MOLECULAR CARCIiVOGENESIS 1:109-115 (1988)

Modified Responsiveness of v-Ha-ras-Transfected Rat Fibroblasts to Growth Factors and a Tumor Promoter Ming Huang, Nobuyuki Karnata.' Kiyoshi Nose, and Toshio Kuroki2 Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan

The correlation of the phenotypic changes of v-Ha-ras transfected cells with the expression o f p21ras and the modified responses t o growth factors and a tumor promoter were examined. Transfedion of the v-Ha-ras gene together with the neomycin-resistance gene into 208F rat fibroblasts yielded transformed clones characterized by morphological changes, anchorage-independent growth, and tumorigenicity in nude mice. The degrees of these biological alterations were parallel with the expression of mRNA and protein of the ras gene. In ras- transformed cells, anchorage-independent growth was stimulated by epidermal growth factor (EGF), insulin, bombesin, and fibroblast growth factor, whereas in the parental 208F cells, anchorage-independent growth was observed only in the presence o f EGF, and there were many fewer EGF-induced colonies than those in the ras-transformed clones. A tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA) also augmented an- chorage-independent growth o f ras-transformed cells and induced morphological changes in monolayer cul- tures without altering the expression of the ras gene or phosphorylation of the p21ras protein. Retinoic acid inhibited the TPA-induced anchorage-independent growth. These results showed a good correlation of the expression o f p21 ras with the phenotypic changes and the increased sensitivity of the p21raS-expressing cells to the stimulation of growth factors and tumor promoter.

Key words: ras oncogene, transformation, growth factor, tumor promoter, anchorage-independent growth

INTRODUCTION

The current enthusiasm for the study of cell growth control is mostly based on the discovery of the link be- tween oncogenes and growth factors or their receptors [I-31. A suggestion of this link was first provided by the findings that transformation by retroviruses carrying on- cogenes, e.g., Moloney or Kirsten murine sarcoma virus, results in secretion of growth factors, termed transform- ing growth factors (TGFs), by which anchorage-indepen- dent growth of cells is stimulated (4, 51. The concept of an autocrine model of transformation is based on these findings. Direct support for this model was obtained by studies on the sis oncogene, which encodes a growth factor that is structurally and functionally homologous to the B chain of platelet-derived growth factor (PDGF), indi- cating that transformation by v-sis is regulated by auto- crine secretion of PDGF [6-81. The finding of sequence homology between the v-erbB gene and the cellular gene of the epidermal growth factor (EGF) receptor also pro- vided evidence for the link between oncogenes and growth factors 19, lo].

On the other hand, growth factors are known to induce transcription of certain oncogenes [I]. Growth factors increase the levels of mRNAs of the c-fos and c-myc genes most frequently (11, 121. In AKR-2B cells, TGFP induces expression of the c-sis gene, resulting in secretion of PDGF into the medium [13].

Furthermore, oncogenes may modify the responses of cells to signals generated by a growth factor (14-161. These modified responses also afford an insight into the

cellular and molecular mechanisms of transformation of cells.

The ras family of oncogenes has been implicated in the development of a wide array of spontaneous and chemi- cally induced tumors of animals and humans [17-201. The function of the products of these oncogenes, termed p21, has been studied intensively. However, GTP-binding and GTPase activity are the only biological characteristics known for p21 [21-241, suggesting the involvement of this protein in the transduction of extracellular signals [25]. Thus, the p21 protein may sensitize the cells to growth factors and other related compounds. In the present study we examined the responses of ras-transfected cells to growth factors and a tumor promoter. We found that the ras gene is capable of potentiating the responses. These modified responses may constitute one of the mechanisms by which ras causes transformation.

'Present address: Department of Oral-Maxillo-Facial Surgery, Fac- ulty of Dentistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan.

ZCorresponding author: Department of Cancer Cell Research Insti- tute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai Minato-ku, Tokyo 108, Japan.

Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; TGF, trans- forming growth factor; TPA, 12-O-tetradecanoylphorbol-l3-acetate; PAGE, polyacrylamide gel electrophoresis; neo', neomycin resistant; SDS, sodium dodecyl sulfate

0 1988ALAN R. LISS, INC.

7 10 HUANG ETAL.

