Clin. Exp. Metastasis, 1992, 10,211-220

Expression of gelatinase/type IV collagenase in tumor necrosis correlates with cell detachment and tumor invasion

R. Daniel Bonfil*t, Paula A. Medina*t, Daniel E. G6mez$, Eduardo Farias$, Alberto Lazarowski*, M. Fernanda Lucero Gritti*t, Roberto P. Meiss* and Oscar D. Bustuoabad*

*IIHEMA, Academia Nacional de Medicina; ?Fundaci6n de Investigaci6n del Cdncer; and ~:Instituto de Oncologfa A. H. Roffo, Facultad de Medicina, Universidad de Buenos Aires, Argentina

(Received 13 May 1991; accepted 5 March 1992)

We have previously observed that acellular extracts from necrotic areas (NE) of the non-metastatic murine mammary adenocarcinoma M3, enhance in vitro cell detachment and spontaneous lung metastases. In the present study, using different proteinase inhibitors along with NE, only the calcium chelator EDTA could significantly abrogate the enhanced cell detachment from M3 produced by NE. The typical cleavage products of type IV collagenase were detected inside the tumor necrotic area, mainly in association with necrobiotic cells, as evaluated by Western blot analysis and immunohistochemical assays. Zymography revealed the presence of 72- and 92-kDa gelatinase/type IV collagenase in NE. Moreover, NE increased the in vitro invasive ability of cultured M3 cells. The use of specific antibodies against both 72- and 92-kDa type IV collagenases in the invasion assay showed that only the latter was able to revert the enhanced invasiveness to the baseline. It can be concluded that tumor necrosis is an important source of gelatinase/type IV collagenase, mainly in its 92 kDa form, and plays a major role in tumor invasion.

Keywords: gelatinase, metalloproteinases, tumor invasion, tumor necrosis, type IV collagenase

Introduction

Neoplastic cells need to detach from the primary tumor in order to reach their target organ and establish secondary tumor foci. An increased rate of malignant cell detachment has been associated, among other factors, with tumor necrosis [1, 2], decreased cellular expression of fibronectin or la- minin [3, 4] and enhanced activity of different enzymes [5, 6]. Serine, thiol-, carboxy-, and metalloproteinases have been implicated in the invasive process [7]. Since type IV collagen is exclusively found in basement membranes [8], type IV collagenase been the object of studies which

Correspondence to: Dr R. Daniel Bonfil, Fundaei6n de Investi- gaci6n del C~incer, F. D. Roosevelt 2408 1 ° "C" , 1428 - Buenos Aires, Argentina.

strongly correlated increased levels of this protease with enhanced invasion and/or metastasis [9-12]. MetaUoproteinase inhibitors [13-15] or type IV collagenase antibody [16] abrogated the in vitro invasive capacity of some tumor cells, as a conse- quence of the absence of enzymes available to degrade extracellular matrix.

We have previously reported that acellular ex- tracts from necrotic areas (NE) of both M3 (non- metastatic) and MM3 (metastatic) murine mam- mary adenocarcinomas, increased in vitro cellular detachment from the primary tumor [1]. Moreover, inoculations of NE into M3 tumors led to the formation of spontaneous lung metastases in 100% of the mice so treated [1].

In the present paper, we have used different

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enzyme inhibitors in order to determine which protease, if any, might be present in this acellular extract. We also have investigated the type IV collagenolytic activity in NE, as well as its effect on the in vitro invasive capacity of cultured M3 cells. Our results demonstrate the existence of gelatinase/type IV collagenase with a molecular weight of 92kDa in NE; this may explain the increased cellular detachment and invasive poten- tial seen in vitro.

Materials and methods

Mice Female BALB/c mice, 10-weeks-old, raised in our Laboratory Animal Facility were used throughout. They were maintained on specially formulated pel- lets (Nutric, C6rdoba, Argentina) and water acl libitum, and were microbiologically tested for pathogenic murine virus and bacteria.

Tumor M3 is a BALB/c mammary adenocarcinoma of spontaneous origin [17]. It has been maintained in our laboratory by serial s.c. passages in syngeneic female mice for 6 years without any apparent behavioral change. It does not give rise to meta- stasis and kills the mice 35-40 days after s.c. inoculation [18].

