Vol. 178, No. 2, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS July 31, 1991 Pages 453-459

Temperature-Sensitive Synthesis of A Metalloproteinase in

tsllO-MSV-M-Transformed NRK Cells

J. C. Chanl , M. Scanlon, H.Z. Zhang, and J. L. Murray III

University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

Received June 5, 1991

SUMMARY: Previously, we reported that transformation associated protein (TAP) was over- expressed in the 6m2 line, but not in theii normal counterparts (1,2). 6m2 is a culture of NRK cells transformed by the ts-110 mutant of MSV-M. The synthesis of TAP and the expression of transformation properties in the 6m2 cells are all temperature-sensitive ( 2; 3; 4). TAP is secreted as two polypeptides of 64 kD and 68 kD (P64 and P68) (2). Experiments were carried out to determine whether any metalloproteinase (MP) activity was associated with TAP. Results of zymograms indicated that the two forms of purified TAP (P64 and P68) had MP activity, using gelatin or collagen type IV as substrates. Serum-free medium (SFM) of 6m2 cells incubated at 330C also showed two bands of MP activity, while the corresponding SFM from 6m2 cells at 390C lacked such MP activity, indicating that the synthesis of MP was temperature-sensitive. The association of MP activity with the P64 and P68 bands of TAP (purified or in SFM) was confiied by simultaneous Western blot analysis, which showed the reactivity of the two MP bands with monoclonal or polyclonal antibodies to TAP. Accordingly, what we previously designated as TAP is apparently one form of MP, which are known to be involved in tumor cell metastasis. 0 1991 Academic Press, Inc.

Utilizing monoclonal antibodies, we previously detected TAP in four different cultures of

rat cells transformed by Moloney-murine sarcoma virus (MSV-M) (l- 4). These cultures include:

the temperature-sensitive (ts) mutant MSV-transformed- NRK cells @x12; 5). MSV-M-induced rat

bone tumor (RBT) cells, MSV-M-transformed rat fibroblast cells (78AlWRC) (1) and in 54-5A4

cell line (a revertant of the 6m2 cells; 3). TAP was not detected in the normal counter parts of these

transformed cultures. TAP differs from P58gag and P37mos in terms of molecular weight,

antigenic determinants, and functions (2). TAP also differs in terms of molecular weight, biological activities and antigenic determinants from the TGF (TGF-a and TGF-R) that are also produced by

MSV-transformed mouse and rat cells (6,7). Using the 6m2 cell line, we found that the synthesis

of both TAP and the v-mos gene product (a se&e/ threonine kinase) was temperature-sensitive and

1 To whom correspondence and reprint requests should be addressed at the Department of Tumor Biology, Box 79, The University of Texas M.D. AndersonCancer Center, 1515 Holcombe Blvd., Houston, TX 77030.

Abbreviations: MSV-M, Moloney murine sarcoma virus; NRK, normal rat kidney cells; RBT, MSV-M-induced rat bone tumor cells; TAP, transformation- associated protein; TGF, transforming growth factors; SPP, secreted phosphoprotein; IP, immuno-precipitation; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide ge&electrophoresis; MAb, monoclonal antibody; SFM, serum -free medium; MEM, minimum essential medium; MP, metalloproteinase;-Mr, molecular weight.

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Vol. 178, No. 2, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

their synthesis closely accompanied the expression of transformed phenotypes by the 6m2 cells,

which is also temperature-sensitive (2). Therefore, TAP represents an over-expressed factor in

MSV-M-transformed rat cells and potentially plays an important role in cellular transformation.

More recently, we showed that TAP is glycosylated (4) and is weakly-phosphorylated in vitro (8).

TAP also differs significantly from SPP which are also found within the same cells in terms of

temperature-sensitivity in synthesis, antigenic determinants, methionine-content, and cellular origin

(8).

Although TAP had been purified by MAb-affinity column chromatography (2), the

functions of TAP have remained unknown. To understand better the functions of TAP, we

undertook two approaches. First, we have undertaken the cloning and the sequencing of the

TAP gene. This approach is now underway in our laboratory, and initial nucelotide sequencing data

indicates certain degree of homology with human and rat metalloproteinases. Second, on the basis

of some reports that the expression of metalloproteinases is induced by certain oncogenes or growth

factors in transformed cells (9), we investigated the possibility that TAP might have proteinase

activities and found that indeed TAP behaves very much like a form of MP. In this communication,

we show by zymogram and Western blot analysis that MP activities are indeed associated with what

we previously designated as TAP. We also report that the synthesis of MP in 6m2 cells was

temperature-sensitive.