MATERIALS AND METHODS

Materials

The following materials were used: G418 (GIBCO, Grand Island, NY), 12-0-tetradecanoylphorbol-13-acetate (TPA; Consolidated Midland Co.), retinoic acid (Wako Pure Chemicals Co., Tokyo, Japan), insulin, EGF and TGFP Uak- ara Shuzo Co., Ltd., Japan), PDGF (Collaborative Research, Lexington, MA); fibroblast (FGF; Biomedical Technologies Inc., Cambridge, MA); bombesin (Peptide Institute Inc.), affinity-purified sheep anti-Ha-ras p21 antibody (Triton Bioscience, Alameda, CA); ABC kit (Vector Laboratories, Burlingame, CA); and agar (Noble; Difco, Detroit, MI).

Cell Culture

208F Rat fibroblast cells [26] and their oncogene-trans- fected clones were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37Oc in a humidified incubator under 5% C 0 2 in air.

Gene Transfer Technique Plasmid DNA was introduced into cells by the conven-

tional method of calcium-phosphate precipitation. Clone H-1 v-Ha-ras DNA (20 pg) 1271 was cotransfected with 5 pg of pSV-2-neo DNA I281 into 3-5 x lo5 cells in a 100- mm dish. The precipitated DNA was left in contact with the cell layer for 12 h. Neomycin-resistant (neo') clones were selected with 400 pgimL of G418 for 14 d.

Hybridization Anafyses of RNA

Cellular RNA was prepared by the guanidium-hot phenol method, followed by electrophoresis on 1.5% aga- rose gel containing 2.2 M formaldehyde and transferred to a nitrocellulose filter (northern blot) [29]. Filters were hybridized to 32P-labeled cDNA probes, e.g, clone BS-9 [27] for v-Ha-ras. Conditions for hybridization have been described elsewhere 1301.

Detections o f p21 Protein

We used the method of Haid and Suissa 1311 with a slight modification to detect p21 protein. Cellular protein was extracted with lysis buffer consisting of 2% sodium dodecyl sulfate (SDS), 0.1 M Tris-HCI (pH 6.8), 2 mM EDTA, 5% 0-mercaptoethanol, 20% glycerol and 0.02% bromphenol blue. The cell lysate was subjected to SDS- polyacrylamide gel electrophoresis (PAGE) on 10-20% polyacrylamide gel and transferred to a nitrocellulose filter (western blot). After precoating with bovine serum albu- min (20 mg/mL), filters were incubated with affinity-puri- fied sheep anti-Ha-ras p21 antibody for at least 1.5 h and then with vectastain biotinylated rabbit anti-sheep immu- noglobulin G antibody for 1-2 h. After final incubation with vectastain reagent, proteins that reacted with anti- bodies were detected by soaking the filters in a 1:l (vlv) mixture of 4-chloro-I-naphthol solution and hydrogen per- oxide solution for 10 min.

TPA-Induced Phosphorylation of p21 and lmmunoprecipitation

Subconfluent cultures were incubated in phosphate-free Eagle's minimum essential medium containing 0.5% bo-

vine serum albumin and 0.1 mCilmL of [32P]ortho- phosphate for 2 h at 37OC and then changed to the fresh medium with or without TPA (100 ng/mL) for an additional 30-240 min. After being washed with phosphate-buff- ered saline for four times, a lysis buffer containing 0.01 M Tris-HCI, 0.15 M NaCI, 1% sodium deoxycholate, 1% Tri- ton X-100, 0.1% SDS and 1 mM phenylmethylsulfonyl- fluoride was added. The extracts were centrifuged at 15,000 rpm for 30 min at 4OC, and the supernatant was incubated with normal sheep serum (30 min) and protein A-cellulofine (30 min) to precipitate nonspecific protein. The solution was then incubated with anti-Ha-ras p21 antibody or normal sheep serum for 2-10 h at 4OC and protein A-cellulofine for another 2 h. The immunoprecipi- tates were collected by centrifugation and washed five times with the same lysis buffer. The pellets were prepared for SDS-PAGE by adding 0.1 M Tris-HCI (pH 6.8) containing 2% SDS, 2 mM EDTA, 5% 0-mercaptoethanol, 20% glyc- erol and 0.02% bromphenol blue and boiling for 3 min.

