Tumor Cell-Derived Collagenase-Stimulatory Factor Increases Expression of Interstitial Collagenase, Stromelysin, and 72-Kda Gelatinase1

[CANCER RESEARCH 53. 3154-3158. July 1. 1993] Tumor Cell-derived Collagenase-stimulatory Factor Increases Expression of Interstitial Collagenase, Stromelysin, and 72-kDa Gelatinase1

Hiroaki Kataoka,2 Rosana DeCastro, Stanley Zucker, and Chitra Biswas3

Department of Anatomy and Cellular Biology. Tufts University School of Medicine, Boston, Massachusetts 021II Â¡H. K., R. D., C. B.Â¡, and Departments of Research and Medicine, Veterans Affairs Medical Center. Northpon, New York 11768 Â¡S.ZJ

ABSTRACT MATERIALS AND METHODS

The tumor cell-derived collagenase-stimulatory factor (TCSF) was pre Cells and Culture Conditions. The LX-1 human lung carcinoma cells viously purified from human lung carcinoma cells (S. M. Ellis, K. Na- were originally isolated from a tumor grown in the nude mouse by Mason beshima, and C. Biswas, Cancer Res., 49: 3385-3391, 1989). This protein Research Institute, Worcester, MA, and maintained in this laboratory (4). The is present on the surface of several types of human tumor cells in vitro and human fetal lung fibroblast cell line, HFL, and the colon fibroblast cell line, in vivo and stimulates production of interstitial collagenase in human CCD-18, were obtained from the American Type Culture Collection, Bethesda, fibroblasts. In this study it is shown that TCSF stimulates expression in MD; the HF line was isolated from human skin in this laboratory (4). These cell human fibroblasts of niKN Vfor Stromelysin 1 and 72-kDa gelatinase/type lines were maintained in Dulbecco's modified Eagle's medium containing 10% IV collagenase, as well as for interstitial collagenase. Measurement of fetal bovine serum and antibiotics. Experimental cultures containing fibro enzyme protein by immunoassay showed that the amounts of interstitial blasts or tumor cells were established in 150-mm tissue culture dishes in collagenase and Stromelysin 1 were increased in TCSF-treated fibroblasts; Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, gelatin zymography indicated that there was an increase in the 72-kDa penicillin (100 units/ml), and streptomycin (100 /ig/ml). The plating density gelatinase. These results indicate that tumor cell interaction with fibro for each cell type was 1 x I07/dish for HFL, 1 x IO7 for HF, 0.3 x IO7 for blasts via TCSF could lead to increased degradation of interstitial or CCD-18. and 0.5 x IO7 for LX-1. The cells were allowed to attach for 24 h at basement membrane matrix components and thus to enhanced tumor cell 37Â°Cin a humid atmosphere of 5% CO? and 95% air, after which the cell layers invasion. were washed with serum-free media and further incubated with 15 ml Dul becco's modified Eagle's medium containing 0.2% lactalbumin hydrolysate