MATERIALS AND METHODS

Cells. The 6m2 cell clone was provided by Dr. Donald Blair, National Cancer Institute, Bethesda, Maryland. The isolation and the properties of 6m2 have been described (5). Dulbecco’s minimum essential medium (MEM) plus 10% inactivated fetal bovine serum (Fl3S) was used as a growth medium for the propagation of the 6m2 cells. Serum -free medium (SFM) ( serum-free Iscove’s medium supplemented by 5 pglml insulin and 5 pg/rnl transferrin) was used to collect secreted TAP from 6m2 cells for analysis of MP activity and for Western blots.

Monoclonal Antibody MC. MC (IgGl isotype) was produced by a hybridoma clone MC that was generated by us (1). Balb/c mouse ascites fluid containing MC were purified by column chromatography using Protein A agarose as described (2).

Rabbit (R) anti-TAP. A New Zealand white rabbit was immunized by a series of injections with extracellular TAP purified from the extracellular serum-free medium of 6m2 cells (33oC).Two different preparations represent bleeding from the same rabbit on two separate occasions after boostering.

Purification of TAP by affinity chromatography. Confluent cultures of 6m2 cells were propagated at 33OC in where McCoy’s 5A Supplemented with 5% FBS for 5 days. 750 ml of the cell-free culture medium was mixed with 3.0 ml affinity gel-HAA (Hydrazide AvidGel Ax, BioProbe International), coupled with MAb MC (2) and shaken overnight at 4°C. The affinity gel was collected in a 1.5 cm (diameter) chromatography column and washed with 8 column volumes of storage buffer (0.025M Tris base, 0.15M NaCl, 0.05% sodium azide, pH 7.4), ‘TAP’ was eluted from the column with elution buffer (0.15M NaCl, OJM acetate, pH 4.0). The eluate was monitored for UV absorbance and the peak fractions were pooled. Immediately after elution, the eluate was brought to pH 8.0 with 1.0 M Tris and was either stored at -70°C until use or immediately processed by electro-elution. The purity was confiied by Coomassie blue staining as having two bands immediately after purification (not shown; 2).

Zymogram. Assay for MP activities was carried out as described (18). Briefly, a gel slab containing 9% acrylamide, 0.1% SDS, and 0.10% copolymer&d gelatin was cast. Samples (not heated before running) were mixed with equal parts of 2X sample buffer (0.125 M Tris, pH

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6.8; 4% SDS, 15% glycerol, 0.01% bromphenol blue) and applied to each lane. Electrophoresis was performed at 4°C at a constant current of 30 mA. Following electrophoresis and processing, the gel was fixed and stained for 1 hr at room temperature using 0.1% amid0 black in a mixture of acetic acid:methanol:water (1:3:6) and then destained in the same mixture without amid0 black. The following substrates were used- gelatin (bovine skin,Sigma), and type IV collagen (human placenta, Sigma).

Western blot analysis. Purified TAP and SFM of 6m2 cells at 330C and 390C (not heated) were electrophoresed on a 9% acrylamide gel (non-reducing). After electrophoresis, the gel was blotted overnight at 4°C 3OV, onto a nitrocellulose membrane (pore size, 0.45 mm in diameter). After blotting, the membrane was blocked with 4% non-fat dry milk for 1 hr at room temperature. The membrane was then incubated with either MAb MC ( 1 ug/ml) or rabbit anti-TAP serum (1:200 dilution), in 4% nonfat dry milk for 2 hrs at room temperature. After washing, the membrane was reacted with peroxidase labeled goat anti-mouse IgG (1: l,OOO), in 4% nonfat dry milk for 1 hr at room temperature. Reactivity was detected with the chromogenic substrate TMB (KPL507601) plus enhancer.

RESULTS AND DISCUSSION

Prior to performing certain experiments, the cloned 6m2 cells were examined for their

temperature-sensitivity. It was confirmed that incubation of these cells at the permissive 33oC led to

morphological alteration (Fig lA), showing refractile round cells. Incubation of the same cells at

390C resulted in the reversion to normal morphology of flat cells (Fig. 1B).