Cell line The cell line M3-TC was established from a s.c. M3 tumor (M.F. Lucero Gritti et al., unpublished data) and maintained in RPMI 1640 culture me- dium (Sigma Chemical Co., St Louis, MO, USA) containing 10% fetal bovine serum (FBS; BIO- NOS, Buenos Aires, Argentina) at 37°C in a 5% CO2 humidified incubator. This cell line has been in continuous culture for more than 90 passages and proved tumorigenic in female BALB/c mice. M3-TC was routinely screened and found to be negative for Mycoplasma spp. contamination, us- ing the Gen-Probe Rapid Detection System (Fisher, Pittsburgh, PA, USA).

Acellular tumor necrosis extract (NE) This was prepared as described previously [1]. Briefly, central necrosis from M3 was homo- genized with 4 ml of saline per gram and cen- trifuged at 4000 and 8000 r.p.m. The protein con- centration of the final supernatant was measured

and used as NE; its pH was 7.2. The extract was stored at -18°C until use. In some experiments NE was again centrifuged at 10000 g to obtain a particulate fraction (consisting mainly of plasma membrane and nuclei) and a supernatant. The toxicity of NE on M3-TC was measured using a colony-forming efficiency assay. Subconfluent M3-TC cells were exposed to different dilutions of NE in FBS-free culture medium with 0.1% bovine serum albumin (BSA, fraction V), for 3 h. Once treated, the cells were rinsed with RPMI, harvested, and finally plated at a concentration of 1000 cells/60 mm 2 Petri dish. Seven days later cells were fixed and stained with Leukostat Stain kit (Fisher Scientific, Orangeburg, NY, USA) and colonies of 50 or more cells were counted. The surviving fraction was calculated as a fraction of the control.

Detachment assay Non-necrotic 30 mm 3 M3 tumor fragments were placed in vials containing 2ml of NE with a protein concentration of 3.7 mg/ml, as measured by the method of Bradford with the Bio-Rad protein assay (Bio-Rad Chemical Division, Richmond, CA, USA). After incubation for 45 min at 37°C without shaking, the tumor frag- ments were agitated for 45 min at 29°C and 225 strokes/minute. The number of cells detached in each case was counted with a hemocytometer, and cell viability measured by Tryptan Blue exclusion test. Controls consisted of M3 tumor fragments incubated in saline instead of NE. The role of proteinases in in vitro detachment from M3 was evaluated by incubating NE with different protei- nase inhibitors. The metalloproteinases were inhi- bited with EDTA (Sigma) [7, 19]. Aprotinin and soybean trypsin inhibitor (SBTI) (both from Sigma) were employed as serine proteinase inhib- itors [7, 19]. The concentrations used were based on their documented biologic activity in the abs- ence of cytotoxicity [13, 19, 20].

Attachment assay Multiwell tissue culture plates were coated with 0.5 ml of basement membrane Matrigel (Collaborative Research, Bedford, MA, USA; 0.5 mg/ml), and left at 37°C to polymerize. One day later subconfluent M3-TC cultures were harvested and washed three times in FBS-free culture medium. These cells were resuspended in RPMI-1640 supplemented with 0.1% BSA, seeded

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at a density of 3 x 105 onto the Matrigel-coated wells, and incubated at 37°C in a 5% CO2 humidi- fied atmosphere. After 60 and 120 min, non-at- tached M3-TC cells were collected and counted. Attached cells were enzymatically harvested and the percentage of attachment was expressed as the number of adherent cells divided by the total number of cells in each well.

Type IV collagenolytic assay The ability of NE to degrade type IV collagen was measured using [3H]proline-labeled type IV col- lagen purified from Engelbreth-Holm-Swarm sar- coma as a substrate. The assay was carried out essentially as described by Nakajima [11], but using collagen in suspension instead of collagen films. Briefly, aliquots of trypsin-activated NE were incubated along with the radioactive sub- strate (5000 c.p.m./tube) at 37°C for 18 h. Products which did not precipitate in 10% trichloroacetic acid and 0.5% tannic acid, measured by liquid scintillation counting, represent type IV collagen digests. Bacterial collagenase (Sigma) was used as a positive control.

Invasion and chemotaxis assays These were carried out essentially as described previously [9]. Polycarbonate filters with 8 #m pore diameter (Nuclepore, Pleasanton, CA, USA), were coated with 50/~g total protein of Matrigel (Collaborative Research). The Matrigel-coated filt- ers were used to separate both compartments of a modified Boyden chamber, which contained in its lower portion 5 #g of laminin (Gibco Laboratories, Grand Island, NY, USA) dissolved in RPMI 1640 with 0.1% BSA as a chemoattractant. The upper compartment was inoculated with 1.5 x 105 M3-TC cells suspended in RPMI 1640 plus 0.1% BSA or the same medium containing NE at a final concen- tration of 3.7 mg/ml, and the whole was incubated at 37°C in a humidified 5% CO 2 atmosphere. After an 8-h incubation the polycarbonate filters were removed, their upper surface swabbed, and fixed and stained with Leukostat stain kit to count cells using a light microscope. The number of cells that traversed the filter and remained attached to its lower surface was used as a measure of their invasive ability.

