Btochmuca et BiopiT~ica Acta, 1073 (1991) 107-113 .'~ 1991 ElsevierScience PublishersB.V. (Biomedical Divismn)03134-4165/91/$0350 ADONIS 030441659i0~155U

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BBAGEN 23427

The identification, purification and characterisation of an m. :.rotor of collagenase (20K) produced by neoplastic epithelial cells •

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J o h n M. W h i t e l o c k l, R o b e r t L. O ' G r a d y t a n d J o h n R. G i b b i n s 2 i Department of Cellular Pathology'. Unwersaty of Technology'. Sydney. Gore thll ( 4 itst: altaJ and : Department of Pathvlogv. Unwer~i O" of S)'dne~'. Cumperdown ¢..!tt~trahto

IRecelved 27 Match 1990) Key words: Collagenaseinhibitor: Tumourinvasion: M~talloprotemase:Zymography;(Epithelial cell1 A rat carcinoma cell line ( T 2 / H T ) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of eollagenolytie activity being detected in conditioned medium. Using reverse zymography, the Af, of t.he inhibitor was found to be 20000 which suggests that it may he the rat homologue of inhibi*or of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelminolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from coilagenase by filtration througl', a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCI and was eluted with 300 mM NaCI. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat. Introduction The turnover of collagen is important in both biological and pathological situations. Collagenase, a metalloproteinase wlfich is central to the" catabolism of collagen, is the only known mammalian enzyme which cleaves the triple helical domain of native collagen at physiological pH and temperature. Once this specific cleavage has occurred, the collagen molecule is denatured which renders it susceptible to further digestion by gelatinolytic enzymes. The ratio of active collagenase to its endogenous in,hibitors is of great importance, as this determines the extent of collagenolysis that occurs in tissues. Tissue inhibitor of metalloproteinases (TIMP), a giycoprntein of M, 28000 [1], is thought to play a major

Abbreviations: APMA. 4-aminophenylmercuric acetate: DME. Dulbecco's modified Eagle's medium; DMSO, dimettiyl sulphoxide; ECM. extracellular matrix; FI2/DME, l:l mixture of Ham'~ FI2 and Dulbecco'smodified Eagle's medium; Hepes, N-2-hydroxyethylpiperazJne+N'-2-cthanesulphonicacid; IMP, inhibitor of metalloproteinascs; SDS, sodium dodecyl sulphate; TIMP, ti~ue inhibitor of metalloproteinases; TPA, 12-O-t,-.tradecanoy113-acetate. Correspondence: R.L. O'Grady, Depai:.,'aem of Cellular Pathology, Umversity of Technology, Sydney, WestbourneSt., Gore Hill. NSW 2065, Australia.

ro!e in the extravascular control of collagenase and other metalloproteina~s, and has been demonstrated in the conditioned medium of cultured tumour cells [2]. Other inhibitors with similar activities to TIMP have been identified, based upon the fact that they had lower molecular weights than TIMP [3-5]. Members of this group have been referred to by different names, depending upon their source. They have been termed inhibitors of metalloproteinases (IMPs) with IMP1 and IMP2 exhibiting M r values of 22000 and 19000, respectively [3], and more recently IMP3 with a M r of 16500 [6]. Alternatively, the human homologue of rabbit IMP2 was named TIMP-2, because its amino acid sequence was 41~ homologous with that of kuatan TIMP [4]. A similar inhibitor has been isolated from bovine cartilage and was referred to as metal!oproteinase inhibitor [MI] [5]. In this publication, all of these inhibitors will be referred to as IMPs, as distinct from the larger TIMP. It has been suggested that the IMPs and TIMP are related molecules, and are members of a family of matrix metalloproteinase inhibitors. However, the IMPs are not simply partially degraded or non-glycosylated versioas of the TIMP molecule [3-5]. Even though both bovine IMP and bovine T1MP can inhibit collagenase, gelatinase and type IV collagenase [5], they have been shown to be distinct molecules immunologically, as antibodies raised against human TIMP did not recog-

108 nise human IMP [7], and antibodies against bovine TIMP did not recognise bovine IMP [51. The amino acid sequence of human IMP shared 90% homology to the inhibitor derived from bovine cartilage, with which it shared the characteristic amino-terminal cysteineserine-cysteine-serine sequence [8]. These data suggest that IMP is the same inhibitor which was purified previously from bovine cartilage [9], and later sequenced [10]. It was shown recently that human melanoma cells [4] and rat carcinoma cells [11] secreted IMP. The IMP from the human source was isolated as complexes with type IV collagenase/gelatinase [4]. These were the first demonstrations of an inhibitor, of M r approx. 20000, which was derived from neoplastic cells. Previous to this report, inhibitors of M r 20000 were isolated only from non-neoplastic sources such as cartilage [9,10], body fluids [12], endothelial cells [3,5] and fibroblasts [7,8]. Results presented in this paper demonstrate that rat neoplastic epithelial cells are capable of secreting collagenase, TIMP and IMP simultaneously, suggesting that all three proteins may play a role in the degradation of connective tissues associated with tumour invasion. Furthermore, it may be suggested that turnout cells, in vivo. are capable of determining the extent of collagen catabolism that occurs in the connective tissue adjacent to the tumour mass. Materials and Methods

