©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Synergistic Induction of Anchorage-independent Growth of NIH3T3 Mouse Fibroblasts by Cysteine Proteinase Inhibitors and a Tumor Promoter (*)

(Received for publication, December 11, 1995; and in revised form, January 24, 1996)

Takaki Hiwasa (§) Toshie Sawada Shigeru Sakiyama

From the Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2, Nitona-cho, Chuo-ku, Chiba 260, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously reported that Ras protein is a potent cysteine proteinase inhibitor. In order to examine whether the cysteine proteinase-inhibitory activity of Ras is involved in carcinogenesis, the effects of the following probes were investigated. Cystatin alpha is a cysteine proteinase-specific inhibitor and has some amino acid sequence homology with Ras. Ras has a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) at the carboxyl terminus, which is indispensable for the biological activity. Thus, cystatin alpha carrying a CAAX motif (cystatin alpha-CVLS) was examined. A v-Ha-Ras deletion mutant, RasDelta42-49, has undetectable GTP binding activity, yet it retains a similar protease inhibitory activity to that of wild-type v-Ras. These genes were inserted into a eukaryotic inducible expression vector and transfected into NIH3T3 cells. The expression was effectively induced by treatment with a glucocorticoid hormone, dexamethasone. The expression of cystatin alpha-CVLS or RasDelta42-49 alone induced neither transformation nor morphological changes. However, when their expression was induced in the presence of a tumor-promoting phorbol ester, a remarkable increase in the anchorage-independent growth was observed in cystatin alpha-CVLS- and RasDelta42-49-transfected clones. These results suggest that cysteine proteinase inhibitors and a tumor promoter synergistically transformed NIH3T3 cells. It is thus possible that the cysteine proteinase-inhibitory activity of Ras might play a key role in the early stage of carcinogenesis.


INTRODUCTION

A number of studies have been made on the action mechanism of ras oncogene product (Ras), and multiple proteins have been proposed as the effectors of Ras protein. These are rasGAP, protein kinase C, c-Raf-1, ralGDS, and phosphatidylinositol 3-kinase(1, 2, 3, 4, 5) . Although Ras associates with these proteins in a GTP-dependent manner, the direct effect of Ras remains to be proved. It has been suggested that Ras does not directly activate the kinase activity of Raf but translocates it from cytosol to membrane (6) . We have previously reported that Ras is a potent protease inhibitor(7, 8, 9) . The inhibitory activity of Ras is specific for cysteine proteinases such as cathepsins B and L. Consistently, the amino acid sequence of Ras is partly homologous to those of the cysteine proteinase inhibitor family designated ``cystatin superfamily''(7, 10) . If the cysteine proteinase-inhibitory activity of Ras is involved in the transformation, cystatins might have a similar transforming activity. The present results suggest that inhibition of cysteine proteinases is sufficient for the conversion from a normal cell to an initiated one.


EXPERIMENTAL PROCEDURES

Cell Culture

NIH3T3 mouse fibroblasts and the transfected clones were cultured in Dulbecco's modified medium supplemented with 5% calf serum.

GTP Binding Assay

Ras proteins (1 µg) were electrophoresed through SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane filter. The filter was then incubated with [alpha-P]GTP (5 µCi/ml) in buffer A (50 mM Tris-HCl (pH 7.5), 5 mM MgCl(2), 1 mM EGTA, and 0.3% Tween 20) for 30 min at room temperature according to the method of Lapetina and Reep(11) . The filter was washed extensively with buffer A and exposed on x-ray film.

Raf Binding Assay

-A cDNA fragment (1627 base pairs) of human c-raf-1 (12) obtained by digestion with PvuII and BglII was inserted into a SmaI site of a prokaryotic expression vector, pGEX-2T (Pharmacia, Uppsala). Glutathione S-transferase (GST) (^1)and GST-c-Raf fusion protein were produced by the transformed Escherichia coli treated with isopropyl-beta-D-thiogalactopyranoside for 3 h and were purified by affinity chromatography using glutathione-Sepharose 4B (Pharmacia).

