activator (11, 15) may serve to removethese componentsfrom the matrix. Althoughthe present experiments do not allow us to distinguish between cell-bound

SEPTEMBER1980 3225

Research. on February 23, 2021. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

P. A. Jones and Y. A. DeC!erck

radation of elastin and collagen. The protective effects of thematrix glycoproteins, which are plasmin sensitive, have alsobeen observed during matrix dissolution by stimulated macrophages.3 The increased susceptibility of trypsin-pretreatedmatrices to hydrolysis was probably not due to residual trypsinacting as a zymogen activator, since the elastolytic and collagenolytic activities were unaffected by several known trypsininhibitors.

The localization of the tumor degradative enzymes was,however, markedly different from that of macrophages. Wewere unable to detect significant elastolytic or collagenolyticactivity in the tumor cell supernatant medium, whereas theseenzymes are shed or secreted by macrophages under similarexperimental conditions.3 Other investigators have also failedto detect elastase activity in the supernatant medium of transformed cells (24), but tumor collagenase activity has beendescribed as being a secreted enzyme (14, 16). The elastolyticand collagenolytic activities of the HT1080 cell appeared to belocalized to the cell membrane, although it is possible that theywere very labile or rapidly neutralized by inhibitors on releasefrom the plasma membrane.

The elastolytic and collagenolytic activities of the humanfibrosarcoma cells required active protein synthesis, suggesting that the degradative enzyme(s) were rapidly turning over.The stimulation of degradation in the presence of actinomycinD might be a phenomenon similar to that reported for plasmin

ogen activator secretion by other tumor cells (27). A furtherdescription of these tumor proteases was hampered by theirresistance to a wide spectrum of protease inhibitors, suggesting that the enzymes were neither serine proteases nor lysosomal enzymes. However, since the tumor cells were grown indirect contact with the substrates, we cannot be certain thatthe exogenous inhibitors were able to interact with the enzymesbefore matrix digestion occurred. We are also unable to explainthe inhibition of proteolytic activity caused by dimethyl sulfoxide, although this might be due to an effect of the solvent onthe plasma membrane.

The exact description of the tumor-produced proteases willtherefore require their isolation from the HT1080 cells. Theydo, however, show specificity with regard to their substrates,since the elastolytic and collagenolytic activities were largelyineffective in digesting the glycoprotein components of thematrix, and the accompanying paper demonstrates that theyare not active in the digestion of underhydroxylated elastin (3).

ACKNOWLEDGMENTS

The authors acknowledge the excellent technical assistance of FelicidadGonzales.

REFERENCES

1. Anderson, J. C. Glycoproteins of the connective tissue matrix. Int. Rev.Connect. Tissue Rae., 7: 251—322,1976.

2. DeClerck, Y. A., and Jones, P. A. The effect of ascorbic acid on the natureand production of collagen and elastin by rat smooth muscle cells. Biochem.J., 186: 217-225, 1980.

3. DeClerck, Y. A., and Jones, P. A. The effect of ascorbic acid on theresistance of the extracellular matrix to hydrolysis by tumor cells. CancerRes.,40: 3228-3231 ,1980.

4. Deutsch, D. G.. and Mertz, E. T. Plasminogen: purification from humanplasma by affinity chromatography. Science (Wash. D. C.). 170: 1095-1096, 1970.

5. Dresden, M. H., Heilman, 5. A., and Schmidt, J. D. Collagenolytic enzymesin human neoplasms. Cancer Res., 32: 993-996, 1972.

0

0LULU-J

>-I->

I

DAYS AFTER PLATING

Chart 5. Effect of plasminegen on the rate of digestion of trypsin-pretreatedmatrices in contact or separated from HT1 080 cells. HT1 080 cells were grownin contact with the matrix (A) or with the matrix floating above the cells (B) in thepresence of 10% acid-treated fetal calf serum (I), plasminogen-depieted acidtreated fetal calf serum (0), or plasminogen-reconstituted acid-treated serum(Lx). Medium was changed every 2 days, and the release of radioactivity from thematrix was determined at the indicated times. Results given are the mean valuesfor duplicate samples; similar results were obtained in a separate experiment.

our overall understanding of the mechanisms of tumor-inducedhydrolysis, it is also important that complex substrates, whichpossess some of the architectural characteristics of connectivetissue, be used. The experiments reported in this paper represent a step in this direction and are the first to describe thedigestion by human tumor cells of the extracellular matrixproteins elaborated by cultured normal cells. The system whichwe have developed (i 2, 13) has the advantages that it is easilymanipulated, yields quantitative data, and utilizes a substratecontaining a radioactive label introduced biosynthetically ratherthan chemically.