INTRODUCTION and antibiotics in the presence or absence of TCSF or TPA. Source of TCSF. TCSF was purified from detergent extracts of LX-1 The role of tumor cell-fibroblast interactions in the regulation of membranes on an antibody affinity column as described previously (7). The interstitial collagenase production in neoplasms has been demon purity of the preparation was determined by electrophoresis of the sample on strated by several investigators (1-6). Goslen et al. (6) have isolated a 10% SDS-polyacrylamide gel followed by staining with Coomassie blue. Fig. a collagenase-stimulatory factor with an approximate molecular mass 1 shows that the predominant band in the preparation has an approximate of 19 kDa from tissue extracts of basal cell carcinoma. We have molecular weight of 58,000; this protein is recognized by the monoclonal demonstrated that mouse and human tumor cell lines produce factors antibody El 1F4 in immunoblots thus confirming that the band corresponds to that stimulate production of collagenase by fibroblasts (2, 4) and have TCSF (7). High molecular weight aggregates reactive with E11F4 were also purified a ~58-kDa, tumor cell-derived, collagenase-stimulatory fac observed as reported previously (7). tor, TCSF,4 from cell membranes of a human lung carcinoma cell line, Isolation of RNA. Monolayer cell cultures containing fibroblasts or tumor cells incubated with or without TCSF or TPA were used for RNA isolation. LX-1 (7). Addition of purified TCSF to fibroblasts in culture stimu After the media were removed the plates were washed 3 times with phosphate- lates collagenase production and elevates the level of collagenase buffered saline, and the cells were lysed with guanidinium thiocyanate. The mRNA in these cells (8). lysate was layered over CsCI and centrifuged at 25,000 rpm for 18 h at 20Â°C Several MMPs in addition to collagenase have been implicated in as described elsewhere ( 14). RNA was collected, precipitated with ethanol, and extracellular matrix degradation in cancer. In some cases the regula stored at -20Â°C. tion of these MMPs has been shown to be coordinate (9-11 ); in others Northern Blot Analysis. Whole cell RNA was used for Northern blot this is not the case (12, 13). To investigate whether TCSF affects other analysis. RNA was suspended in a solution of 5 Mformaldehyde, 27% formamide. and 4X morpholinopropanesulfonic acid buffer and incubated at 80Â°C MMPs in addition to interstitial collagenase (MMP-1), we have per formed Northern blot analysis with RNA isolated from human fibro for 10 min. After incubation, the RNA was electrophoresed on a 1% agarose gel in morpholinopropanesulfonic acid-formaldehyde buffer at 100 V and blasts incubated with TCSF. The complementary DNA probes used transferred to nitrocellulose paper (15). Hybridization was performed in 50% were for stromelysin 1 (MMP-3), Stromelysin 3 (MMP-11), and 72- formamide, 5X Denhardt's solution, 25 mm phosphate buffer (pH 6.5), 0.1% kDa gelatinase/type IV collagenase (MMP-2), as well as for MMP-1 SDS, 50 /Â¿g/mlof sonicated and heat-denatured salmon sperm DNA, and 5X and the serine protease, uPA. Our data indicate that TCSF increases SSC at 42Â°Cfor 16 h. The blot was washed as follows: twice in 3x SSC the expression of MMP-1, MMP-2, and MMP-3 in fibroblasts, al containing 0.5% Sarkosyl for 15 min at room temperature; twice in 3 x SSC for though there are considerable differences in the pattern of response in 15 min at 65Â°C;once in 0.5X SSC for 15 min at 65Â°C;and once in 0.2X SSC different fibroblast lines. No effect was observed on the mRNA level for 15 min at 65Â°C.Filters were autoradiographed with Kodak XR-5 film at of uPA or MMP-11 in any of the fibroblast lines studied. -80Â°C. The probes used for hybridization were: pCol 185.2 for human MMP-1; pSL 51.4 for human MMP-3; and pGEL 186.2 for human MMP-2 (16-18) (kindly provided by Dr. G. Goldberg); ZIV for human MMP-11 (19) Received 12/3/92; accepted 4/27/93. The cosls of publication of this article were defrayed in part by the payment of page (kindly provided by Drs. ChambÃ³n and BasseÂ«);pHUK for uPA and pHcGAP charges. This article must therefore be hereby marked advertisement in accordance with for glyceraldehyde-3-phosphate dehydrogenase (American Type Culture Col 18 U.S.C. Section 1734 solely to indicate this fact. lection). The probes were excised from the plasmids, purified by agarose gel 1This study was supported by NIH Grant Ã‡A388I7 (C. B.) and by a Department of electrophoresis and SpinBind cartridge (FMC BioProducts, Rockland, ME) Veteran Affairs grant (S. Z.). filtration, and radiolabeled by nick translation with [12P]CTP. 1 Present address: Department of Pathology, Miyazaki Medical College, Miyazaki 889-16, Japan. Substrate Gels. Culture media from cells incubated in the presence and 3 To whom requests for reprints should be addressed. absence of TCSF or TPA were electrophoresed in 10% SDS-polyacrylamide 4 The abbreviations used are: TCSF, tumor cell-derived collagenase-stimulatory factor; MMP, metalloprotease; SDS. sodium dodecyl sulfate; SSC, standard saline-citrate (8.75 g gels impregnated with 0.5 mg/ml of gelatin (9, 20) for demonstration of the NaCl/4.4lg sodium citrate per liter); TPA. 12-O-tetradecanoylphorbol-13-acetate ester; presence of gelatinase activity. Samples were mixed with Laemmli sample uPA, urokinase-type plasminogen activator. buffer without dithiothreitol and electrophoresed without boiling. After elec- 3154

