[CANCER RESEARCH 33, 2790 2801, November 1973]

Collagenolytic Activities of Squamous Cell Carcinoma of theSkinKen Hashimoto,1 Yuji Yamanishi,1 Edgar Maeyens, ' Mustafa K. Dabbous,2 and Tamotsu Kanzaki '

Memphis Veterans Administration Hospital and Division of Dermatology, Department of Medicine, The University of Tennessee College of MedicineMemphis 38104 [K. H., Y. Y., E. M., T. K.}, and Department of Biochemistry, The University of Tennessee, College of Basic Medical Sciences,Memphis, Tennessee 38103 (M. K. D.]

SUMMARY

Electron microscopic and physicochemical studies oncollagenolytic activities of squamous cell carcinomas ofthe skin were performed. Collagen and basal lamina degeneration was evident in the stroma immediately surrounding the squamous cell carcinomas. With the exception of fine filament aggregations with periodical cross-bands at 1000-A intervals, amorphous debris, and intactelastic Tibers, the peri-squamous cell carcinoma stroma wasempty. A large number of villous projections from the advancing border of the squamous cell carcinomas perforatedthe basal lamina. An amorphous coat of the elastic fibersproduced hemidesmosomes with tumor cells. Electronmicroscopic examination of segment-long-spacing tropo-collagen prepared from squamous cell carcinoma homoge-nate-treated calf skin collagen revealed molecules cleavedat the (822region. Squamous cell carcinoma homogenatesreleased significantly higher radioactivity from proline-14C-labeled guinea pig collagen than did the normal skincontrol. Caseinolytic activity of squamous cell carcinomahomogenate was higher than that of the normal skin, butsoybean trypsin inhibitor inhibited collagenolysis only12.7%. Known collagenase inhibitors such as normal humanserum (10%) ethylenediaminetetraacetic acid (0.01 M), andcysteine (0.01 M), on the other hand, inhibited collagenolysis 75, 70, and 57.2%, respectively. Disc electrophoresisof the reaction mixture showed degradation bands of a andßchains. Specific viscosity of tropocollagen decreased by42% after 24 hr of incubation with squamous cell carcinoma homogenate. The melting temperature midpoint oftropocollagen dropped about 10°as a result of incubation

with squamous cell carcinoma homogenate. The crude enzyme activities, as measured by the radioactivity releasedfrom proline-l4C-labeled salt-extracted guinea pig collagen, increased linearly with respect to the time and theenzyme concentration. The optimal pH for the crude enzyme was determined to be pH 6.5. Elastase activity in thetumor homogenates was not increased above the normal

'Supported in part by Medical Investigator Award, DermatologyTraining Grant, and Designated Component Research Funds from theVeterans Administration.

2Supported in part by American Cancer Society (University of Tennessee) Grant IN 85F and USPHS, University of Tennessee InstitutionalGrant I534R10.

Received March 21, 1973: accepted July 26, 1973.

skin level. Numerous aggregates of fine filaments withperiodical cross-bands were observed in the stroma. Theseresults strongly suggest that squamous cell carcinomas ofthe skin contain specific collagenase that is active in vivo.

INTRODUCTION

In our previous publications reporting the collagenolytic activities in basal cell epithelioma of the skin (22,40), 7 squamous cell carcinomas were analyzed as 1 of thecontrol conditions. It was described briefly that thesetumors released radioactivity from proline-14C-labeledreconstituted collagen at a significantly higher rate thannormal skin (1). Caseinolytic (trypsin-like) activity, inhibitor studies, kinetic studies, and physical determination of collagen degradation by the crude enzyme were,however, not done. In the present investigation, electronmicroscopic and biochemical investigations were performed to establish that such activities are due to specificcollagenase(s).

MATERIALS AND METHODS

One large tumor (5x4 cm) on the forehead and relatively small tumors on the lower lip, left ear, and templewere used individually. Five small tumors from variouslocations were pooled together. All specimens were excisedsurgically under local anesthesia.

