EXPERIMENTALCELL

RESEARCH

186,83-89

(1990)

Epidermal Growth Factor (EGF)-Nonresponsive Variants Kidne ell Line: Response to EGF and Transforming Gr RYOJI HAMANAKA,* MAYUMI ONO,* YUICHIRO KURATOMI,” WIROMOTO REIKO HIRAI,~ KIMITOSHI KOHNO,* ANIPMICHIHIKO KUWANO*~~ *Department

of Biochemistry, Tokyo

Oita Medical Metropolitan

School, Hasama-machi, Institute for Medical

Press,

Inc.

INTRODUCTION

DeLarco and Todaro [l] have found that transformed cells in culture secrete factors which cause transition from the normal to the transformed phenotype. The ability of a cell to produce such transforming growth factors (TGFs) proposes a unique mechanism of “auto’ To whom correspondence dressed.

and reprint

Tokyo

Japan; and TDepartment 113, Japan

of Oncology,

crine” growth control for the unlim of malignantly transformed cells [ of autocrine stimulation of growth formed cells has been strongly sup that growth factors or their recept similarities to various oncogenes [5 pects that transfection of growth factor genes or their receptors might cause transformation. The basic fibroblast growth factor gene with a signal peptide sequence is a potent oncogene when introduced into mouse NIH3T3 cells [X2]. DiFiore et ak. [13] bave also s that transfection of total hum EGF receptor causes transformation of NIH 3 cells under anchorage-independent growth conditions in soft agar in the presence of EGF. Establishment of variants csf~o~tra~sf~rmed growth property with altered responses to growt contribute to an understanding of bow growth factors influence malignant transformation as well as growth behavior of the transformed cells. ~ersbma~ and his colleagues have isolated EGF-nonresponsive variants with altered EGF receptor activity from mouse Swiss3T3 cell line and these variants are unable to show a mitogenic response to EGF [X4,15]. We have isolate terized a monensin (a Na+/K+ ion resistant mutant from mouse Balb/3T3 cell li 5 with almost no EGF receptor activity [Is, 171 dent growth in soft agar by TGF-P was observed at similar levels in MO-5 and Balbj3T3 cells [17] while MO-5 was very resistant to transformation by chemical carcinogens [ 181 polyoma middle T antigen and [19]. TGFs contain TGF-(w and TGF-& functions by binding to EGF-receptors [2 by various virally transformed fibrobl~~ts [1,21]. When normal rat kidney (NRK) cells are expos TGF-a or EGF and TGF-0, they transiently displa ious traits common to their virally transformed counterparts that produce TGFs, including disor~a~i~at~~~ toskeleton, enhanced glucose uptake, an colonies in soft agar [I, 211. EGF Or TGFreceptor appear to play a role in maintaining the transformed phenotype by autocrine stimul However, the precise mechanism of ho

Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-8 (TGF-6) and epidermal growth factor (EGF) (or TGF-(r). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with lz51-EGF showed one-half or fewer EGF receptors in N3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy-D-glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-/3 treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. I9NA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-fi, whereas that of N-3 was significantly enhanced by TGF-@ alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-@ mRNA, but there appeared little expression of TGF-8 mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-@ enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors. o isso Academic

Oita 879-56, Science,

requests should be ad-

83 All

Copyright 0 1990 rights of reproduction

0014.4827/90 $3.00 by Academic Press, Inc. in any form reserved.