In addition to MC, rabbit anti-TAP sera were also prepared. Prior to their use in experiments, both

the MC and the rabbit antibodies were characterized by Western blot analysis. It was demonstrated

that the two extracellular forms of TAP having respective Mr of 64- and 68 kD (P64 and P68)

reacted similarly with both [1251]-labeled-MC and two different preparations of [125yl-labeled

rabbit anti-TAP antibodies (Fig. 2). Having thus affirmed the reactivity of both MC and rabbit anti-

TAP, these antibodies were used in later Western blot analysis.

The possibility that TAP may carry MP activities was investigated using zymogram and

Western blot analysis. When gelatin was used as a substrate in the zymogram, MP activities were

Fig. 1. Temperature-sensitive alteration in the morphology of 6m2 cells. The morphology of 6m2 cells at 33oC (A) or 39oC (B).

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0 2

Fig. 2.

Fig. 3.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-68 kD

-64 kD

C

-68 KD

-64 KD

0 3

Temperature-sensitive synthesis of two extracellular forms of TAP by 6m2 cells. 6m2 cells were incubated at 33oC for 18 hr. tbe cell free medium was concentrated ten-fold and was electrophoresed on SDS-PAGE as described (2) and reacted with either [12$labeled-MC (preparation BlO) (lane a) or two different preparations of [125yl-labeled rabbit anti-TAP antibodies (lanes b and c). Two extracellular bands (P64 and P68) of TAP were detected. In contrast, sister 6m2 cell culture incubated at 39oc did not produce these two bands (results not shown; see 2,3).

Zymogram (panel A) showing digestion of gelatin bv “TAP”and Western blots (nanels B and Cjidentifying two secrea b&ds of TAP (64- and 68&D), using MAb MC‘& rabbit anti-TAP serum. In panel A. a eel slab containine 9% acrvlamide. 0.1% SDS. and 0.10% copolymerized gelat& was cast: Samples (not heited before nmning) were el&rophoresed. Following electrophoresis and processing, the gel was fixed and stained for 1 br at room temperature using 0.1% amido black in a mixture of acetic acid:metbanol:water (1:3:6) and then destained in the same mixture without amid0 black. In panel B, samples (not heated) were electrophoresed on a 9% acrylamide gel (non-reducingj. After elect?oph&sis, the gel was blotted. The membrane was then incubated with MAb MC. In nanel C. another membrane was incubated with rabbit anti-TAP serum and processed as in panel B. In all three panels, purified TAP (lane 3); 10X SFM of 6m2 cells (33oC) (lane 2) and 10X SFM of 6m2 cells (39oc) (lane 1) were analyzed.

found. For example, in Fig 3A; purified TAP exhibited two bands of Mp activities (64- and 68kD,

corresponding to P64 and P68) (lane 3).The SFM of 6m2 (33oC ) showed one intense band (68 kD)

and one faint band (64 kD) of MP (lane 2); four higher Mr bands of MF were visible, which may

represent aggregates of the same MP. The SFM of 6m2 cells (39oc) did not show any ?vlP activity

in the corresponding area of 64-68 m (lane 1, Fig. 3A), indicating the lack of production by 6m2

cells at 39°C. Similar results wete obtained wheat human type IV collagen was used as a substrate in

zymogram (results not shown).

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A simultaneous Western blot analysis was carried out to confii the identity of the MP

bands and the results are shown in panels B and C of Fig. 3. The purified TAP (64- and 68 kD)

reacted with MC. Light reactivity was observed in areas at the top and below the 64/68 kD area.

The top reactive bands may represent aggregates while the lower Mr. band may represent

degradation product (lane 3, Fig. 3B).

Two MC-reactive bands (64- and 68 kTI) were also found in SFM of 6m2 cells (33’C).

(Additional bands were also found at the top of the gel (Again, possibly due to the aggregation of

64-and 68 kD) (lane 2, Fig. 3B); No bands were found in the SFM of 6m2 cells (39”(Z), indicating

that the 64- and 68 kD proteins were not synthesized. (lane 1, Fig. 3B). When rabbit anti-TAP was

used in Western blot analysis, the same results (Pig. 3C) was also obtained.