Tissue Preparation. Excised tumors were prepared individually for light and electron microscopic observations.A part of each tumor was fixed in 10% formalin, embeddedin paraffin, sectioned, and stained with hematoxylin andeosin. Another part was fixed for 3 hr in 5% glutaraldehydein 0.1 M cacodylate buffer, pH 7.4. Some specimens werestained in tissue blocks with 0.01% ruthenium red. Allspecimens were rinsed overnight in the same buffer andpostfixed with 1% osmic acid in the same buffer for 30min, dehydrated routinely, and embedded in Araldite.Thin sections, 400 to 600 A, were placed on Formvar-coated 200 mesh copper grids, stained with 15% uranylacetate in 50% methanol, and stained again with lead citrate (36). Stained sections were examined in a HitachiHU-12 electron microscope at an accelerating voltage of125 kV.

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Collagenase in Squamous Cell Carcinoma

Preparation of Segment-long-spacing Crystallites fromReaction Mixture. After incubation of the tumor homoge-nates with Fraction 2A or 2B of acid-soluble calf skin tro-pocollagen and the confirmation of the initial viscositydrop (see "Viscometric Method"), the reaction mixture

was acidified to pH 3.8 with acetic acid, or EDTA wasadded to a final concentration of 0.01 M, to inhibit theenzyme activity. The acidified solution was filtered andcentrifuged at 25,000 rpm, dialyzed against 0.05% aceticacid, and then lyophilized. A small amount of lyophilizedmaterial (1 to 2 mg) was dissolved in 0.05 M acetic acidand dialyzed against 0.05 M acetic acid for 24 hr. To thisdialyzed solution, 1% ATP was slowly added with constantstirring until the final concentration of 0.4% was reached.The solution showing an ideal turbidity was kept in thecold for 60 min and then dropped by pipet onto Formvar-coated 200 mesh copper grids. The specimens were airdried and then stained with 1% aqueous phosphotungsticacid (pH 3.7) for 5 min and stained again with 1% aqueousuranyl acetate for 3 hr. The stained specimens were observed with a Hitachi HU-12 electron microscope at anaccelerating voltage of 125 kV.

Preparation of Tumor and Control Skin Homogenates.Both pooled and individual tumors were minced and homogenized with a ground-glass homogenizer in 2 to 4 mlof 0.05 M Tris-HCl buffer (pH 7.6) containing 0.001 MCaCl2. Contaminating blood, necrotic areas, and attachedfatty tissues were removed as well as possible. Homogeni-zation was done in an ice bath kept at 0-4°.Normal skin

control specimens were obtained from surgically amputated lower legs. The cause of the amputation was trauma.The epidermis and superficial dermis were removed fromall the specimens. Quantitative determination of proteinof each homogenate was done by the method of Lowry et al.(29).

Preparation of Substrate. Uniformly labeled proline-I4C(Schwarz/Mann, Orangeburg, N. Y.) was injected in 2doses of 25 ¿iCieach i.p. into randomly bred, young albino guinea pigs weighing about 200 g each. The animalswere sacrificed 48 hr after the injection. The skin was removed and cleaned mechanically. Salt-soluble collagenwas extracted by the method of Gross (16). The final products were lyophilized and stored in a desiccator at -20°.

Quantitative determination of tumor protein present in theincubation mixture was done by the method of Lowry et al.(29).

Incubation Mixture and Isotope Release. For production of reconstituted collagen fibers, the method of Nagaiet al. (31) was followed. A highly viscous solution was thusprepared. Aliquots of 0.5 ml of this solution were pipettedinto plastic centrifuge tubes and allowed to gel in a 37°water bath for at least 12 hr. Prior to admixture with thetumor homogenate, the gelatinized substrate was disrupted with a steel needle to ensure a good contact withthe homogenate. An equal amount (0.5 ml) of the tumorhomogenate (enzyme solution) was added to each tube.All tubes were incubated at 37°for 18 hr with constant

agitation. Normal skin homogenate was similarly admixed with the substrate and incubated. After incubation,

tubes were centrifuged at 59,000 x g at room temperaturefor 30 min to sediment undissolved collagen. A 0.5-ml aliquot of the supernatant was added to 10 ml of Insta-Gel(Packard Instrument Co., Downers Grove, 111.),and theradioactivity was counted in a liquid scintillation spectrometer.

Caseinolytic Activity. Caseinolytic activity of the sametumor homogenates was measured by the methods ofKunitz (27) and Nagai et al. (31).