The extracellular matrix elaborated by the R22 strain of ratsmooth muscle cells contains glycoproteins, elastin, and collagen (i 3) and was digested completely by only one of the 4tumor lines tested. The low collagenolytic activity of some ofthe tumor lines may have been due to the fact that the matrixcollagen was highly cross-linked in contrast to the extractedcollagen substrates used in other investigations (5, i 4, 16).The finding that all of the lines removed glycoproteins from thematrix (possibly through the mediation of plasmin) may, however, have significance in tumor invasion. The connective tissueglycoproteins have often been suggested to play the part ofmolecular ‘‘cements'‘(1) so that their digestion may lead tosufficient loss of matrix structural integrity to allow infiltrationby tumor cells. Alternatively, our investigations on the digestionof the extracellular matrix by stimulated macrophages,3 as wellas the present studies, have demonstrated that glycoproteinremoval markedly increases the susceptibility of elastin andcollagen to hydrolysis. Glycoprotein removal from the matrixby tumor cells may therefore rapidly accelerate the degradationof elastin and collagen by tumor-associated inflammatory cells.

The HT1080 line, which was originally derived from a highlymetastatic fibrosarcoma (23), was capable of the completedigestion of all of the matrix components. It therefore demonstrated elastolytic and collagenolytic activity in addition to theability to hydrolyze glycoproteins from the matrix. Our experiments with medium containing different plasminogen levelsstrongly suggest a major role for the HT1080 plasminogenactivator (1 i , 15) in glycoprotein removal. The removal of theglycoproteins was essential for the efficient subsequent deg

2 4 6 2 4 6

3226 CANCERRESEARCHVOL. 40

Research. on February 23, 2021. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

6. Fldler, I. J., Gersten, D. M., and Hart, I. A. The biology of cancer invasionandmetastasis.Adv.CancerRes.,28:149-250,1978.

7. Gordon, S., and Werb, Z. Secretion of macrophage neutral proteinase isenhanced by coichicine. Proc. NatI. Acad. Sci. U. S. A., 73: 872-876, 1976.

8. Harris, E. D., and Krane, S. M. Collagenases. N. EngI. J. Med., 291: 557-563, 605—609,652-661, 1974.

9. Hartley, B. S.. and Shotton, D. M. Pancreatic elastase. In: P. D. Boyer (ed).The Enzymes. vol. 3, pp. 323—373.New York: Academic Press, Inc., 1971.

10. Jones, P. A., Benedict, W. F., Strickland, S., and Reich, E. Fibrin overlaymethods for the detection of single transformed cells and colonies oftransformed cells. Cell, 5: 323—329.197S.

11. Jones, P. A., Laug, W. E., and Benedict, W. F. Clonal variation of fibrinolyticactivity in a human fibrosarcoma cell line and evidence for the induction ofplasminogen activator secretion during tumor formation. Cell, 6: 245—252,1975.

12. Jones, P. A., and Scott-Burden, T. Activated macrophages digest the extracellular matrix proteins produced by cultured cells. Biochem. Biophys. Res.Commun.,86: 71-77, 1979.

13. Jones, P. A., Scott-Burden, T., and Gevers, W. Glycoprotein, elastin andcollagen secretion by rat smooth muscle cells. Proc. NatI. Acad. Sci. U. S.A., 76: 353-357, 1979.

14. Kuettner. K. E., Soble, L., Crexen, R. L., Marczynslea, B., Hiti, J., andHarper, E. Tumor cell collagenase and its inhibition by a cartilage-derivedprotease Inhibitor. Science (Wash. D. C.), 196: 653—654,1977.