Both the zymogram and the Western blot analysis also showed that the synthesis of TAP (or

ML’) in 6m2 cells was temperature-sensitive (Fig. 3A and 3B). At 33”C, ME’ was found to be

produced and reacted with MC in Western blot analysis. No MP activity was produced by 6m2 cells

at 39°C and therefore was not detected in terms of MP activity or by Western blot.

We chose to focus our further characterization of TAP in the 6m2 cell system because the

6m2 cells are temperature-sensitive in terms of their expression of various transformation

properties, such as loss of topoinhibition, altered cytoskeleton, ability to form colonies in agar-

medium, autonomous growth in serum-poor media, etc (2,5, 12). The splicing mechanism of the v-

mos mRNA in 6m2 cells is also temperature-sensitive (13,14) and the cells revert to normal

phenotypes at non-permissive temperatures of 39oC or higher. Within the 6m2 cells resides the

partial MSV genome (gag and v-mos) of the tsll0 mutant of MSV-M that encodes respectively two

viral proteins: P58gag and P37mos (15). The v-mos-gene product (P37mos) has been identified as

having serine/threonine kinase activities and is believed to be involved in cellular transformation

(16). Recent results also indicate the involvement of v-mos in the regulation of cell cycle by

phosphorylation of cyclin B2 subunit of the maturation-promoting factor (17).

Most interestingly, we found that within the 6m2 cells, the synthesis of TAP, the synthesis of v-mos

gene product (P37mos), and the expression of transformation properties were all temperature-

sensitive, implicating a strong possibility that TAP and the v-mos-gene product might both be

involved in cellular transformation (3). When the temperature was shifted from 39oC to 33OC, the

reappearance of v-mos gene product in 6m2 cells appeared to precede that of TAP, suggesting that

v-mos gene product might activate the synthesis of TAP (3).

Although we had previously isolated purified TAP by affinity column chromatography, the

biological functions of TAP had remained obscure (2). Since the production of MP by transformed

cells can be induced by certain oncogenes or growth factors ( 9), we initiated studies to look for the

synthesis of MP by 6m2 cells .

In gelatin zymogram, affinity chromatography-puritled TAP exhibited MP activities

corresponding to P64 and P68 of TAP (Fig. 3A). These proteins, as measured by MP activity,

were synthesized by 6m2 cells at 33°C but not at 39OC. MP activities were also found in the SFM of

6m2 cells 33°C , but not at SFM of 39°C. These two bands (P64 and P68) were identified as two

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forms of TAP by their reactivity in Western blot analysis, using polyclonal and monoclonal anti-

TAP antibodies (Fig. 3B). The demonstration of the temperature-sensitive synthesis of MP in the

6m2 system is new and suggests that this system will provide unique opportunities to evaluate the

interplays of oncogene and growth factors, in the induction of metalloproteinases, as well as in

tumor cell proliferation and tumor metastasis. In our initial studies, gelatin and human type IV

collagen were used as substrates for MP in the zymograms. Initial results indicated that both gelatin

and type IV collagen (not shown) were degraded by “TAP”. Testing for a variety of other substrates

has been planned.

Previously, Spinucci et al (19) found that the transformation of NM-3T3 mouse fibroblasts

by the c-Ha-ras oncogene leads to highly invasive and metastatic tumor cells that produce high levels

of type IV collagenase (one form of Mp, 18). In gelatin zymogram, the type IV collagenase was

resolved into two-gelatin-degrading polypeptides of 67- and 62 kD bands. Whether the 64- and 68

kD proteins of TAP resolved in our gelatin-zymogram are related to the two mouse MP reported by

Spinucci et al., (18) remains to be investigated.

It is now well established that MI’ (such as stromelysin, collagenase, gelatinase) play a very

important role in the invasion and metastasis of the cancer cells ( 19-23) by degradation of the

extracellular matrix.