Cell culture T 2299F/H7 ( T 2 / H 7 ) was a clonal cell line, derived from a tumour which arose following the subcutaneous injection of A5P/B10 cells which had been co-transfected with BC1 D N A and pSV2neo. These cells form highly invasive and metastatic tumours, as distinct from the parent line (A5P/BI0), in syngeneic DA rats (Dl. J. Gibbins; personal communication). These cells were maintained in a medium consisting of a 1 : 1 mixture of Joklick's (Gibco) and Ham's F12 (Gibco) supplemented with 10% calf serum (Flow) O o k / F 1 2 / C S ) . The ceils were depleted of serum by washing twice with sterile PBS, and then cultured in a i : 1 mixture of DME (Gibco) and Ham's FI2 ( D M E / Fi2) which was buffered using Hepes (Sigma). To maintain the viability of the cells, every second feed consisted ~f J o k / F 1 2 / C S . A stock solution of TPA (1 m g / m l in DMSO)(Sigma) was added tc the cultures to provide a final concentration of 5 • 10 -7 M

Assays Collagenase. This was performed as described previou,,~ly [13]. Trypsin (25 p.g/ml) (Sigma) was used to activate the proenzyme. The relative collagenolytic activity of collagenase ( U / m l ) was defined as the amount of collagen degraded (p.g) per min per ml at 35°C.

Inhibitor. Thc inhibit,'~r assay was developed as an extension of the collagenase assay. Serial dilutions of active collagenase, purified from neoplastic epithelial cells [141, were incubr.ted with collagen for 2 h at 35°C. The collager.olytic activi:y of the diluted sample of enzyme, which lysed 50% of the collagen in the well, was calculated. This dilution was then used in each collagen-containing well, for the detection of inhibitory activity of various samples in 2h assays at 35°C. Following the incubation, the wells were stained with Coomassie blue R250 (0.25%) (Bio-Rad) in 50% methanol, 10% acetic acid for 25 min, and the A~o0 was read using a Multiskan spectrophotometer. The inhibitory activity present in each sample was quantitated by comparing the absorbance of each well. which contained the unknown sample, with the absorbance of an enzyme-only control (for which the relative collagenolytic activity had been calculated previously). 1 unit of inhibitor was defined as the amount of inhibitor which inhibited 2 units of collagenase by 50%. SDS gel electrophoresis and gelatin zymography SDS gel electrophoresis was performed using discontinuous gels [15]. Gelatin zymography was performed essentially as described previously [16]. Gelatin (Sigma) was incorporated into the polyacrylamide matrix at a final concentration of 1 m g / m l . Samples for zymography were incubated in non-reducing sample buffer at room temperature. After electrophoresis, the gel was washed in 2.5% Triton X-100 (Bio-Rad) for 1 h with gentle rocking, placed into 50 m M Tris, 75 m M NaCI, 10 m M CaCI 2, 0.02% NaN3 (pH 7.5) and incubated for 16 h at 35°C. Gels were stained with 0.25% Coomassie blue R250 in 50% methanol, 10% acetic acid and destained using 50% methanol, 10% acetic acid.

Reverse zymography Reverse zymography was performed as described in Ref. 3. Gelatin was incorporated into polyacrylamide gels at a final concentration of 1 m g / m l . Samples for reverse zymography were incubated in non-reducino sample buffer at room temperature. After eleetrophoresis, the gel was removed and incubated, with gentle rocking, in an aqueous solution of 2.5% Triton X-100 for 30 rain at room temperature. After a second 30 min wash in 2.5% Triton X-100, the gel was removed and incubated in purified collagenase (10 # g / m l ) at 35"C. The time the gels were incubated in collagenase depended upon both the size and thickness of the gels. Gels were stained with 0.25% Coomassie blue R250 in 50% methanol, 10% acetic acid and destained using 50% methanol, 10% acetic acid.