The chemotaxis assay (non-invasive migration assay), was carried out under the same conditions described for the invasion assay, except that 5 #g of type IV collagen (Gibco) was used to coat the filters to permit cell attachment. Unlike Matrigel,

type IV collagen does not cover the filter pores, thus allowing free movement of the cells through the pores towards the chemoattractant.

All assays were performed in quadruplicate.

Gelatin zymography Gelatinolytic activity in NE was identified by an assay based on the method of Heussen and Dowdle [21], and adapted to use a Mini Protean II dual slab system (Bio Rad). Non-heated NE ali- quots were mixed with SDS sample buffer without fl-mercaptoethanol, and applied to 7.5% acry- lamide separating gels co-polymerized with 1 mg/ml of gelatin (Sigma). After electrophoresis, gels were rinsed with 2.5% (v/v) Triton X-100 in 50 mM Tris-HC1 buffer, pH 7.5, to remove SDS, and then incubated overnight in 0.15MNaC1, 10 mMCaC12 and 50 mMTris-HC1 buffer, pH 7.5. Gels were stained with 0.05% (w/v) Coomassie brilliant Blue G-250 in a mixture of methanol: acetic acid:water (5:1:4), and destained in the same solution without dye. Gelatinolytic enzymes were detected as transparent bands on the back- ground of Coomassie Blue-stained gelatin. Pre- stained standard molecular weight marker proteins (Amersham Corp., Arlington Heights, IL, USA) were used as reference.

Western blot analysis NE aliquots were analyzed as for zymography, except that they were previously reduced with /~-mercaptoethanol followed by boiling for 3 min, and then transferred to nitrocellulose filters (Schleicher and Schuell, Keene, NH, USA) at 100 V for 1 h at 4°C. Non-specific binding sites were blocked with 5% skim milk. The nitrocellu- lose sheets were then incubated overnight at 4°C with rabbit antibodies raised against type IV col- lagen (used at 1:60) or against 92-kDa type IV collagenase (1:250). In the first case NE was electrophoresed in a 7.5% polyacrylamide gel, while for antibodies to type IV collagenase a 6% polyacrylamide gel was used. After washing in phosphate buffer solution (PBS), the sheets were treated for 1 h at room temperature with horse- radish peroxidase-conjugated goat anti-rabbit IgG (Sigma) at a dilution of 1:2500. Peroxidase activity was visualized using as chromogen substrate 3,3'-diaminobenzidine tetrahydrochloride (S~gma) in the presence of hydrogen peroxide. Affinity purified antibodies to type IV collagen were kindly supplied by Dr Hynda Kleinman (NIDR, NIH). Anti-92-kDa type IV collagenase was a gift from

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Dr Gregory Goldberg (Washington University School of Medicine).

Immunohistochemical technique The classical 3-step peroxidase-anti-peroxidase (PAP) staining method was used to localize 92-kDa type IV collagenase on frozen sections from M3 tumors. Briefly, after blocking of endo- genous peroxidase activity, sections were incubated with anti-type IV collagenase for the first anti- body, anti-rabbit IgG for the bridge antibody, and finally the PAP complex. Diaminobenzidine (con- taining hydrogen peroxide) was used as chromogen substrate. In some cases, before mounting, slides were counterstained for 30 seconds with hematoxy- lin.

Statistical analysis Non-parametric Mann-Whitney U-test, Student's t-test and variance analysis were used; P < 0.05 was considered to indicate statistical significance. In the latter the Bonferroni T-test was performed to identify which pairs of groups were significantly different (on the ranks, P -- 0.05).

Resul ts

Effect of proteinase inhibitors on in vitro M3 tumor cell detachment Figure 1 summarizes the degree of cell detachment seen when non-necrotic M3 tumor fragments were treated with the different agents. As previously observed NE enhanced spontaneous cell release [1]: this was not due to a cytotoxic effect of NE, since there was no alteration in colony-forming ability at the doses used. Furthermore, the viabil- ity of cells released from the tumor fragments, as evaluated by Trytpan Blue, was always greater than 90%, with or without NE. We studied whether this cell detachment enhancement pro- duced by NE could be caused by enzymatic action, using proteinase inhibitors. Only the metal chela- tor EDTA could abrogate the effect induced by NE on cell detachment; SBTI and aprotinin had no inhibitory effect. We speculate that the en- hanced number of cells released by these serine proteinase inhibitors when used together with NE, was not due to their own effect since they did not alter the baseline cell detachment when used alone.