Assay of Anchorage-Independent Growth

Anchorage independent growth of cells was assayed by the simplified soft agar microassay of Morita et al. [32]. In brief, 0.5 mL of 0.33% agar medium containing 1-5 x lo3 cells was poured onto a basal layer of 0.5% agar in wells of 24-well plates. Growth factors and other test chemicals in 0.1 mL of solution were added on top of the agar layers. Colonies larger than 100 pm were counted on day 14 under a dissecting microscope.

Transplantation into Nude Mice

Suspensions of lo6 cells in 0.15 mL phosphate-buffered saline were injected subcutaneously into untreated nude mice. The animals were examined for tumor formation over a period of 8 wk.

RESULTS Transfection of Oncogenes

The v-Ha-ras oncogene was cotransfected with the neo' gene into 208F rat fibroblasts, and G418-resistant clones were isolated. Five of these clones were found to express high levels of ras mRNA upon northern blot analysis (Fig- ure 1A). Among them, clones 6, 11, and 14 gave a strong band at a molecular weight of about 21,000 in western blotting, using a p21-specific antibody (Figure 1 B); in clones 22 and 24, less p21 protein was detected. No p21 was demonstrated in parental 208F cells or cells transfected with pSV2-neo DNA alone.

Phenotypic Properties o f v-Ha-ras-Transfected Cells

Morphological changes were observed in these ras- transfected clones: the cells became refractile and rounded and grew with a criss-crossed arrangement. These changes varied to some extent in different clones, clones 11 and 14 showing typical transformed morphology (Figure 26 and C) and other clones showing less pronounced changes (Figure 2D). In contrast, the parental 208F cells and neo' clones were sensitive to contact inhibition and formed flat monolayers at confluency (Figure 2A).

GROWTH CONTROL IN ras-TRANSFECTED CELLS 7 1 1

Augmentation of Anchorage-Independent Growth by Growth Factors A

4

B 4 4 4

4

p2 1, 4

4

Figure 1. (A) Northern blot assay of RNA and (B) western blot assay of protein products of the v-Ha-ras transfected 208F rat fibroblasts. In the panel A, the arrow at the left side indi- cates the c-Ha-ras transcript, and upper and lower arrowheads at the right side indicate the positions of 285 and 185 rRNA, respectively. In the panel B, arrowheads a t the right side indi- cate the positions of molecular-weight markers (from the top, 92,500, 66,200, 45,000, 31,000, 21,500 and 14,400).

The ras-transfected cells reached a higher saturation density than the parental 208F cells. Some variations were noted in the growth rates of the ras-transfected clones: clone 11 grew much more slowly than the other clones and than normal 208F cells although the clone 11 had a high level of p21 protein and a fully transformed phenotype.

We found that all of the ras-transfected clones formed colonies to various extents in soft agar medium. The fre- quency. of anchorage-independent growth was much higher in clones 6 and 14 than in others, whereas the parental 208F cells and neo' clones did not grow in soft agar (Table 1).

The ras-transfected cells formed tumors on injection into nude mice, but again at varying degrees among the clones: at higher frequency and after shorter latent pe- riods in clones 6, l l, and 14. No tumors were formed by parental 208F cells (Table 2).

Based on the degree of these phenotypic changes, clones 6, 11, and 14 are regarded as fully transformed cells and clones 22 and 24 as partially transformed ones. The degree of transformation seems to correlate well with the expression of p21ras among these clones.

Oncogenes may modulate the response to growth fac- tors by interacting, directly or indirectly, with signal trans- duction pathways [33,34]. We therefore examined anchorage-independent growth of ras-transformed cells in the presence of various growth factors such as bombesin (1-20 pglml), EGF (1-10 nglmL), FGF (10-100 nglml), insulin (5-50 pglmL), PDGF (1-6 UlmL) and TGFP (0.5-5 nglml). As shown in Table 1. addition of EGF greatly increased the anchorage-independent growth of ras-trans- formed cells, but the degree of stimulation varied in dif- ferent clones. It caused more than a 10-fold increase with partially transformed clones 22 and 24 but only about 2- to 3-fold increase with fully transformed clones 6 and 14.

Insulin increased colony formation of ras-transformed cells 1.3-3.7 times. Bombesin showed a similar effect (1.5-3.6 times increase for clones 11, 22, and 24). FGF also caused a 1.3-1.9-time increase in clones 6, 14, and 22 (Table 1). Other growth factors, e.g., PDGF (1-6 UlmL) and TGFP (0.5-5 nglml), caused little or no increase of anchorage-independent growth (data not shown).