The plates were then incubated for 2 h at 37Â°Cfollowed by washing and 1 addition of a second antibody corresponding to the particular MMP. The antibodies used in the above step were biotinylated monoclonal to MMP-1 (MAC 066), unconjugated monoclonal to MMP-2, and unconjugated polyclonal to MMP-3. The plates were incubated for l h at 37Â°Cand washed. The MMP-1 plates were then incubated for 30 min at 37Â°Cwith alkaline phosphatase-conjugated streptavidin followed by washing and color develop ment with p-nitrophenyl phosphate. The MMP-2 plates were incubated with biotinylated goat anti-mouse immunoglobulin at 37Â°Cfor l h and then reacted with alkaline phosphatase conjugated to streptavidin and processed for color 97Â»- development as for MMP-1. The MMP-3 plates were incubated with peroxi- dase-conjugated donkey anti-rabbit antibody and processed in the same way as for MMP-2. After color development the plates were read in a microplate 66** reader (BioTek EL 309, Winooski, VT) at /4405 nm. The concentrations of MMPs were quantitated by extrapolation from a log-log linear regression curve constructed from values obtained with varied concentrations of purified MMPs (22). The ELISAs for MMP-1, MMP-2, and MMP-3 are monospecific for the 43Â»- corresponding antigen and do not detect a 100-fold excess of unrelated MMP antigen. The ELISA for MMP-1 and MMP-3 detects both activated and latent forms of the corresponding enzymes (21). The ELISA for MMP-2 detects latent but not aminophenyl mercuric acetate-activated MMP-2.

RESULTS AND DISCUSSION

31Â»- Northern Blot Analyses. The effect of TCSF on the mRNA level in fibroblasts for several MMPs was investigated by Northern blot analysis. Fibroblasts were incubated with purified TCSF and RNA was isolated from these cells as described in "Materials and Methods." As shown in Fig. 2, 1.0 /xg TCSF/ml of culture media was found to increase the level of MMP-1 mRNA, whereas 0.1 fig had little or no 21Â»- effect in all three cell lines. For comparison, phorbol ester was added to one of the cell lines and, as expected, it caused marked stimulation of the MMP-1 mRNA level. As also can be seen in Fig. 2, the endogenous level of MMP-1 and Fig. 1. SDS-polyacrylamide gel electrophoresis gel of purified TCSF. Samples were the extent of MMP-1 stimulation by TCSF varied between the three analyzed by SDS-polyacrylamide gel electrophoresis and the protein bands were detected cell lines; this may be a reflection of their passage number or tissue of by Coomassie blue staining. Lane 1, molecular weight standards; arrowheads, positions of origin. Variation with passage number in the level of production of phosphorylase b {97 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21 kDa) and lysozyme (14 kDa). Lane 2, MMPs by fibroblasts has been observed in previous studies (23, 24). 25 u,g of purified TCSF. Large arrowhead, major TCSF band corresponding to a molec We have observed a decreased response to TCSF with fibroblast ular weight of ~-58,000; this band is also recognized by the monoclonal antibody El 1F4 passage number,5 and this may explain the relatively low response of in parallel Western blots (7). The small amount of protein of ~-55 kDa, the faint band at â€”¿30kDa (small arrowhead), and the aggregated protein at the top of the gel, all of which the HF line, which was in its 20th passage. CCD-18, on the other are reactive with El 1F4, are often observed in the purified preparations (7, 43). Asterisks, hand, was only in its 8th passage. Also, variations in response to TCSF anifactual bands present in all lanes of the particular gel. and other collagenase-stimulating agents by fibroblasts from different origins have been observed previously (8, 25). The reason for varia trophoresis, SDS was eluted from the gel in 2.5% Triton X-100 for 30 min at tions in response to TCSF is not clear but two obvious possibilities 25Â°Cand incubated overnight in substrate buffer containing 50 min Tris-HCl would be variation in the expression of cell surface receptor for TCSF buffer (pH 8.0) and 5 mM CaCl2. The gel was stained with Coomassie blue and or in the intracellular responses to signal transduction subsequent to the presence of gelatinolytic activity was detected by the appearance of clear interaction of TCSF with the cell surface. zones. Analyses similar to the above, performed with the MMP-3 probe, Enzyme-linked Immunoassays for MMPs. The murine monoclonal anti revealed that TCSF also caused a large increase in the level of MMP-3 bodies to human MMP-1 (MAC 064 and MAC 066) and human MMP-3 (MAC in CCD-18 and a modest increase in HFL and HF cells (Fig. 3A ; data 078) and the rabbit polyclonal antibody to MMP-3 were kindly provided by Dr. not shown). With the MMP-2 probe, stimulation was observed only in T. Baker (Cell Tech Ltd., Slough, England). Rabbit polyclonal and murine CCD-18 cells and not in the other two cell lines (Fig. 3ÃŸ).For a monoclonal antibody to human MMP-2 were kindly provided by Dr. W. Stetler Stevenson (National Cancer Institute, Bethesda, MD) and Dr. H. Birkedal- positive control, we incubated HF cells with phorbol ester which stimulated MMP-3 but not MMP-2 (Fig. 3) as reported previously in Hansen (University of Alabama, Birmingham, AL). The sandwich assays used are based on previously published methods (21, other systems (13). No effect of TCSF on the MMP-11 or uPA mR- 22). Microtiter plates containing 96 wells (Dynatech Immulon II, Alexandria, NAs was apparent in any of the three cell lines (data not shown). None VA) were coated with antibody corresponding to individual MMPs in buffer of the five mRNAs measured was detected in LX-1 cells (Figs. 2 and containing 0. l MNaHCO, (pH 9.0) for 18 h at 4Â°C.The antibodies used for this 3; data not shown). The above observations were confirmed in three step were monoclonals to MMP-1 (MAC 064) and MMP-3 (MAC 078) and separate experiments. polyclonal to MMP-2. Unbound antibody was removed and excess binding To determine the extent of stimulation by TCSF, the radioactive sites were blocked by addition of 1% BSA in bicarbonate buffer. The plates bands of the Northern blots were quantitated by densitometry, and the were washed three times with buffer containing 50 HIMsodium phosphate (pH 7.2), 0.1 M NaCI, and 0.05% Tween 20, and the test samples or purified, standard MMP samples, at various dilutions, were added to duplicate wells. 5 Unpublished data. 3155