NE

NE+APROTiNIN

SALI NE-~APROTINtN

NE+EDTA

SALJNE+EDTA

(39 -59)

(28-42)

(18 -87)

102-123)

(61-140)

NE+SBTI (64-109)

SALI NE+SBTI (28-44)

SALINE 30-65)

I I I I

0 20 40 60 80 100 120 140

MEDIAN NUMBER OF CELLS (range) x 103

Figure 1. Cell detachment from M3 tumor fragments treated in vitro with NE and proteinase inhibitors. Doses used were: aprotinin 500 KIU/ml, EDTA 0.5 mg/ml, SBTI 100 #g/ml, NE 3.7 mg protein/ml. Results were analyzed by non-parametric Mann-Whitney U-test: *P = 0.0095, **P = 0.0011 compared to NE values.

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Gelatinase/type IV collagenase in tumor necrosis

Influence of NE on in vitro tumor invasiveness, adhesion to extracellular matrix (ECM), type IV collagenolysis and chemotaxis Since many metaUoproteinases have shown to be of importance in invasion and metastases [22, 23], we decided to study the effect of NE on tumor invasive ability. When fibronectin and laminin were tested as chemoattractants in the chemo- invasion assay, M3-TC cells only migrated in re- sponse to the latter. Those cells, when suspended in culture medium containing NE, showed a signi- ficantly enhanced invasion with respect to the control (median number of invasive cells 143 (87-180) vs. 45 (21-69), P = 0.028 by non-para- metric Ma nn-Whi tney U-test). The invasive pro- cess is supposed to occur in at least three steps: adhesion to ECM, degradation of ECM, and cell motility [24]; therefore we studied the effect of the acellular extract in each one of these. NE, used at the same concentrat ion as in the chemoinvasion assay, did not modify the adhesive rate of M3-TC cells to Matrigel: 8 4 . 1 + 0 . 5 vs. 79 .4+ 1.4 at 60 min, and 76.9 + 1.8 vs. 87.0 + 1.4 at 120 min, NE and control respectively 0 ] _ DS, expressed as percentage of attached cells). Moreover , the che- motactic response promoted by laminin did not significantly differ in NE-treated (median number of cells 194 (84-230)) or non-treated M3-TC cells (180 (75-201)). However , NE showed a type IV collagenolytic activity that represented 62% of that generated by bacterial collagenase, and was almost three times higher than the control (Table 1). This effect could be reversed by preheating NE at 100°C or incubating it together with E D T A , which again demonstrates that the factor responsible is a

Table 1. Type IV collagenolytic activity of NE

Treatment a Type IV collagen degrading activity (c.p.m.) b

NE 2187.0 + 125.3" NE + EDTA 699.1 + 72.3 NE pretreated at 100°C 813.6 + 42.0 Bacterial collagenase 3505.7 ___ 108.8"* Saline + EDTA 742.5 ___ 37.2 Saline 784.5 + 54.5

aNE: 3.7 mg protein/ml; bacterial collagenase: 0.1 mg/ml; EDTA: 0.5 mg/ml. bData represent mean + S.D. of triplicate samples. Total c.p.m./tube = 5000. *P = 0.003; **P = 0.0003 compared to saline by Stud- ent's t-test.

metalloproteinase. To determine more accurately which collagenolytic metalloproteinase was re- sponsible for the enhancement of invasion shown by NE, rabbit antibodies against 72- or 92-kDa type IV procollagenases, kindly supplied by Dr Motowo Nakajima, were added at a dilution of 1:250 to the NE-treated groups. As can be seen in Figure 2, only the antibody against the 92-kDa form was capable of inhibiting the NE- related enhanced invasiveness; no alteration in chemotaxis was observed.