Kinetic Studies. Aliquots of 0.5 ml of either pooled orindividual tumor homogenates were added to the sameamount of substrate solution in the studies of pH optimum,enzyme concentration, and time dependency of collageno-lytic activities as measured by the radioactivity releasedfrom the substrate. For the study of pH dependency, acetate buffer was used for pH's 5 and 5.6; Tris-maleatebuffer for pH's 6.0 and 6.6; Tris-HCl buffer for pH's 7.0,

7.6 and 8.3; and glycylglycine buffer for pH 9.O.Inhibition Studies. To 0.5 ml of substrate solution, 0.1

ml of pooled tumor homogenate and the following collagen-ase inhibitors were added to make the final reaction mixture of 1.0 ml: (a) normal human whole serum at final dilutions of 10, 50, 250, 500, and 1000; (b) EDTA at a finalconcentration of 0.01 M; (c) cysteine, 0.01 M; and (d) soybean trypsin inhibitor, 100 /¿g(Sigma Chemical Co., St.Louis, Mo.). As an inhibitor of nonspecific protease, DFP3was used at the final concentration of 1, 2, and 3 mM in 1.0ml of 0.05 MTris-HCl buffer (pH 7.6), containing 1.0 mgof proline-14C-labeled guinea pig collagen, 1.45 mg of

tumor supernatant, and 0.001 M CaCl2. The reaction mixture was incubated at 37°for 18 hr.

Disc Electrophoresis. Polyacrylamide gel electrophoresiswas used. The analysis was carried out on 9% gel accordingto the method of Dabbous el al. (4). An aliquot of thepooled tumor homogenate was incubated with purifiedFraction 2A or 2B of acid-soluble tropocollagen preparedfrom calf skin by the method of Rubin et al. (38). The purity of these fractions was checked by amino acid analysisand has been published elsewhere (22). The actual procedures were the same as previously published (40).

Viscometric Method. Viscosity was measured in Ost-wald viscometers as previously described (40). A controlsample was similarly prepared, as were the expérimentais,except that the tumor homogenate was placed in a boilingwater bath for 5 min, cooled to 27°,then incubated withthe same tropocollagen substrate at 27 ±0.1°.

Optical Rotation and Melting Curves. A Zeiss polarim-eter with a hydrogen light source (365 nm) was used tomeasure optical rotations. The procedure was the same aspreviously described (40).

Elastase Assay. Elastase activity of the tumor homogenate was measured by the method of Janoff (23) usingbenzyloxycarbonyl-L-alanine p-nitrophenyl ester (Sigma)as substrate. Controls were run with normal skin homogenates as control tissue and with swine pancreas elastase(Worthington Biochemicals Corp., Freehold, N. J.) ascontrol enzyme.

'The abbreviation used is: DFP, diisopropyl fluorophosphate.

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Hashimoto, Yamanishi, Maeyens, Dabbous, Kanzaki

RESULTS

Histology. Pooled tumors showed histological featuresof squamous cell carcinoma Grade I to Grade IV inBroders' classification (3). The tumor excised from the

forehead was Grade III squamous cell carcinoma and hadinfiltrated the dermis to the level of the eccrine sweatglands (Fig. \A). Rarefaction of the stroma was noted surrounding many tumor islands (Fig. l, B and C). In all invasive tumors, Verhoeffs elastic stain revealed the presenceof elastic fibers between tumor cells (Fig. 1, A and B).

Fine Structure of the Tumors. All tumors used in thepresent study showed essentially the same features. Tumorcells were abnormally keratinized or nonkeratinized. Alarge number of vacuoles were present in the cytoplasmof most tumor cells (Figs. 2A and 3A). They were looselyconnected with each other by interdigitation of the peripheral villi on which desmosomes and gap junctions werepresent. The continuity of the basal lamina surroundingthe periphery of the tumor parenchyma was interruptedand the peripheral cytoplasm of tumor cells projectedthrough such breakages (Fig. 2A). The stromal collagen inthe immediate vicinity of such areas showed disarray ordisruption and often disappeared partially or completely.Between the tumor cells there were empty spaces whichwere often occupied by intact elastic fibers and/or aggregations of fine filaments (Fig. 3/4). These elastic fibers werethought to be entrapped between tumor cells as the tumorinfiltrated the dermis. When these elastic fibers made contact with the tumor cells, hemidesmosomes were producedalong the periphery of the tumor cells (Fig. 3Ä). Denseamorphous or fuzzy substances coating the surface of theelastic Tiber (which also invaginate into the fiber, becomedenser, or aggregate to form dense layers) played a basallamina role (Fig. 3B). Between this "basal lamina" pro