In the neo' clone, anchorage-independent growth was observed only in the presence of EGF, and the number of EGF-induced colonies was much smaller than for the ras- transformed clones.

In monolayer cultures, EGF did not stimulate the growth of ras-transformed cells or the parental 208F cells (data not shown). However, clones partially transformed by the ras gene, i.e., clones 22 and 24, showed further morpho- logical changes when treated with EGF at 100 nglmL for 7 d (Figure 2E). No morphological changes were induced by EGF in the control 208F cells

Augmentation of Anchorage-Independent Growth by TPA

We found that like growth factors, TPA also increased anchorage independent growth of the ras-transformed cells 2--10 times but did not affect that of neo' cells (Table 3). The extent of increase again varied in different clones: partially transformed clones 22 and 24 were the most sensitive, and their colony formation was increased 8-10 times by TPA, as observed with EGF.

Retinoic acid at 100 nM inhibited anchorage-indepen- dent growth of the ras-transformed cells in the presence and absence of TPA, except for clone 11 (without TPA) and clone 24 (with and without TPA).

In monolayer cultures, TPA did not stimulate the growth rate, but caused a marked change of morphology in ras- transformed cells, especially partially transformed clones, resulting in piling up and random orientation of the cells (Figure 2F). These morphological changes were not asso- ciated with increased expression of the ras gene at either the RNA level or protein level even after treatment with TPA for 2 mo (Figure 3A and B). Ballester et al. 1351 reported phosphorylation of p21 protein of c-Ki-ras by the treatment with TPA of mouse adrenocortical cell lines. In the present cell lines, however, no phosphorylation of the p21 protein was observed in the absence or presence of

I I2 HUANG ETAL.

Figure 2. Morphology of the ras-transfected cells and their responses to EGF and TPA in monolayer culture. (A) Parental 208F cells, (B) clone 11, fully transformed by v-Ha-ras, (C) clone 14, fully transformed by v-Ha-ras, (D) clone 24, partially trans-

formed by v-Hams, (E) partially transformed clone 24, treated with 10 nglmL EGF for 7 d, (F) partially transformed clone 24, treated with 100 ng/mL of TPA for 7 d.

100 nglmL TPA for various times from 30-240 min (data not shown).

DISCUSSION

In this study we examined the correlation of the phe- notypic changes of v-Ha-ras-transformed cells with the expression of p21ras and their modified responses to growth factors and a tumor promoter. We used 208F rat fibroblasts, which are good recipients and also good ex- pressors of transfected genes.

Transfection with the ras gene resulted in transforma- tion of 208F cells characterized by morphological changes, anchorageindependent growth, and tumorigenicity. It is

intriguing to note that the degree of these phenotypic changes correlated well with the expression of p21 pro- tein: clones giving a strong band of 21,000 daltons in western blot assay show pronounced transforming characters.

We report here that the ras-transformed cells showed modified responsiveness to growth factors and a tumor promoter. EGF considerably potentiated anchorage-inde- pendent growth of the cells and caused morphological changes of cells in monolayer culture. Insulin, bombesin, and FGF had similar effects on these cells. These effects varied in different clones. In general, the responsiveness was greater in partially transformed clones and less in the

GROWTH CONTROL IN ras-TRANSFECTED CELLS

Table 1. Anchorage-independent Growth of the ras-Transformed Cells in the Presence and Absence of Growth Factors

7 13

Number of colonieslwell EGF Born besin FGF Insulin

Cells Control (10 nglmL)* (4 pglmL) (50 nglmL) (50 pglml)

Control neo-1 0 42 f 5 0 0 0 ras-Transf ected clone 6 104 f 11 224 f 7 121 f 15 140 & 5 210 f 4 clone 11 13 f 2 78 f 7 39 f 1 9 k 2 17 + 2 clone 14t 250 f 16 780 f 80 320 f 14 470 5 80 675 + 60 clone 22 16 f 1 160 f 2 24 f 4 30 f 8 60 f 10 clone 24 8 f 1 138 f 32 29 f 6 9 i 1 24 f 2

Colonies larger than 100 pm were counted 2 wk after seeding 5000 cellsll0-mm well. Data are means * S.D. for two independent experiments. To avoid complication of the table, data at a certain concentration are included. *Concentrations are indicated in parentheses. tBecause of high frequency of anchorage-independent growth, 1000 cells were seeded per well, and data are presented after normalizing to 5000 cellsll0-mm well. EGF, epidermal growth factor; FGF, fibroblast growth factor.