Binding between Ras proteins and GST-c-Raf was investigated as described previously(13) . Purified GST or GST-c-Raf protein (1 µg) was first incubated at 4 °C for 30 min with glutathione-Sepharose in binding buffer (100 mM KCl, 6.33 mM MgCl(2), 20 mM Tris-HCl (pH 7.4), 1.0 mg of bovine serum albumin/ml, 25 µM ZnCl(2)), and the unbound proteins were removed by washing with binding buffer. Guanine nucleotides prebound to Ras were exchanged by incubation of Ras at 30 °C for 15 min in loading buffer (50 mM HEPES (pH 7.4), 5 mM EDTA, 1 mM dithiothreitol, 75 µg of bovine serum albumin/ml, and 1 mM GDP or GMP-PNP. GST or GST-c-Raf immobilized on glutathione-Sepharose was then incubated with Ras (1 µg) at 4 °C for 30 min in binding buffer and washed 4 times with wash buffer (100 mM KCl, 6.33 mM MgCl(2), 20 mM Tris-HCl (pH 7.4) and 0.25% Triton X-100). The bound materials were analyzed by Western blot using an anti-Ras monoclonal antibody, NCC-RAS-001(14) .

Construction of Plasmids

Rat cystatin alpha cDNA was provided by Drs. Nobuhiko Katunuma (Tokushima Bunri University) and Yoshimasa Ike (Mitsui Pharmaceutical Co. Ltd.)(15) . A gene for cystatin alpha bearing a CAAX motif (Tyr-Cys-Val-Leu-Ser) at the carboxyl terminus was also constructed as described previously (16) . This cystatin alpha variant was designated ``cystatin alpha-CVLS'' in the following part. v-Ha-ras gene (v-Ras(WT)) was provided by Dr. Berthe M. Willumsen (University Institute of Microbiology, Copenhagen). Two deletion mutants were constructed as described(17) . RasDelta43-45 and RasDelta42-49 have deletions in amino acids 43-45 and 42-49, respectively. Cystatin alpha, cystatin alpha-CVLS, v-Ras(WT), RasDelta43-45, and RasDelta42-49 DNAs were inserted into a SmaI site of an inducible eukaryotic expression vector, pMSG (Pharmacia). Using this vector, the inserted genes are effectively induced by dexamethasone (Dex).

Transfection

Cystatin alpha or cystatin alpha-CVLS DNA (10 µg) was co-transfected with neo (1 µg) into NIH3T3 cells (5 times 10^5 cells) using Lipofectin (Life Technologies, Inc.). Transfected clones were selected in the presence of G418 (400 µg/ml). NV, NY, NS, NR, NII, and NIII represent the transfectants of vector, cystatin alpha, cystatin alpha-CVLS, v-ras(WT), rasD42-29, and rasDelta43-45 DNAs, respectively.

Anchorage-independent Growth

Cell growth in soft agar medium was examined as described previously(18) . Five thousand cells were plated in soft agar medium, which contained 0.4% SeaPlaque-agarose, 10% calf serum, Dulbecco's modified Eagle's medium, and Dex (1 µM) or the solvent, dimethyl sulfoxide (0.1%). After culture at 37 °C for 3 weeks, colonies whose diameters were larger than 0.1 mm were scored. The colony-forming efficiency was calculated by dividing the colony number by the cell number plated.


RESULTS AND DISCUSSION

The cysteine proteinase-inhibitory activity of cystatin alpha was more potent than that of Ras(7, 19) . However, cystatin alpha possesses neither guanine nucleotide binding activity nor a CAAX motif. A CAAX motif at the carboxyl-terminal end of Ras is necessary not only for post-translational farnesylation but also for the biological activity(20) . Thus the biological activity of cystatin alpha carrying the CAAX motif (cystatin alpha-CVLS) was also investigated.

Other probes used were two deletion mutants of v-Ha-Ras, RasDelta43-45 and RasDelta42-49. The most conserved amino acid sequence among the cystatin superfamily is Gln-Val-Val(10) , which is also found in Ras at amino acid positions between 43 and 45(7) . These Ras proteins were bacterially expressed and purified. In spite of the deletion of the conserved amino acid sequence, RasDelta43-45 and RasDelta42-49 showed similar protease-inhibitory activities as that of v-Ras(WT)(17) . For example, the K(i) values of v-Ras(WT), RasDelta42-49, and RasDelta43-45 were 48, 102, and 40 nM, respectively, toward bovine cathepsin B and 14, 11, and 8 nM, respectively, toward papain. These results are consistent with the previous report that point mutants in the Gln-Val-Val region did not significantly affected the protease-inhibitory activity of cystatin A (21) . Thus, the Gln-Val-Val sequence might be important but not essential for the interaction with cysteine proteinases.

Guanine nucleotide binding activities of these two deletion mutants were also compared with that of v-Ras(WT). Fig. 1shows the results of GTP binding examined by the filter overlay assay using [alpha-P]GTP(11) . v-Ras(WT) showed a potent GTP binding activity while RasDelta42-49 and RasDelta43-45 showed no detectable GTP binding activity. The GTP binding activity was also examined by rapid filtration according to the method of Gibbs et al.(22) , and the results showed both RasDelta42-49 and RasDelta43-45 had less than 1% guanine nucleotide binding activity of that of v-Ras(WT)(17) .