84

HAMANAKA

p work in concert to induce growth of NRK is not clear. In EGF-nonresponsive variants of their transformation properties TGF-P. MATERIALS

AND

anchorage-independent this study, we isolated NRK and characterized in response to EGF and

METHODS

Materials. Epidermal growth factor (EGF) was obtained from Toyobo Corp; transforming growth factor-p (TGF-/3) was from Collaborative Res., Inc. (Bedford, MA). [3H]Thymidine (120 Ci/mmol), 2[3H]deoxy-D-glucose (10 Ci/mmol), and 32Pi (8500-9120 Ci/mmol) were obtained from New England Nuclear (Boston, MA). [Wlcysteine (988 Ci/mmol) was obtained from ICN Biochemicals (Irvine, CA). YEGF was iodinated with Nalz51 by the chrolamine T method as described previously [17, 241. Anti-rat EGF monoclonal antibody IgG (HM-56) was purchased from Ohtsuka Pharmacent. Co., Tokushima, Japan. Anti-phosphotyrosine antibody was obtained from Amersham Japan Co., Chiba, Japan. Murine TGF-0 cDNA [25] probe was obtained from R. Derynck (Genentech, Inc., South San Francisco, CA). Cell cultures and soft agur gromth assuys. NRK cell clone 49F and its EGF-nonresponsive variants, N-3 and N-9, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS). N/MT-6, a malignant transformed cell line of NRK, was selected after transformation with polyoma middle T antigen according to the procedure described previously [19]. One milliliter of cell suspension (3 X 103/ml) of NRK and its variants in 0.3% agar (Difco, agar noble) in DMEM containing 5% FBS and additional growth factors was pipetted onto a l-ml base layer (0.5% agar in the supplemented medium) in 35-mm petri dishes. Plates were incubated at 37°C for 14 days, and unstained colonies greater than 50 pm were scored as positive [ 171. Immunoprecipitation of secretory proteins with anti-EGF antibody. The cells were treated with or without growth factors for 18 h after seeding into loo-mm dishes and then washed with phosphatebuffered saline (PBS) (g/liter:NaCl, 8.0; Na2HP04. 12Hz0, 2.9; KCl, 0.2; KH2P04, 0.2) and subsequently incubated with 100 j&i of [?S]cysteine for 20 h in MEM containing 2% FBS. The media were collected and immunoprecipitated with anti-EGF antibody as described elsewhere [26]. Labeled medium (7.5 X lo6 cpm) was boiled for 4 min and then immunoreacted with 10 yl anti-rat EGF monoclonal antibody. The immunoreactive EGF was recovered by precipitation with Protein A and eluted by boiling in a buffer containing 0.125 M Tris, 2% SDS, and 5% /l-mercaptoethanol. The samples were electrophoresed in a 15% polyacrylamide gel [27]. The gels were fixed, enhanced with 2,5-diphenyloxazole dissolved in dimethylsulfoxide, dried, and exposed to Kodak XAR-5 X-ray film at -80°C. ‘251-EGF-binding. NRK and its variants cells were seeded in 35. mm dishes and grown to a density of 1 X lo6 cells/dish. Cells were washed twice with PBS and then incubated with 500 pl of serum-free MEM containing 20 n&f Hepes, 5 mg/ml bovine serum albumin, and iz51-EGF in the presence or absence of loo-fold excess EGF [17]. Cells were then washed with PBS three times and lysed with 1 N NaOH. The lysates were measured by gamma counter. Specific ‘s51-EGF binding was determined as the difference between the counts in the absence and presence of excess unlabeled EGF. DNA synthesis. Cells were grown in 35.mm plastic dishes in 2 ml of MEM containing 5% FBS. At confluence, the medium was removed and replaced with 1 ml of MEM containing 0.5% PBS, and the cells were incubated for 48 h at 37°C. EGF at indicated concentrations and 0.1 &i/ml of [3H]thymidine were simultaneously added to each dish followed by incubation for an additional 18 h at 37°C. The cells were then washed with ice-cold PBS, incubated with ice-cold 5% trichloroacetic acid for 30 min, and then solubilized in 0.5 N NaOH. An aliquot of the acid-insoluble material was counted in a Beckman LS-9000 liquid scintillation counter.

ET

AL.