While this work was in progress, we have recently cloned the TAP-gene and determined

the nucleotide sequences of one of the TAP-cDNA clones. A search through the GenBank revealed

that this TAP-cDNA clone is highly related to that of rat transin gene family, which encodes for a rat

metalloproteinase. Thus, the association of MP activity with TAP is confiied by results of the

molecular cloning experiments as well. Results of the molecular cloning and analyses are being

prepared and will be submitted elsewhere as a separate manuscript. However, it is exciting to learn

that the synthesis of MP (previously designated as TAP by us) in 6m2 cells is also temperature-

sensitive. Whether MP synthesis is activated by the v-mos gene product, P37mos, and if so, how,

also remains to be determined. Furthermore, it would be important to investigate what other

functions in cellular transformation, if any, besides tumor cell invasion and metastasis, are provided

by the MP.

ACKNOWLEDGMENTS

This work was supported by the Council for Tobacco Research-USA, Inc., Grant 2264M

(to JCC) and NC1 Contract C M #97910 (to JLM).

REFERENCES

1. Chan, J.C., Keck, M.E., and Li, W. (1986) B&hem. Biophys. Res. Commun. 134,1223- 1230.

2. Li, W.J., Chi, K., Gallick, G.E., and Chart, J.C.(1987a) Virology 156, 91-100. 3. Li, W.J., Bowen, J.M., and Chan, J.C. (1987b) Intervirology 2850-56. 4. Li, W.J., and Chan, J.C. (1989) Cancer Res.49, 1746-1751. 5. Blair, D.G., Hull, M.A., and Finch, E.A. (1979) Virology, 95,303-316. 6. Todaro, G.J. and DeLarco, J.E. (1980) Control Mechanisms in Animal Cells, pp. 223-

243.Raven Press New York.

458

Vol. 178, No. 2, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7. Anzano, M.A., Roberts, A.B., Smith, J.M., Sporn, M.B., and Larco, J.E. (1983) Proc. Natl. Acad. Sci. USA, 80,6264-6268.

8. Chan, J.C., Scanlon, M., Denhardt, D.T., Singh, K., Mukherjee, B.B., Farach-Carson, M.C., and Butler, W.T. (1990) Int. J. Cancer, 46,864-870.

9. Matrisian, L.M., Leroy, P., Ruhlmann, C., Gesnel, M.-C., and Breathnach, R. (1986)

10.

11.

12.

13.

14.

15. 16. 17.

18.

19.

2 22:

23.

Mol.Cell. Biol. 6, i679- 1686. Breathnach, R., Matrisian L.M., Gesnel, M.-C. Staub, A., and Leroy, P. (1987) Nucleic

Acids Res. 151139-l 150. Zhang, H-Z., Grdonex, N.G., Bats&is, J.G., and Chan, J.C. (1989) Cancer Res. 49,5766-

5773. Brown, R.L., Horn, J.P., Wible, L., Arlinghaus, R.B., and rinkley, B.R. (1981) Proc. Natl.

Acad. Sci. USA 78,5593-5597 Nash, M., Brown, N., Wong, J., Arlinghaus, R.B., and Murphy, Jr., E.C. (1984) J. Virol.

50,478-488. de Mars, M., Sterner, D.A., Chiocca, SM., Biggart, N.W., and Murphy, Jr. E.C. (1990) J.

Virol. 64, 1421-1446. Maxwell, S.A. and Arlinghaus, R.B. (1985) Virology 143,321-333. Kloetzer, W.S., Maxwell, S.A., and Arlinghaus, R.B. (1984) Virology 138,143-155. Roy, L.M., Singh, B., Gautier, J., Arlinghaus, R.B., Nordeen, S.K., and Maller, J.L. (1990)

Cell 61,825-831. Spinucci, C., Zucker, S., Wieman, J.M., Lysik, R.M., Imhof, B., Ramamurthy, N., Liotta,

and L.A., Nagase, H. (1988) J. Natl. Can. Inst. 80,1416-1420. Nicolson, G.L. (1982) B&hem. Biophys. Acta 695,113-174. Liotta, L.A., Rao, C.N., and Barsky, S.H. (1983) Lab. Invest. 49,636~649 . Chin, J.R., Murphy G., and Werb, Z. J. (1985) J. Biol. Chem. 260, 12367-12376. Whitham, S.E., Murphy, G., Angel, P., Rahmsdorf, J., Smith, B.J., Lyons, A., Harris, T.J.,

Reynolds, J.J., Herrlich, P., and Dockerty, A.J.P. (1986) Biochem. J. 240,913-916. Khokha, R. and D. T. Denhardt. (1989) Invasion Metastasis 9,391-405.

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