Figure 1: GTP binding activity of v-Ras(WT), RasDelta43-45, and RasDelta42-49. GTP binding activity was investigated by the filter overlay method. v-Ras(WT) (lane 1), RasDelta43-45 (lane 2), and RasDelta42-49 (lane 3) were electrophoresed and blotted on a nitrocellulose filter, which was then incubated with [alpha-P]GTP and washed extensively. The autoradiogram is shown. An arrow indicates the position of v-Ras(WT).



We have also investigated the in vitro association of Ras and c-Raf-1. c-Ha-Ras bound to GST-c-Raf only in the presence of GMP-PNP, a non-hydrolyzable GTP analogue (Fig. 2). No significant binding was observed toward GST. Under the same conditions, v-Ras(WT) bound to GST-c-Raf irrespective of the GDP- or GMP-PNP-bound form. Guanine nucleotides sometimes do not affect the association between Ras and c-Raf under certain experimental conditions as reported previously(23, 24) . RasDelta42-49 showed similar results as those of v-Ras(WT), suggesting that the region between amino acid positions 42 and 49 is not necessarily required for Raf binding. However, RasDelta43-45 failed to bind to GST-c-Raf probably because the deletion caused some additional alteration in the three-dimensional conformation of Ras.


Figure 2: Binding of Ras proteins and c-Raf-1. Ras proteins, which had been loaded with GDP or GMP-PNP, were mixed with GST-c-Raf or GST immobilized to glutathione-Sepharose. The bound proteins were examined by immunoblot analysis using anti-Ras monoclonal antibody. The lowest bands correspond to Ras monomers while the upper bands represent Ras dimers(31) . The mobility of RasDelta42-49 in the gel was higher than that theoretically expected, and thus trimer-like complexes were also observed. The mobility of RasDelta43-49 was almost the same as that of v-Ras(WT), yet there was no detectable signal.



DNAs for cystatin alpha, cystatin alpha-CVLS, v-Ras(WT), RasDelta42-49, and RasDelta43-45 were inserted into a downstream region of mouse mammary tumor virus-long terminal repeat of a eukaryotic expression vector, pMSG, and transfected into NIH3T3 cells. Western blot analysis demonstrated that the transfected genes were effectively induced after treatment with Dex(9, 16, 17) . Induction of cystatin alpha, cystatin alpha-CVLS, RasDelta42-49, and RasDelta43-45 did not cause any significant morphological changes whereas induction of v-Ras(WT) resulted in drastic morphological changes as well as the appearance of dense transformed foci (not shown).

Because anchorage-independent growth is one of the specific phenotypes observed in malignant cells(18) , growth of transfected clones in soft agar medium was investigated. Vector-transfected clones, NV1, NV2, and NV3, as well as the parent NIH3T3 cells did not grow in soft agar medium while v-ras(WT)-transfected clones, NR24 and NR37, showed high colony-forming efficiencies in the presence of Dex (Fig. 3). These clones grew in soft agar medium even in the absence of Dex probably due to the leaked expression of Ras in the absence of Dex(9, 16) . On the other hand, induction of cystatin alpha, cystatin alpha-CVLS, RasDelta42-49, and RasDelta43-45 by Dex did not result in any discernible change in the colony-forming ability. This implies that induction of cystatin alpha, cystatin alpha-CVLS, RasDelta42-49, or RasDelta43-45 alone is insufficient to induce anchorage-independent transformation.


Figure 3: Colony-forming efficiency of transfected clones in the presence or absence of Dex and/or TPA. Anchorage-independent growth in soft agar medium was investigated for vector-transfected control clones (NV-1, -2, and -3), cystatin alpha-transfected clones (NY-9, -21, and -23), cystatin alpha-CVLS-transfected clones (NS-13 and -18), v-ras(WT)-transfected clones (NR-24 and -37), rasDelta42-49-transfected clones (NII-2 and -3), and rasDelta43-45-transfected clones (NIII-6 and -7). Column values and error bars represent the average percentage ± S.E., respectively, over three independent experiments.