Assay of 2-deoxy-D-glucose uptake. Uptake of 2-deoxyglucose was measured as described previously [19,28]. Cell monolayers grown exponentially in 5% FBS containing DMEM were washed with PBS once and then 1 &i/ml of 2-[3H]deoxy-D-glucose (1 Ci/mmol:NEN; Boston, MA) was added and the reaction was allowed to proceed at 37°C for up to 15 min, during which time uptake was linear. Uptake was terminated by three washes with cold PBS containing 25 mMglucase and the cells were solubilized in 0.05% SDS; radioactivity was then measured in the solubilized fraction. TGF-p mRNA by Northern analysis. Cytoplasmic RNA was prepared as described [29] with minor modification [30]. Briefly, the cells were rinsed twice with cold PBS, scraped in PBS, and pelleted at 1000 rpm for 5 min. The cell pellet was resuspended in cell lysis buffer (1% NP-40, 5 n&f NaCl, 5 mM Tris-HCl, pH 7.5, 0.5 mA4 MgCl*) and centrifuged at 3000 rpm for 10 min. The supernatant was mixed with an equal volume of solution containing 1% SDS, 7 Murea, 0.3 MNaCI, 20 mM Tris-HCl, pH 7.5, and 2 mM EDTA. After serial extractions with phenol, phenol-chloroform, and chloroform, RNA was precipitated with ethanol. Twenty micrograms of RNA was fractionated on 1% agarose containing formaldehyde. After gel electrophoresis, the gel was stained by ethidium bromide, photographed, and transferred to nitrocellulose filter (Zeta probe, Bio-Rad) with 10X ssc (1.5 M NaCI, 150 mMsodium citrate, pH 7.0). The filter was prehybridized for 3 h at 30°C with buffer containing 50% deionized formamide, 5X Denhardt’s solution, 0.1% SDS, and 100 @g/ml salmon sperm DNA. Hybridization was carried out for 36 h at 30°C in the same buffer. Filters were washed at room temperature twice in 2X ssc and 0.1% SDS. Autoradiography was carried out using Kodak XAR film. Phosphorylation of membrane’proteins. Cells at confluent state were incubated for 24 h with phosphorus-free DMEM containing 0.5% FBS, incubated for 18 h in the absence or presence of 5 rig/ml EGF, subsequently exposed to 0.2 mCi/ml s2P for 6 h, harvested, and centrifuged [28]. Cell pellets were suspended in 1 ml of 50 mM Tris-HCl (pH 7.4) containing 1 m&f EGTA, 5 mM MgClz, 10 fig of leupeptin, 10 fig of pepstatin, 200 pg of bacitracin, 0.5 fig of aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate (Buffer I). The suspended samples were then rapidly frozen and thawed and centrifuged at SOOg for 5 min at 4°C. The supernatant was further centrifuged at 45,000g for 30 min at 4°C [31,32]. The resulting pellets were solubilized in Buffer I, immunoprecipitated with antiphosphotyrosine antibody, run on 6% SDS-PAGE, and exposed to Kodak XAR-5 X-ray film at -80°C.

RESULTS

Isolation of EGF-Nonresponsive Variants of NRK Cells Isolation of EGF-nonresponsive variants was done according to the procedure of Pruss and Hershman [14]. In brief, exponentially growing NRK cells in loo-mm petri dishes were treated with 500 yg/ml ethyl methane sulfonate (EMS) for 24 h at 37°C. By this procedure cell survival was reduced to about 20% of the initial cell number. NRK cells were then cultured in the absence of EMS for 10 days in 10% NCS-containing MEM, and the cells at confluent state were further cultured in the presence of 0.5% NCS for 2 days. EGF at 10 rig/ml and colchicine at 1 @g/ml were added, the cultures incubated for an additional 3 days, and EGF-responsive cells removed by a shearing stream of medium, leaving only the EGF-nonresponsive cell population. The remaining cells were grown to confluence and the above selection was repeated four times. We have selected and cloned two EGF-specific nonproliferative variants of NRK cells, N-