Table 2. Tumorigenicity of ras-Transformed Cells

Wk after transplantation* Cells 4 5 6 8

Control 208F Ol5t 015 015 015 ras-Tra nsformed clone 6 015 015 215 315 clone 11 015 1 I5 315 315 clone 14 315 515 515 515 clone 22 015 115 1 I5 1 I 5 clone 24 015 015 015 1 I5

'Subcutaneous injection of l o 6 cells. tNumber of mice with tumorslnumber of mice examined.

fully transformed clones expressing large amounts of p21 protein, probably because in the latter, the transformed characters were already clear enough even without fur- ther stimulation.

Hsiao et al. (361 and Dotto et al. [37] reported further morphological change of the ras-transformed cells on

treatment with TPA, a finding that they interpreted as being analogous to two-step carcinogenesis, i.e., initiation by the ras gene and subsequent promotion by TPA. We also found that TPA potentiates the phenotypic changes of ras-transformed cells, especially partially transformed clones. These changes were not accompanied by amplifi- cation or increased expression of the transfected ras gene.

We found that in the same transformed clones as used in this study, the activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases were increased, whereas the activity of diacylglycerol-kinase was less than that in untransformed cells [38]. The imbalance of these kinases resulted in accumulation of diacylglycerol in the ras-transformed cells, which in turn caused intracellular translocation, activation and downregulation of protein kinase C. Further treatment of these ras-transformed cells with TPA resulted in complete translocation of protein kinase C from the cytosol to the membrane fraction. These results indicate that activation of protein kinase C may be involved, at least in part, in expression of transformed phenotypes. No TPA-induced phosphorylation of p21 pro- tein was detected as reported by Ballester et al. in c-Ki-ras

Table 3. Anchorage-independent Growth in ras-Transformed Cells Treated With TPA andlor Retinoic Acid

TPA (10 nglml) &

TPA Retinoic acid retinoic acid (10 nglmL)* (100 nM) (100 nM) Cells Control

Control

ras-Transfected neo- 1 0 0 0 0

clone 6 104 f 11 194 & 6 34 f 0 59 + 12 clone 11 13 f 2 60 + 2 10 f 3 11 f 3 clone 14t 250 f 16 645 + 45 25 f 10 120 f 0 clone 22 16 & 1 164 f 15 0 64 + 4 clone 24 8 + 1 67 f 13 8 + 3 61 f 19 Colonies larger than 100 pm were counted 2 wk after seeding 5000 cellsllO-mm well. Data are means ~t S.D. for two independent experiments. *Concentrations are indicated in parentheses. tBecause of the high frequency of anchorageindependent growth, 1000 cells were seeded per 10-mm well and data are presented after normalizing to 5000 cells/lO-rnm well.

114 HUANG ETAL.

C T C T C T C A T

C T C T C T C T

B a 4

C T C T C T C T

B a 4

4

p21, 4

4

Figure 3. Expression of the ras gene in the presence and absence of TPA at 100 nglrnL; (A) northern blot assay, (B) western blot assay. T, TPA-treated cultures, C, control cultures. (See Figure 1 for explanation o f arrows.)

[35], suggesting the biological effects of TPA in the pres- ent study may be achieved by a pathway other than p21.

The results presented here indicate that v-Ha-ras gene sensitizes 208F cells to the stimulation of growth factors and a tumor promoter. It is likely that this oncogene causes transformation either by a high level of its expres- sion, by synergistic effect with the modified responses to extracellular signals, or by both.

ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid for Special Project Research, Cancer-Bioscience, from the Ministry of Education, Science, and Culture of Japan. The work by Ming Huang reported in this paper was done during his tenure as a Research Training Fellow from the International Agency for Research on Cancer, Lyon, France (1986) and the Fujisawa Foundation, Tokyo, Japan (1987).

Received October 26, 1987; revised April 8, 1988; accepted April 18, 1988.

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