Detection of 92-kDa type IV collagenase/gelatinase enzyme in NE Matrix metalloproteinases with type IV collagen- degrading ability include not only 72- and 92-kDa type IV collagenases but also stromelysin and stromelysin-2 (also known as transin and transin-2) [25, 26]. The type IV collagenases degrade the basement membrane collagen as well as having gelatinolytic activity [27-29]. Thus we decided to study the gelatin degrading capacity of NE by zymography. Two major negatively stained bands of 72 and 92 kDa were observed when whole NE or particulate fraction from NE, obtained at 10000 g, were electrophoresed in the SDS-poly- acrylamide system including the gelatin substrate (Figure 3). The gelatinolysis shown by the super- natant obtained by centrifugation of NE at 10000 g was only due to a band of 72 kDa. No activation by aminophenylmercuric acetate (APMA) or trypsin was necessary for the gelati- nase activity. Gelatinolytic activities were com- pletely inhibited by including E D T A in the incu- bation buffer used to reveal the enzymatic reac- tion. Immunoblott ing of the gelatin/type IV col- lagen degrading enzyme detected in NE, using polyclonal antibodies to 92-kDa type IV collage- nase, recognized two proteins of approximately 72 and 92 kDa (Figure 4).

Type IV collagenase degrades basement membrane collagen in vivo In order to demonstrate that the collagen degrad- ing activity effectively occurs in vivo, NE-reduced aliquots were run in a S D S - P A G E system and antibodies to type IV collagen were added for Western blot analysis. As can be seen in Figure 5, degradation of collagenous proteins naturally tak- ing place in tumor necrotic areas could be detected by the antibody. The typical cleavage products [30, 31] of approximately 135 and 125 kDa correspond- ing to 75% from a~l (185 kDa) and or2 chains (170 kDa) were obtained. Other type IV collagen

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R. D. Bonfi l et al.

800

m I,¢1

600

.~, ~ 400 4-1 (D I X _ j , ~ ._1 LI.I 0

200

0 CONTROL NE

_T_

NE 4.

a n t ; - 7 2 K d

_I.

NE 4.

a n t ; - g 2 K d

NE 4.

n o r m a l IgG

m INVASION f ~ l CHEMOTAXIS

Figure 2. Effect of rabbit antibodies against 72- and 92-kDa type IV procollagenases on the in vitro invasion and chemotaxis of M3-TC cells treated with NE. Antibodies and rabbit non-immune IgG were used at a dilution of 1:250, NE at a concentration of 3.7 mg protein/ml in RPMI-1640 supplemented with 0.1% BSA. Each bar represents mean number of cells migrating through porous filter. The values are calculated from quadruplicate determinations. Difference in invasion among the groups is extremely significant (variance analysis, P = 0.0001). The Bonferroni T-test showed significant differences only between control and NE, and NE and anti-92-kDa groups (P < 0.05).

A B C D 97K

'q69K Figure 3. Zymographic analysis of NE. Negative-stained bands represent proteinases with gelatinolytic activity, and their disappearance indicates specific inhibition. Lane A: whole NE; lane B: supernatant obtained after centrifuging NE at 10000 g; lane C: pellet obtained at 10000 g from NE; lane D: whole NE when EDTA was added to the incubation buffer. Positions of the mi- gration of standard protein markers are indicated ac- cording to their molecular weight: Phosphorylase b = 97 kDa; bovine serum albumin = 69 kDa.

216 Clinical & Experimental Metastasis Vol 10 No 3

Gelatinase/type I V collagenase in tumor necrosis

-97K -69K

Figure 4. Western blot analysis of type IV collagenase in NE. Bands detected represent 72-kDa and 92-kDa type IV collagenases. Positions of 69-kDa and 97-kDa weight markers are shown.

185kD 17ok1>

135kD 125k1>

o200K

the dilutions used the ant ibody only detected the enzyme in necrobiotic regions (Figure 6).

Discussion

Necrosis is a relatively common event in solid tumors. Although this can be induced by different factors, as tumors grow larger oxygen and nutr- ients become unavailable to central areas because of peripheral vascularization: the max imum dist- ance across which oxygen and nutrients can diffuse would be 100-150/~m f rom blood vessels [32]. This results in a peripheral well-perfused region, a central necrotic area and an intermediate semi- necrotic zone [33]. We have previously observed that acellular extracts f rom necrotic areas of the M3 murine m a m m a r y adenocarc inoma could enh- ance in vitro cell de tachment and even generate spontaneous lung metastases in this non-metastat ic tumor model [1]. In the present study, we have examined the nature of N E obtained f rom M3, concluding that its cell-detaching capacity could

o97K

Figure 5. Western blot analysis of reduced NE using antibodies against type IV collagen. Conversion of type IV collagen to lower molecular species is observed. 170 and 185 kDa represent o:1 and 0:2 chains; 125 and 135 kDa corresponding to the typical cleavage products generated by type IV collagenase are indicated. 97 and 200 kDa represent phosphorylase b and myosin, used as molecular weight standards.

degradat ion products were observed; these could be due to other enzymes present in physiological conditions but not active in vitro. I t is important to emphasize that all the degradations detected oc- curred inside the tumor necrotic area in the ab- sence of any enzyme inhibitor, and that type IV collagen was par t of the tumor extracellular mat- rix.