vided by the elastic fiber and the hemidesmosomes, therewas a subepithelial dense layer, rather closely apposed tothe tumor cells (Fig. 3B). This dense layer was equivalentto the subbasal cell dense layer of the epidermis (20). Thefilamentous components of the intercellular spaces weremore often than not organized and arranged in parallelfashion (Fig. 3C). They ran in the same direction as collagen fibrils, which were often admixed in small numbersand showed periodical, cross-banded, dense areas at intervals of approximately 1000 A (Fig. 4A). These bodies,particularly the cross-band region, were stained denselywith ruthenium red (Fig. 4/1). In addition, ruthenium red-

stainable pericollagen substance and fine filaments couldbe demonstrated in a large amount in the vicinity of thesebodies as well as near the collagen degeneration (Fig. 4,B and C).

Segment-long-spacing of Crude Enzyme-treated Tropo-collagen. A number of cleaved molecules of tropocollagenwere found. When matched with the full molecule, it wasseen that they measured 75% of the full molecular lengthfrom the A end, namely, TC75A(Fig. 5). However, the remaining 25% pieces from the B end, namely, TC25B

could not be found.Isotope Release. The amount of collagen substrate lysed

by enzyme activities was counted by the isotope releasedinto the supernatant of the reaction mixture. Thus, collagen gel lysis per mg protein of each tumor homogenateand that of the normal skin control was calculated (Table1). The pooled tumor homogenate lysed the substrate gel9.4% and the homogenate of the forehead tumor 4.4%,respectively, whereas the normal skin lysed only 0.8%(Table _1).

Caseinolytic Activity. As an index of nonspecific protease activity such as that of trypsin, caseinolytic activityof both pooled and forehead tumor was measured. Expressed as ßgtrypsin equivalence, the activities in bothspecimens were 3 to 4 times higher than the normal skincontrol (Table 1).

Inhibition Studies. Normal human sera at a dilution of1:10 inhibited collagenolysis of the tumor homogenates75%. Further dilutions down to 1:1000 decreased gradually the degree of inhibition (Table 2). Other known col-lagenase inhibitors such as EDTA and cysteine inhibitedcollagenolysis by the crude enzyme 70.4 and 57.2%, respectively (Table 2). Soybean trypsin inhibitor inhibitedcollagenolysis by tumor homogenate only 12.7%, whereascollagenolysis by 50 ^g of pure trypsin was inhibited upto 88.3% by the same amount of soybean trypsin inhibitor(Table 2), thus indicating that the inhibition system usedwas effective. Pure trypsin does not cleave the collagenmolecule but erodes the telopeptide region (6) and thusseems to release radioactivity (proline-14C) contained inthis region. A relatively low level of inhibition by trypsininhibitor in contrast to a high level of inhibition (70 ~75%) by collagenase inhibitors such as normal serum andEDTA may mean that contribution by nonspecific proteases resembling trypsin to the total collagenolysis of thetumor homogenates was not very significant. Similarly,serine-containing proteases, which are susceptible to DFP

Table 1Collagenolylic activities on salt-extracted, proline-"C-iabeled guinea pig skin collagen and caseinolytic activities

SpecimenTumorTumorNormal

skinLocationPooledForeheadLegTissue

protein(mg/0.2ml)1.82.94.7cpm"solubilized(aboveblank)55040048Collagengel17.012.71.5%

lysis/mgprotein9.44.40.8Caseinolyticactivity"11.18.53.0

" Total radioactivity per incubation mixture was 3240 cpm." Expressed as pig trypsin equvalence per mg crude enzyme protein.

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inhibition, did not seem to contribute very significantly tothe observed collagenolysis, since DFP at the final concentration of 1 mM inhibited collagenolysis only 3.4%. Thedegree of inhibition, however, increased up to 15.7% with3 mM and reached a plateau above the concentration of3 mM (Table 2).

Kinetic Studies. The collagenolytic activity increasedlinearly with time (Chart 1) and with an increase of the

Collagenase in Squamous Cell Carcinoma

crude enzyme concentration (Chart 2). Optimal pH was6.5 (Chart 3); below and above this range enzyme activitydropped sharply (Chart 3).