Purification of T2 / H7 IMP Heparin-Sepharose chromatography. This was performed essentially as described for the purification of

109 collagenase from rat neoplastic epithelial cells [141, except that 0.05% Brij-35 was included in all of the buffers. Samples were applied to the column in 50 mM Tris, 75 mM NaCI, 10 mM CaCI,, 0.02% NAN3, 0.05% Brij-35 (Sigma) (pH 7.5). Proteins were eluted from the column by increasing, stepwise, the NaCI concentration in the starting buffer to 300 mM NaCI, 800 mM NaCI and 1 M NaCI. Con A-Sepharose chromatography• The column (10 ml) was equilibrated with 50 mM Tris, 75 mM NaCI. 10 mM CaCI 2, 0.02% NaN.t, 0.05% Brij-35 (pH 7.5). Samples were applied in this buffer, and the bound protein was ehited with 0.1 M a-D-methylmannoside. Gelatin-Sepharose chromatography. This was performed according to the method of Moore et al. [8] with slight modifications. The column (10 ml) was equilibrated with 50 mM Tris, 75 mM NaCI, 10 mM CaCI,, 0.02% NaN 3, 0.05% Brij-35 (pH 7.5). Samples were applied, and the bound protein was eluted sequentially with 0.2 M acetic acid followed by 7.5% DMSO in the same buffer. The pH of both ehiates was adjusted to 7.0 by the addition of 12.5 M NaOH.

Characterisation of the inhibitor Inhibitor was incubated with trypsin (25 ~tg/ml in 50 mM Tris, 10 mM CaCI 2, 75 mM NaCI, 0.02% NAN,, 0.05% Brij-35 (pH 7.5)(TCN) at 35°C. After 10,15 and 30 rain, an equal volume of soy-bean trypsin inhibitor (Sigma) (125 tLg/ml in TCN) was added. Controls received an equal volume of TCN instead of both trypsin and soy-bean trypsin inhibitor. After trypsin treatment, residual collagenase inhibitory activity was measured in a 2 h assay. Inhibitor was incubated with APMA (Sigma) (1 mM) for 15 and 30 rain at 350C. Controls received an equal volume of 10 mM NaOH. Residual inhibitory activity was measured in a 2 h assay, Inhibitor was heated at 95°C for a maximum of 60 min and assayed for activity in a 2 h assay. Results

Identification of the inhibitor Medium conditioned by T 2 / H 7 cells contained typically 0.3-0.4 units/mi of collagenase activity. When these cells were incubated with 5 . 1 0 - 7 M 1?-O-tetradecanoylphorbol 13-acetate (TPA), the relative coilagenolyric activity decreased to 0.05 units/ml. To investigate whether or not TPA had induced the synthesis a n d / o r secretion of an inhibitor, conditioned medium (TPAstimulated) was fractionated on an Amicon YM30 membrane. The volume of the retentate was adjusted to the original volume of medium, with 50 mM Tris, 75 mM NaCI, I0 mM CaCI 2, 0.02% NaN3 (pH 7.5), and assayed for collagenolytic activity. The relative collagenolytic activity present in the retentate was 0.2

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. . . . x?. . ~.~ .:~: , ,~ ,~ ,..~. ~,.. Fig. I. Inhibitoryacti'.ily of the YM30 filtrate. Conditioned medium from TPA-stlmulated cultures was fractionated on a YM30 membrane The filtrate was concentrated 10-fold on a YMI0 membrane and dial'.sed against 50 mM Tris 75 mM NaCI 10 mM CaCl:. 0.05% Brw35 and 002% Nz.N~(pH 7.5). Increasing,.'olumesof the filtrate ~'ere assayed, in trtplicate, for inhibitoryactivity in a 2 h a~say. •

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units/mL which was 4-times greater than that measured in the unfractionated conditioned medium in the presence of TPA. This suggested that the phorbol ester had induced the production of an inhibitor, and that it had been separated from the proenzyme by filtration. To support this, when an increasing volume of the YM30 filtrate was added to purified collagenase, a corresponding decrease in collagenase activity was demonstrated (Fig. 1). Reverse zymography was performed on the YM30 filtrate to elucidate the M r of the inhibitor. Using this technique, an inhibitory band of M~ 20000 was detected (Fig. 2).

Partial purification of the inhibitor Partial purification of the inhibitor was achieved using the following protocol. Step I. Conditioned medium (batch size of 1 litre) was fractionated on an Amicon YM30 membrane to separate the inhibitor from procollagenase. The enzyme was retained, whilst the inhibitor passed through in the filtrate. Inhibitory activity, per se, could not be detected in unfractionated medium due to the presence of proteinases, including collagenase, which were secreted concomitantly with the inhib'~tor. If T 2 / H 7 conditioned medium was concentrated over an Amicon YMI0 membrane and subjected to gelatin zymography, gelatinases with molecular weights of 95000 and 64000 were evident. The relative amounts of these enzymes did not appear to alter when the cells were incubated with TPA. If reverse zymography was performed on the YMI0 retentate (concentrated medium) an inhibitory, band at 20 000 was demonstrated (data not shown). Step 2. The volume of the YM30 filtrate was reduced to approx. 100 ml via further fractionation on an Amicon YMI0 membrane. This step concentrated the inhibitor present in the YM30 filtrate, The YM10 retentate (100 ml) was then dialysed extensively against 50 mM Tris,

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The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells.

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol ...
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