These proteins might play a role at a certain stage in multistage carcinogenesis. Two-stage carcinogenesis, which consists of initiation and promotion, was first proposed by Berenblum(25) . Initiated cells produced by irradiation or treatment with chemical carcinogens are easily transformed in the presence of tumor promoter phorbol ester, TPA, and the transformation can be evaluated by focus-forming assay (26, 27) . Some of the chemical carcinogens that induce initiation have been shown to induce specific mutations in ras protooncogenes, and thus the involvement of Ras in the early stage of carcinogenesis was suggested(28, 29, 30) . Consequently, cystatin alpha might have a role at the stage of initiation and cooperate with a tumor promoter to induce full transformation. Thus, the anchorage-independent growth in the presence of TPA was also investigated (Fig. 3). The culture of cystatin alpha-CVLS- and RasDelta42-49-transfected clones in the presence of both Dex and TPA resulted in a remarkable increase in colony-forming efficiency, which was significantly higher than that in the absence of either TPA or Dex (p < 0.01). The presence of TPA alone was not sufficient to induce the anchorage-independent transformation in these transfectants. Vector-transfected control clones and NIH3T3 cells showed no significant increase in colony numbers. Induction of cystatin alpha showed the intermediate results and the colony-forming efficiencies of NY clones in the presence of Dex and TPA were between 10 and 30% (0.01 < p < 0.1) versus that in the absence of TPA. This suggests that the expression of cystatin alpha partly induces the changes corresponding to initiation.

The transformed colonies formed by NY, NS, and NII clones were similar to those formed by NR clones. However, the sizes of the former were smaller than those of the latter (Fig. 4), i.e. the diameters of most colonies of NY, NS, and NII clones were between 0.05 and 0.2 mm while those of NR clones were larger than 0.2 mm. The colony-forming efficiency of NR clones could be underestimated because some colonies were overlapped.


Figure 4: Phase morphology of transformed colonies. Colonies formed by NY-21 (A), NS-13 (B), NII-3 (C), and NR-37 (D) in soft agar in the presence of both Dex and TPA are shown. Original magnification was times60. The bar in C represents 0.2 mm.



The results obtained in the present study are summarized in Table 1. Although cystatin alpha-CVLS alone cannot induce full transformation, it induced anchorage-independent transformation in the presence of a tumor promoter. RasDelta42-49 also induced anchorage-independent growth at a high efficiency in the presence of TPA in spite of the drastic decrease in the GTP binding activity. This mutant retains the cysteine proteinase-inhibitory activity, which is similar to that of v-Ras(WT). Consequently, suppression of cysteine proteinases may be sufficient for the conversion of a normal cell into an initiated one. The specific role of cysteine protease inhibitors at the stage of initiation is consistent with the possible involvement of ras oncogenes in the stage of initiation(28, 29, 30) .



Cystatin alpha-CVLS also induced initiation without any GTP binding activity. The expression of RasDelta43-45 resulted in a slight increase in the colony formation even in the presence of TPA. Since this mutant cannot bind to c-Raf-1, association with Raf protein might be necessary for the transformation. Cystatin alpha-CVLS induced similar transformation without binding to c-Raf-1, possibly because the protease-inhibitory activity of cystatin alpha is higher than that of v-Ras(WT)(7, 19) .

Even if the suppression of cysteine proteinases induces initiation, the present results cannot identify the target protease of Ras and cystatin alpha. Higher colony-forming efficiencies of NS clones as compared with those of NY clones suggest that the target protease is localized at a membrane fraction. In order to identify the substrates of the target protease, we have investigated the intracellular contents of Ras-binding proteins in the transfected cells before or after treatment with Dex. They were c-Raf-1, A-Raf, MEK, MEK2, protein kinase C, rasGAP, and phosphatidylinositol 3-kinase p85. However, none of them increased reproducibly after induction of Ras or cystatin alpha (data not shown). It is thus unlikely that Ras and cystatin alpha protect these proteins from degradation. Further study is necessary to determine the target protease and its substrates, which are involved in the induction of initiation.


FOOTNOTES

*
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-43-254-5431 (ext. 5502); Fax: 81-43-265-4459; thiwasa{at}biolab.kazusa.or.jp.

(^1)
The abbreviations used are: GST, glutathione S-transferase; CVLS, Cys-Val-Leu-Ser; Dex, dexamethasone; GMP-PNP, guanylyl 5`-(beta,-imino)diphosphate; TPA, 12-O-tetradecanoylphorbol-13-acetate.


ACKNOWLEDGEMENTS

We are grateful to Drs. Berthe M. Willumsen (University Institute of Microbiology, Copenhagen), Nobuhiko Katunuma (Tokushima Bunri University), and Yoshimasa Ike (Mitsui Pharmaceutical Inc.) for providing plasmids. Human c-raf-1 gene was obtained from the Japanese Cancer Research Resources Bank.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.




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