Disc Electrophoresis. Denatured control tropocollagensolutions gave the usual electrophoretic pattern; the a-bands (ai, a2) with the highest mobility, followed by theß-bands(ß12,/3n) with intermediate mobility and thenthe slow-moving -y-bands and higher-molecular-weight

Table 2Effects of coliagenase, trypsin, and protease inhibitors on squamous cell carcinoma collagenolysis of salt-

extracted, proline->4C-labeled guinea pig skin collagen

Squamous cell carcinoma with:SerumSerumSerumSerumSerumEDTACysteine

Soybean trypsininhibitorTrypsin

with:Soybean trypsin inhibitorConcentration1:101:501:2501:5001:10000.01

M0.01

M100

Mg50

Mg100 ngcpm

(aboveblank)"514

126218374404432148214428298

60%

inhibition75.056.025.019.213.570.457.2

12.788.3

Squamous cell carcinoma completewith:

1 mM DFP2 mM DKP3 mM DFP

4058»

392434683422

3.414.615.7

" Total radioactivity per incubation mixture was 3240 cpm.»Totalradioactivity per incubation mixture for this experiment was 8150 cpm.

0LÃœCO

Utet

o.o

200

150

100

50

M

UJ

co

UJcr

Q-O

500

400

= 300

200

N-z.ÃœJ

10005mg I.Omg 1.5mg 2.0mg

HOURChart 1. A linear increase of radioactivity released from proline-"C-

labeled, salt-soluble collagen is seen in relation to the length of the incubation. Supernatant of pooled specimens was used.

CRUDE ENZYME PROTEIN

Chart 2. A linear increase of radioactivity released from proline-14C-labeled, salt-soluble collagen is seen in relation to the increase of the crudeenzyme concentration. Supernatant of pooled specimens was used.

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Hashimoto, Yamanishi, Maeyens, Dabbous, Kanzaki

100

80

60

40v

> 20 -h-o

0

< 100

x

| 80

60

40

20 -

5.5 6.5 75 5.5 65 75 85

5.5 6.5 75 85

pHChart 3. Peaks of enzyme activity as measured by the radioactivity re

leased from proline-"C-labeled, salt-soluble collagen are seen at pH 7.0

in Specimen A and at pH 6.5 in Specimens B and C. The enzyme activitiesbelow and above the peaks are expressed as percentage of the peak values.

components closer to the top of the upper section of theseparating gel (Fig. 6A). Crude enzyme-treated tropocol-lagen similarly denatured after incubation showed a relatively smaller amount of a, ß,and 7 components thanthose of the control (Fig. 6B). Compared with the bandsseen in the control, a fast-moving set of bands with mobilityhigher than that of a2 appeared, representing «Acomponents. A broad-band region of low intensity, moving fasterthan the a Aband, was also seen and might represent a Bcomponents. In the /3-band region another set of bandscorresponding to ßcomponents appeared with mobilitieshigher than that of ßl2-An additional band (x) appearing near or above the original ßlt might represent cleavage products of y and/or high-molecular-weight components. Close to the bottom section of the separating gel,another broad band (Y) of high intensity appeared andmay represent further breakdown materials of low molecular weight, derived from preceding bands.

Viscosity Change. The supernatant of tumor homoge-nates decreased the viscosity of acid-soluble calf skin collagen during incubation at 27°.The specific viscosity of

dilute tropocollagen solutions decreased by 42% after 24hr of incubation. However, when incubation was continued,the loss in specific viscosity reached 72% of the originalvalue at 0 incubation time (also with respect to the control sample). Chart 4 shows the progress of loss of specificviscosity of tropocollagen as a function of incubation time.The loss of viscosity occurred without an apparent lag

period. The viscosity loss was attributed to the cleavageof the tropocollagen molecule into TC76Aand TC25B(Fig.

5). Spontaneous denaturation of the cleaved products,particularly the shorter TC25B pieces, and further degra

dation of cleaved and denatured products by nonspecificproteases were also considered to be contributory factors tothe observed viscosity loss, especially after a long incubation time, toward the end of the experiments.