In order to localize enzyme activity in situ, M3 frozen sections were incubated with ant ibody to 92-kDa type IV collagenase and immunohisto- chemically detected using the PAP technique. At

Figure6. Immunodetection on frozen sections (PAP technique) of type IV collagenase: a = positivity around nest of ceils in necrobiotic areas; b = negative in well- preserved areas. Hematoxylin counterstain, x40.

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only be reversed using a metalloproteinase inhibi- tor. More detailed studies showed that NE con- tained 72- and 92-kDa gelatinases/type IV colla- genases; the 92-kDa species was mainly located in the particulate fraction of NE, presumably bound to the plasma membrane debris. Unlike lysosomal enzymes, collagenases are secreted by the cell or localized on its surface [6, 7]. In a recent study no collagenolytic activity could be detected in condi- tioned media from M3 cultures [34]. However, focal clearing of matrigel could be observed in the vicinity of M3 pseudopodia by electron microscopy [34], which supports our presumption that mem- brane-bound collagenase is involved. Moreover, NE was able to enhance in vitro invasiveness of cultured M3 cells and to degrade type IV collagen. That effect would be due mainly to the activity of 92-kDa type IV collagenase, since the use of the specific antibody could revert the effect of NE. This would be of importance, taking into con- sideration that the 92-kDa form of the enzyme was essentially associated with malignant transforma- tion [19, 27, 35], while it was only found in a few normal cells [22, 27]. Immunohistochemical analy- sis detected the gelatinase/type IV collagenase en- zyme mainly in semi-necrotic areas and not in regions composed of morphologically well- preserved cells. Under no circumstance can it be assumed that viable cells cannot express the same enzyme activities as cells in necrobiosis, but it is possible that higher levels become concentrated in the latter and are therefore detected more easily than in other areas. Hypothetically, it could be that acquisition of collagenolytic abilities would be of great advantage to cells with a deficit of oxygen and nutrients, permitting cells in necrobiosis to escape from such an inadequate environment. Their metastatic success would depend on the availability of lymphatic or blood vessels close to the necrotic areas. This was confirmed, in part, by the fact that MM3 tumor, which alternates necrotic and well-perfused non-necrotic areas, gives rise to metastases, while M3, which does not contain vessels near the necrotic zones, does not [1]. It is difficult to imagine how cells committed to die are able to synthesize and/or secrete collagenase in huge amounts. However, Young [36, 37] observed that tumor hypoxia induces DNA over-replication and enhances metastatic potential in murine sys- tems, supporting our hypothesis.

It is important to emphasize that type IV collagen degradation could be detected in NE without previous activation and without adding exogenous type IV collagen, as in in vitro assays.

Collagenases have been found secreted or mem- brane-bound in transformed cells [6, 9-12, 19, 22, 23, 27], macrophages [38], polymorphonuclear leukocytes [39], or even in serum from tumor- bearing hosts [40]. However, to our knowledge, none of the above studies demonstrated in vivo collagen-degrading activity, as observed herein.

B a s e d on these results, one can assume that tumor necrosis could enhance invasion and metas- tasis through gelatinase/type IV collagenase activ- ity, mainly in its 92-kDa form. Thus, if 99.9% of the cells contained in a 1 cm 3 tumor, which repre- sents approximately 109 cells [41], are killed by any therapeutic modality, the necrosis generated would be sufficient to enhance the invasive and metastatic potentials of the 106 cells remaining alive. Perhaps this could explain the many cases in which initially successful radiotherapy or chemo- therapy are followed by a prompt metastatic development.

Acknowledgements

We thank Dr Christiane Dosne Pasqualini for her critical reading of this manuscript and helpful suggestions; Dr Hynda Kleinman for providing the antibody to type IV collagen, Dr Gregory Gold- berg for the anti-92-kDa type IV collagenase anti- body and Dr Motowo Nakajima for the polyclonal anti-72- and 92-kDa type IV coUagenase anti- bodies. This work was supported by CONICET (Consejo Nacional de Investigaciones Cientificas y Trcnicas).

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