CCD-18 _ HF HFL LX-1

123456 Fig. 2. Northern blot analysis of collagenase (MMP-1) mRNA. Total RNA was isolated from cells incubated for 48 h without or with TCSF or TPA. Ten (Â¿gof total RNA were loaded in each lane. Total radioactivity used for hybridization for 2-4 pCol 185.2 (A) was 0.5 X IO6cpm/ml and that for the pHacGAPD probe for glyceraldehyde-3-phosphate dehydrogenase (ÃŸ)was 1 x IO6cpm/ml; the duration of the film exposure was 1 week. After autoradiography the probes were stripped from the 1.4 fillers and rehybridized with a glyceraldehyde-3- phosphate dehydrogenase probe (ÃŸ).Results were confirmed in three separate experiments. Lanes 1, 2, and 3, CCD-18 fibroblasts; Lanes 4 and 5, HF fibroblasta; Lanes 6, 7, and 8, HFL fibroblasts; Lane 9, LX-1 carcinoma cells. Lanes I, 4, and 6, with 1 jig/ml TCSF; Lane 2, with 0.1 /Â¿g/mlTCSF; Lanes 3. 5, 7. and 9. no addition; Lane 8, with 100 ng/ml TPA.

values obtained for each MMP were normalized against the mRNA CCD-18 HFL LX-1 level for glyceraldehyde-3-phosphate dehydrogenase. Table 1A shows -I - TPA! -I the data from such analysis for the response of the 3 fibroblast lines r+ + to TCSF. An increase in MMP-1 mRNA was observed in all three cell lines and the highest stimulation (4.7-fold) was obtained in HFL A 1 2 3 456 fibroblasts. A large increase in MMP-3 mRNA ( 13-fold) was obtained 2.4 in CCD-18 cells and approximately a 2-fold increase in HFL cells. A 3-fold increase in MMP-2 mRNA was obtained in the CCD-18 fibro 1.4 blasts, but not in HFL, in the experiment shown. Similar degrees of stimulation were obtained in two separate experiments. Analyses per formed with the uPA and MMP-11 probes confirmed that TCSF had no effect on their mRNA levels. The above results indicate that TCSF stimulates mRNA levels for MMP-1, MMP-2, and MMP-3 in responsive cells. To determine whether TCSF also stimulates expression of the enzymes themselves we analyzed the amounts of enzyme protein in the conditioned media of the CCD-18 and HFL cells by immunoassay. B Enzyme-linked Immunoassay of MMPs. MMPs were analyzed in the culture media of HFL and CCD-18 cells incubated with and without TCSF. Table IB shows the results of immunoassay for 2.4 MMP-1, MMP-2, and MMP-3. Each assay was performed at three different dilutions in duplicate. The amount of MMP-1 increased in 1.4 both cell lines with HFL cells showing the largest increase (10-fold). An increased stimulation of MMP-3 (6-fold) was also observed in CCD-18 cells, and there was a modest increase in HFL cells. These results are in agreement with the data obtained in Northern blots and indicate that increased expression of mRNA for MMP-1 and MMP-3 is associated with increased production of the corresponding protein, although the extent of stimulation measured by the two separate techniques varies somewhat. To our surprise, TCSF appeared to produce only a small increase in MMP-2 protein secretion by CCD-18 cells when measured by the immunoassay, as compared to a large increase in MMP-2 mRNA expression. Consequently we used substrate gel zymography in an Fig. 3. Northern blot analysis of stromelysin-1 (MMP-3) (A) and 72-kDa gelatinase attempt to resolve this apparent discrepancy. (MMP-2) (B) mRNAs. Total RNA was isolated from cells incubated for 48 h without or with TCSF or TPA. Ten fig of total RNA were loaded in each lane. Total radioactivity used Substrate Gel Zymography. Fig. 4 shows gelatin zymography of for hybridization was 0.5 X IO6 cpm/ml for the pSL 51.4 probe for MMP-3 (A) and for the culture media of CCD-18 and HFL cells incubated with TCSF. As the pGEL 186.2 probe for MMP-2 (B) and 1 X IO6 cpm/ml for the pHcGAP probe for expected from the mRNA results, there was little difference in the glyceraldehyde-3-phosphate dehydrogenase (C). The duration of film exposure was 1 week in each case. Results were confirmed in three separate experiments. Lanes 1 and 2, level of gelatinolytic activity in medium from HFL cells treated or not CCD-18 cells; Lanes 3, 4, and 5, HFL cells; Lane 6, LX-1 cells; Lanes I and 3, with 1 with TCSF. However, a significant increase in gelatinolytic activity at Hg/ml TCSF; Lanes 2. 4, and 6, no addition; Lane 3, with 100 ng/ml TPA. 3156