Melting Curve.The supernatant solution of tumor homo-genate did not alter the optical rotation of acid-solublecalf skin tropocollagen to any significant extent after incubation at 27°for 24 hr. This implies that during this

treatment the native triple helical structure of tropocollagen is not denatured. However, the stability of the reaction products was decreased when the temperature wasincreased in a stepwise manner; namely, the negative opticalrotation of the reaction products decreased (Chart 5).This decrease was first gradual, at temperatures up to29°,and then sharp as the temperature was raised above29°.The melting temperature midpoint in the denaturationcurve (Tm) of the reaction products was almost 10°lower

than that of the native tropocollagen macromolecule.Chart 5 shows the course of the denaturation curves: 7,for control collagen solutions; and //, for the products ofincubation of tropocollagen with the tumor homogenates.Curve II showed a plateau region at a point correspondingto about 72% denaturation. This biphasic nature of themelting curve has been previously reported by us and byother investigators for the products of collagen digestion bytissue and tumor collagenases (5, 10, 25, 40) and was interpreted to represent the presence of a fraction of unaltered tropocollagen macromolecules admixed with thecleavage products. We measured an average change ofmultiple Tm's, since the digested collagen substrate con

tained at least 3 components, namely, unaltered collagen,TC75A, and TC25 Bwith their own Tm's. Such a change

took the form of a biphasic curve and hence resembledthose previously found with other tissue collagenases.

Elastase Activity. The change of absorbance due to hy-

o.a>

o(O

o

0 4 8 12 16

TIME (hr )Chart 4. The viscosity of normal acid-soluble calf skin collagen de

creased by 25% after 16 hr incubation with the supernatant of pooled specimens. r),p, specific viscosity.

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Collagenase in Squamous Cell Carcinoma

Ecin

-1300-'

-l 100-

-900-

-700 -

-50032 36 40

TEMPERATURE

Chart 5. Melting curve of normal acid-soluble calf skin collagen control (/) is compared with the melting curve of tumor-treated calf skin collagen (//). Denaturation of collagen molecules was measured by the decrease of negative optical rotation. It is seen that the melting temperaturemidpoint of Curve I (40°)is dropped almost 10°in Curve ¡I(30°)as the

result of incubation with the pooled tumor homogenates.

drolysis of the benzyloxycarbonyl-L-alanine p-nitrophenylester substrate could not be detected in the tumor homoge-nate, whereas benzyloxycarbonyl-L-alanine p-nitrophenylester substrate treated with 6.2 ¿¿g(0.4 unit) of swinepancreatic elastase showed an increase of absorbance(0.061). A similar range in the increase of density usingthe same test system was reported by Janoff (23).

DISCUSSION

Whatever the real cause of the squamous anaplasia ofthe skin and mucous membrane might be, it is reasonable to hypothesize that collagenolytic enzyme(s) producedby squamous cell carcinomas facilitates the breakage ofthe basal lamina, which contains collagen (26), and facilitates the invasion of the upper dermis or submucosa,which are mainly composed of collagen and ground substances. In this study, such enzyme activities were indeeddemonstrated in the tumor by various means.

In contrast to most of the animal collagenases (1, 2, 7,8, 10, 12-18, 24, 25, 37, 39), squamous cell carcinoma col-lagenase is active in vivo. Thus, the enzyme activities couldbe detected directly in the tumor homogenate. In this respect, squamous cell carcinoma collagenase is similar tothose of basal cell epithelioma of the skin (21, 40) andmalignant melanoma (41). The specificity of this collagenase could be shown by the electron microscopic demonstration of a molecular cleavage at the ß22locus; thismode of scission is specific for many animal collagenases,including those of basal cell epithelioma (21) and melanoma (41). Although TCjs could not be found in the re

action mixture, the presence of TC,\ indicates that themolecular scission took place at this region. Degradationbands that seem to correspond to smaller B end cleavageproducts such as a", however, could be shown in discelectrophoresis of the reaction mixture. Some of the TC26B

could have been further degraded by nonspecific proteases.Contamination by nonspecific proteases is always a prob