EGF-NONRESPONSIVE

NRK

CELL

LINES

85

-0 could stimulate the glucose N-3, and N-9 cells. In NRK cells E hanced cellular uptake of 2-L coadministration of both EGF ditive effect cm the uptake (Fig. 2). In contrast, additnon of EGF caused only a slight or le enhancement of glucose uptake in both N-3 9 cells, whereas TGF-/I? enhanced the uptake in variants as well as in their parental NRK cells. C inistration of EGF and TGF-P caused effect compared to that of TGF-8 in Comparison TGF-R

0

0

-f--f-

oo++

oo++

FIG. 1. DNA synthesis of NRK, N-3, and N-9 cells in the presence of EGF or TGF-P. NRK (A), N-3 (B), and N-9 (C) cells at confluent state were freshly incubated for 2 days with DMEM containing 0.5% serum, followed by exposure to [3H]thymidine for 18 h in the absence or presence of 5 rig/ml EGF and/or 1 rig/ml TGF-/3. Radioactivities in the acid-insoluble fraction were measured and each value (lirSD) is the average of triplicate dishes.

3 and N-9, independently: N-3 and N-9 were isolated from different dishes for cell culture. Growth curves of NRK, N-3, and N-9 cells were followed in 35-mm dishes in the presence of 10% FBS at 37°C: N-3 and NRK showed a doubling time of 24 h while N-9 showed a slower growth rate with a doubling time of about 35 h. Saturation densities were about 1 X lo6 cells per dish in all three NRK, N-3, and N-9 cell lines. Saturation densities of N-3 and N-9 cells were similar to that of their nontransformed counterpart, suggesting a nontransformed property for N-3 and N-9. Efiect o,‘EGF or/and Glucose Uptake

TGF-fl on. DNA

Synthesis

and

We examined whether DNA synthesis of N-3 and N9 responded to exogenously added EGF when the cells were challenged in the quiescent state by decreasing serum concentration to 0.5%. DNA synthesis of the parental NRK and its EGF-nonresponsive variants, N-3 and N-9, resumed 15 h after addition of 10% serum, suggesting the onset of §-phase of the cell cycle (R. Hamanaka and M. Ono, data not shown). As seen in Fig. 1, an approximately five-fold increase in DNA synthesis was observed in the parental NRK when incorporation of [3H] thymidine was measured after a 24-h incubation in the presence of 5 rig/ml EGF, but there appeared only a slight, if any, stimulation of DNA synthesis by exoge110~5 EGF in N-3 and N-9. TGF-P at 1 rig/ml alone did not stimulate DNA synthesis in NRK or in N-3 and N9 cells. EGF or TGF-P enhances glucose transport in mammalian cells in culture [33]. We examined whether EGF

of EGF-Receptor

Aetiuity

We examined whether N-3 and N-9 were altered in their EGF receptor binding properties. As seen in Fig. 3, NRK, N-3, and N-9 demonstrated both high- and lowaffinity receptors for EGF. N-3 an creased EGF binding capacity compa to that of NRK cells. The number of EGF receptors N-9 was 3.6 X 104, 1.8 X 104, and I.5 X IO” per cell for high-affinity receptors and 6.9 X P04, 4.8 X IQ*, and 4.8 X lO* per cell for low-affinity receptors. Scatchard analysis also showed that the & values for the high-affinity binding were 1.0 X 10e9 M for N K, N-3, and N-9 an the low-affinity I& values HP9 44 for NRK and 6.2 X 19’ M for N-3 igh affinity values of EGF receptor binding ar among the three cell lines, but the numbe ceptors appearing on the cell surface of N-9 or 3 was less than one-half that of NRK cells. Anchorage-Independent Presence of Growth