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1993 American Association for Cancer Research. TCSF STIMULATES COLLAGENASE. STROMELYSIN. AND OELATINASE both 66 kDa and 59 kDa was observed in the media from CCD-18 3 4 cells treated with TCSF as compared to the untreated cells. The 66-kDa band corresponds to the molecular mass of the proform of MMP-2 in the absence of dithiothreitol and the 59-kDa band corre sponds to the activated form (26). Thus these results, as opposed to those obtained by immunoassay, support the Northern blot analyses showing increased stimulation of MMP-2 mRNA by TCSF in CCD-18 cells but not HFL cells (Fig. 3). It can be seen clearly in Fig. 4 that TCSF caused a far greater increase in the activated form of MMP-2 than the precursor form. The most likely explanation, then, for the disparate results obtained by immunoassay and zymography is the fact that the ELISA used for MMP-2 does not detect the activated form of enzyme, whereas zy mography is even more sensitive for detecting activated MMP-2 than latent MMP-2 (26). The mechanism whereby treatment of fibroblasts with TCSF leads to increased activation of latent MMP-2, in addition to an increase in total amount of the enzyme, is unknown but is of considerable interest since a physiological activator of MMP-2 has long been sought. Comparable to our observations, Overall and Sodek (26) have dem onstrated that concanavalin A treatment of fibroblasts leads to in creased production of the activated form of MMP-2 which is more readily demonstrated by zymography than by MMP-2 protein mea surement. Since the level of stromelysin (MMP-3) is stimulated by TCSF (see Fig. 3 and Table IB) and since MMP-3 is a known activator of MMP-1 (27,28), one possibility is that this increased level Fig. 4. Gelatin zymogram of conditioned medium from CCD-18 and HFL fihroblasts. of MMP-3 also leads to the increased activation of MMP-2. Recent The cells were incubated with or without TCSF and the conditioned media were collected. Forty /il of conditioned media from CCD-18 cells and 20 Â¿ilfrom HFL cells were studies by Miyazaki et al. (29) indicating that MMP-3 activates a electrophoresed on a gelatin-containing gel as described in "Materials and Methods." Lane MMP tissue inhibitor-2-bound progelatinase complex support this I. CCD-18 cells incubated without TCSF; Lane 2. CCD-18 cells with TCSF (1 jig/ml); idea. However, the role of MMP-3 in the activation of MMP-2 has yet Lane 3. HFL cells without TCSF; Lane 4. HFL cells with TCSF (I ng/ml); Lane 5. medium alone incubated with TCSF (I /ig/ml). Arrows, 66-kDa proform (above) and to be fully established. 59-kDa active (below) gelatinase bands, respectively. Although TCSF stimulated MMP-2 production, it did not appear to have any effect on the 92-kDa form of gelatinase (MMP-9) activity not MMP-3 (26), whereas transforming growth factor ÃŸstimulates (Fig. 4). On the other hand, phorbol ester stimulated MMP-9 but not MMP-2 and inhibits MMP-1 production in both fibroblasts and tumor MMP-2 production (data not shown), as has been observed previously cells (13, 35). To the best of our knowledge, the work described here by others (30). These results indicate that the mechanism of stimula is the first report of stimulation of MMP-1, MMP-2, and MMP-3 tion of these MMPs by TCSF is different from that by phorbol ester. production at both the mRNA