lem in crude enzyme preparations such as ours. Contribution of trypsin and serine-containing proteases versus collagenase to the observed collagenolysis can, however, beassessed indirectly by inhibition studies; soybean trypsininhibitor and serine protease inhibitor (DFP) inhibited13 ~ 16% of the total collagenolytic activity of the tumorhomogenates, whereas known collagenase inhibitors suchas human serum, EDTA, and cysteine inhibited the activity significantly more (57 to 75%). Although there is a possibility that general proteases other than trypsin and serine proteases are involved, these proteases usually erodethe ends of the molecule but do not cleave it (6). A cleavageacross the molecule such as shown in Fig. 5 requires specific collagenase (18). Since the molecules once cleavedand denatured become susceptible to nonspecific proteases,it is conceivable that after the primary scission of collagenmolecules by the specific collagenase a secondary systemof nonspecific proteases completes the stromal collagendegradation in vivo. Specific viscosity drop and loweringof denaturation temperature midpoint as the result ofincubation with tumor homogenate support the presenceof active collagenase in squamous cell carcinoma.

Direct observation of the tumor by the electron microscope revealed a number of features suggestive of collagenolysis. In the immediate vicinity of the advancingborder of the tumor, loosening and fragmentation of collagen islands were frequently seen. The basal laminas surrounding such borders were more often disrupted or completely absent than normal. The presence of intact elasticfibers and the absence of collagen fibrils between tumorcells suggested that the stromal components were entrapped between invading tumor cells. Subsequently, collagen was dissolved but elastic fibers remained intact. Thelevel of elastase in the tumor homogenate was not increasedabove the normal skin level. Although the proportion varies according to location, the number of elastic fibers inthe skin never exceeds one-tenth of the collagen strands(islands), each strand consisting of numerous fibrils. Thepreservation of elastic fibers and disappearance of collagen fibrils from the spaces between tumor cells, therefore, indicates a disposal of numerous collagen fibrils.

The fine filament aggregations with periodical cross-bands at intervals of about 1000 A have been found in avariety of nerve organs (21, 32, 34) and its tumors (30).They are often referred to as Luse's (30) body or zebra

body. They have recently been reported in connective tissue disorders such as systemic sclerosis (28) and varioustypes of amyloidoses of the skin (K. Hashimoto and H.Ohyama, manuscript in preparation). It is interesting thatskin tumors that produce collagenases also contain thesebodies; thus, basal cell epithelioma (19), melanoma (K.

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Hashimoto, Yamanishi, Maeyens, Dabbous, Kanzaki

Hashimoto and H. Ohyama, manuscript in preparation),and squamous cell carcinoma all contain them. On theother hand, other data indicate that acid mucopolysac-charides are increased in those tumors which contain theseaggregates (33) and that they are related to "fibrous-long-spacing" collagen whose formation may also be related to

the presence of mucopolysaccharides (35). Randall et al.(35) demonstrated in phosphate-extracted long-spacingcollagen variations of pattern and periodicity and presumedthat the proportion of mucopolysaccharides admixed inthe extracts were important in determining which patternwill be produced. Ruthenium red-stainable pericollagensubstance and filaments are present in the normal dermalcollagen but in small amount. An increased amount ofthese substances and heavy staining of the cross-bandedfilament aggregates suggest that ground substance mucopolysaccharides are increased in the neighborhood of tumorcells, perhaps due to collagen degeneration, and that theformation of the cross-banded filament aggregates is indeed influenced by such mucopolysaccharides.

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4. Dabbous, M. K., Seif, M., and Brinkley, E. The Action of Tetranitro-methane on Acid-Soluble Tropocollagen. Biochem. Biophys. Res.Commun., 48: 1586-1591, 1972.

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15. Grillo, H. C., Lapiere, C. M., Dresden, M. H., and Gross, J. Collagenolytic Activity in Regenerating Forelimbs of the Adult Newt(Triturus viridescens). Develop. Biol., ¡7:571-583, 1968.

16. Gross, M. Studies on the Formation of Collagen. I. Properties andFractionation of Neutral Salts Extracts of Normal Guinea Pig Connective Tissue. J. Exptl. Med., 107: 247-263, 1958.

17. Gross, J., and Lapiere, C. M. Collagenolytic Activity in AmphibianTissues: A Tissue Culture Assay. Proc. Nati. Acad. Sei. U. S.,' 48:

1014-1022, 1962.18. Gross, J., and Nagai, Y. Specific Degradation of the Collagen Mole

cule by Tadpole Collagenolytic Enzyme. Proc. Nati. Acad. Sei. U. S.,54: 1197 1204, 1965.