15. Laug, W. E., Jones, P. A., and Benedict, W. F. Relationship betweenfibrinolysis of cultured cells and malignancy. J. NatI. Cancer Inst., 54: 173—179, 1975.

16. Liofta, L A., Abe, S., Robey, P. G., and Martin, G. R. Preferential digestionof basement membrane collagen by an enzyme derived from a metastaticmurine tumor. Proc. NatI. Acad. Sci. U. S. A., 76: 2268-2272, 1979.

17. Loskutoff, D. J., and Edgington, T. S. Synthesis of a fibrinolytic activatorand inhibitor by endothelial cells. Proc. NatI. Acad. Sci. U. S. A., 74: 3903-3907, 1977.

18. McAllister, A. M., Gardner, M. B., Greene, A. E., Brodt, C., Nichols, W. W.,and Landing, B. H. Cultivation in vitro of cells derived from a humanosteosarcoma. Cancer (Phila.), 27: 397—402,1971.

19. McAllister, R. M., Melnyk, J., Finkelstein, J. Z., Adams, E. L., and Gardner,M. B. Cultivation in vitro of cells derived from a human rhabdomyosarcoma.Cancer (Phila.), 24: 520—526,1969.

20. McDonald, J. A., Baum, B. J., Rosenberg, D. M., Keiman, J. A., Brin, S. C.,and Crystal, R. G. Destruction of a major extracellular adhesive glycoprotein(fibronectin) of human fibroblasts by neutral proteases from polymorphonuclear leukocyte granules. Lab. Invest., 40: 350—357,1979.

21. Poole, A. R. Tumor lysosomal enzymes and invasive growth. In: J. T. Dingie(ed), Lysosomes in Biology and.Pathology, vol. 3, pp. 303-337. Amsterdam: North Holland Publishing Co., 1973.

22. Poole, A. R., Tiltman, K. J., Recklies, A. D., and Stoker, T. A. M. Differencesin secretion of the proteinase cathepsin B at the edges of human breastcarcinomas and fibroadenomas. Nature (Lend.), 273: 545—547,1978.

23. Rasheed, S., Nelson-Rees,W. A., Toth, E. M., Arnstein, P., and Gardner, M.B. Characterization of a newly derived human sarcoma cell line (HT1080).Cancer (Phila.), 33: 1027-1033, 1974.

24. Rifkin, D. B., and Crowe, R. M. A sensitive assay for elastase employingradioactive elastin coupled to sepharose. Anal. Biochem., 79: 268—275,1977.

25. Seeger, R. C., Rayner, S. A., Banerjee, A., Chung, H., Laug, W. E., Neustein,H. B., and Benedict, W. F. Morphology, growth, chromosomal pattern, andfibrinolytic activity of two new human neuroblastoma cell lines. Cancer Res.,37: 1364—1371,1977.

26. Unkeless. J. C., Tobia, A., Ossowski, L., Quigley, J. P., Rifkin, D. B.. andReich, E. An enzymatic function associated with transformation of fibroblastsby oncogenic viruses. J. Exp. Med., 137: 85—111. 1973.

27. Wigler, M., Ford, J. P., and Weinstein, I. B. Glucecorticoid inhibition of thefibrinolytic activity of tumor cells. In: E. Reich, D. B. Rifkin, and E. Shaw(eds.), Proteases and Biological Control, pp. 849-866. Cold Spring Harbor,N. Y.: Cold Spring Harbor Laboratory, 197g.

3227

Matrix Hydro!ysis by Tumor Ce!!s

SEPTEMBER1980

Research. on February 23, 2021. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from

1980;40:3222-3227. Cancer Res   Peter A. Jones and Yves A. DeClerck  Elastin, and Collagen by Metastatic Human Tumor CellsDestruction of Extracellular Matrices Containing Glycoproteins,

  Updated version

  http://cancerres.aacrjournals.org/content/40/9/3222

Access the most recent version of this article at:

   

   

   

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

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/40/9/3222To request permission to re-use all or part of this article, use this link

Research. on February 23, 2021. © 1980 American Association for Cancercancerres.aacrjournals.org Downloaded from