and protein levels by a single biological Regulation of Matrix Degradation by TCSF. Stimulation of both MMP-1 and MMP-3 production by a variety of reagents has been factor, TCSF. TCSF exhibits the potential to regulate degradation of both the observed previously. For example, phorbol esters (10), cytochalasin B stromal and basement membrane compartments of the extracellular (10), interleukin 1 (31), heat shock (32), and some growth factors (33, milieu since the combination of MMP-1, MMP-2, and MMP-3 is 34) have been shown to stimulate MMP-1 and MMP-3 production in capable of degrading the collagenous and noncollagenous components fibroblasts. Concanavalin A treatment of fibroblasts, however, gives rise to elevated levels of MMP-1, MMP-2, and MMP-7 (PUMP-1 ) but of these two compartments. This is especially significant since the stimulatory agent, TCSF, is a biological factor associated with tumor cells in vitro (7) and in vivo0 but not with fibroblasts or leukocytes Table I Quantilalion of Northern blots and ELISA (7).6 Although tumor cells themselves are clearly capable of produc A. Quantitation of Northern blot data by densitometry: Autoradiograms were analyzed using the Biolmage Model 110S-2D system (Millipore. Bedford, MAI. Whole band ing MMP-1 (36, 37), MMP-2 (38), and MMP-3 (39), the interaction analysis software was used to define the boundaries of individual bands and to determine of tumor cells with fibroblasts may amplify MMP production in the the integrated absorbance of each band. Values are expressed as a ratio of the integrated absorbance of the test band with that of the corresponding glyceraldehyde-3-phosphate region of the tumor-stroma interface to the point where the tissue dehydrogenase band. The results were confirmed in two separate experiments, n.d., not inhibitors of MMPs no longer balance their activity. Investigators determined. from several laboratories have shown by in situ hybridization that the B. Quantitation of MMPs by ELISA: Immunological reactivities of MMP-1, MMP-2. and MMP-3 were assayed using a sandwich assay (see "Materials and Methods") and are strongest signal for mRNA to MMP-1 (40) and MMP-2 (41, 42), expressed as ng/ml enzyme. within a variety of malignant human cancers, is in fibroblasts at the Numbers in parentheses, extent of stimulation obtained in the presence of 1 ng/ml tumor-stroma interface rather than in the tumor cells themselves. This TCSF; -. +, cells incubated without and with TCSF. evidence, combined with the low levels of MMP-1 and MMP-2 found MMP-1 MMP-2 MMP-3 in normal fibroblasts (40, 41), strongly suggests that peritumoral Cell type fibroblasts are induced to produce higher levels of these enzymes, A.blotsHFCCD-18HFLB. Northern presumably by tumor cell-derived TCSF. It is postulated then that TCSF is an important regulator of tissue destruction during tumor cell (13)0.03(1.9) invasion through extracellular matrices. (4.7)0.50.8ND1.5(3.0)0.6 (0.0)0.060.02ND0.8 ELISA0.70.20.061.8(2.5)0.7(3.5)0.3 CCD-18 30 95 (3.2) 625 788 (1.3) 35 212 (6.0) 6 K. Muraoka. K. Nabeshima. T. Murayama. M. Koono. and C. Biswas, unpublished HFL 129 1292 (10) 2658 4055 (1.6) 31 58 (1.8) observations. 3157

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Hiroaki Kataoka, Rosana DeCastro, Stanley Zucker, et al.

Cancer Res 1993;53:3154-3158.

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