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Collagenase in Squamous Cell Carcinoma

Properties of Tropocollagen Macromolecules. Biochemistry, 4: 181- lagenolytic Activity in Basal Cell Epithelioma of the Skin on Re-190, 1965. constituted Collagen and Physical Properties and Kinetics of the

39. Walker, D. G., Lapiere, C. M., and Gross, J. A. Collagenolytic Factor Crude Enzyme. Cancer Res., 32: 2551-2560, 1972.in Rat Bone Promoted by Parathyroid Extract. Biochem. Biophys. 41. Yamanishi, Y., Maeyens, E., Dabbous, M. K., Ohyama, H., and Ha-Res. Commun., 15: 397 402, 1964. shimoto, K. Collagenolytic Activity in Malignant Melanoma. Physico-

40. Yamanishi, Y., Dabbous, M. K., and Hashimoto, K. Effect of Col- chemical Studies. Cancer Res., 33: 2507 2512, 1973.

Fig. 1. The tumor shows characteristics of Grade III squamous cell carcinoma of the skin. There is a tendency to minimal keratinization and individual cells are variable in shape and size, with dense nuclear chromatin. Notice rarefaction (asterisks) of the stroma in the immediate vicinity oftumor islands. Elastic fibers (arrows) are either intermingled with tumor cells or abundantly present in the periparenchymal clear spaces. E, eccrinesweat glands. Forehead lesion. A and B, Verhoeff elastic fiber stain. C, H & E. A, x 25; B, x 100; C, x 250.

Fig. 2. Periphery of tumor mass is surrounded with interrupted basal lamina (arrows). Villous projections of various sizes (asterisks) of tumor cellsprotrude through such perforations of the basal lamina. Except poorly stained sporadic remnants, (c), collagen is largely absent, whereas elastic fibers(E) are intact. Spaces that immediately surround the tumor are largely empty except where amorphous and filamentous materials (/) and cellulardebris are present. 7",tonofibrils; V, cytoplasmic vacuole, x 20,000.

Fig. 3. A number of elastic fibers (asterisks) are entrapped between tumor cells or along the periphery. Hemidesmosomes (B and C) are producedwhere the dense amorphous coat (B, aslerisks) of elastic fibers come into contact with the tumor cells. Such amorphous substances play a basal laminarole. A subepithelial dense layer (B, arrowheads) is produced between the amorphous substance and the tumor cells. Except for several aggregations offine filaments (C, f) stremai collagen is entirely dissolved. These aggregates can be organized and periodically cross-banded (C, p) producing cross-banded filament aggregations. Suggestions of basal lamina are seen at hemidesmosomes (A, d) but most of the tumor periphery lacks it. T, tonofibrils;V, cytoplasmic vacuole. A, x 150,000: B. x 100,000; C, x 30,000.

Fig. 4. In A, high magnification of a ruthenium red-stained cross-banded filament aggregation reveals component fine filaments (arrows) andamorphous dense substance mainly at cross-bands at intervals of 1000 A. A small number of collagen fibrils (C) are admixed; some of them show degenerative changes (hollow arrows). In B and C, ruthenium red-stained dense substance, often filamentous (arrows), is increased between collagen fibrilsnear the tumor mass. Striations of some of these collagen fibrils became obscure (asterisks), x 150,000.

Fig. 5. Scission products of segment-long-spacing tropocollagen molecules, produced by the squamous cell carcinoma homogenate, were matchedwith an intact full length segment-long-spacing molecule at the bottom. It is seen that the cleavage across the molecule at the /322region producedTC75. Major dense bands such as ß,1,¿,,and ¿3are marked lor reference. A, amino acid terminal of the molecule; B, carboxylic acid terminal of

the molecule, x 300,000.Fig. 6. In A, disc electrophoresis of calf skin tropocollagen control shows typical bands representing 7- and high molecular bands (//), /3U, 012,

«i,and «2chains. In B, after incubation with squamous cell carcinoma homogenate, all original bands become less dense or obscured and degradationbands such as |8A, aA, and a" appear in front of the y and/or high-molecular-weight components. Y, further breakdown products of low molecularweight; X, x-band.

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1973;33:2790-2801. Cancer Res   Ken Hashimoto, Yuji Yamanishi, Edgar Maeyens, et al.   SkinCollagenolytic Activities of Squamous Cell Carcinoma of the

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