Growth Factors

in Soft Agar in the

Anchorage independent growth o cells in soft agar is dependent on both EGF and [34,35]. we examined the effect of EGF and TGIF-0 on the anchorage-independent growth of N-3 and -9 by scoring colonies with a diameter greater than 5 pm that appeared 14 days after inoculating 3000 cells in soft agar (Fig. 4). Dose-response curves of colony formation of the three cell iines to TGF-fl were compared when 5 rig/ml EGF was absent or present. Colony formation of NRK was significantly enhanced with increasin only when EGF was present, appeared in the absence of EG [36, 371. Colony formation of N-3 was enhanced as a function of the amount of TGF-8 in the absence of EGF and was apparently increased by further addition of EGF in the presence of TGF-P. A small number of colonies (3 to 7) of N-3 EGF alone. No co10 was inoculated even in Dose-response curves 0 to EGF were compared for the ancho presence or absence of complete dependence of the

86

HAMANAKA

I

I

5

IO

I

15

/

ET

AL.

I

I

I

5

IO

15

Minutes FIG. 2. Glucose uptake of NRK, N-3, and N-9 cells in the presence of EGF or TGF-P. NRK (A), N-3 (B), and N-9 (C) cells were grown in DMEM containing 5% FBS in the absence or presence of 5 rig/ml EGF and/or 1 rig/ml TGF-P for 18 h. The cells were washed twice with PBS, exposed to 2-[3H]deoxy-D-glucose, followed by incubation at 37°C for up to 15 min. Uptake was terminated at 5, 10, and 15 min, and cellassociated radioactivities were measured. Each point is the average of duplicated dishes. None (O), EGF alone (A), TGF-/3 alone (A), and EGF plus TGF-P (0).

growth of NRK on both EGF and TGF-P. By contrast, 30 to 40 colonies of N-3 already appeared in the presence of 1 rig/ml TGF-P alone and an approximately twofold increase in the colony number was observed by further addition of TGF-/3. TGF-/3 requires synergistic interaction with EGF for anchorage-independent growth of NRK whereas N-3 requires only TGF-@ for its anchorage-independent growth. However, another EGF-nonresponsive variant, N-9, failed to show anchorage-independent growth in the presence of EGF and TGF-0.

Tyrosine Phosphorylation in NRK and N-3 Cells by EGF EGF causes stimulation of tyrosine phosphorylation of EGF receptor after binding to the cell surface EGF receptor [38-401. We thus compared tyrosine phosphorylation activity of membrane proteins of NRK and N-3 cells when EGF was absent or present. NRK and N-3 cells at confluence were incubated for 18 h in MEM containing 0.5% FBS with or without EGF and phosphorylated; then membrane proteins were immunoprecipitated with anti-phosphotyrosine antibody. SDS-PAGE patterns show apparently increased phosphorylation by EGF of a band corresponding to 170 kDa of NRK cells

I

2

0

I TGF-R

to NRK, N-3, and FIG. 3. Scatchard analysis of i” I- EGF binding N-9 cells. Saturation binding kinetics for ‘251-EGF binding was determined for the three cell lines at 4°C (see insert). NRK (0), N-3 (o), and N-9 (m).

2

0

1

2

(nglml)

FIG. 4. Anchorage-independent growth in soft agar of NRK, N3, and N-9 cells. One milliliter of cell suspension (3000 cells per milliliter) of NRK (A), N-3 (B), and N-9 (C) in 0.3% Difco Nobel agar in DMEM containing 5% FBS and various doses of TGF-P with (0) or without (0) 5 rig/ml EGF was pipetted onto a l-ml base layer (0.5% agar in the supplemented medium) in 35.mm petri dishes. Plates were incubated at 37°C for 14 days, and colonies greater than 50 pm in diameter were scored as positive. Each value is the average of duplicate dishes.

EGF-NONRESPONSIVE

NRK

CELL

LINES

87

(data not of 1 pg/ml of nonradioactive atterns of secretory proteins similar to those .7B were also observed by using anti-rat polyclonal antibody, which was supplied by T. Kato at Nagoya City University School of~edici~e, Nagoya, Japan (R. Hamanaka and M. One, ~~~~b~isbe~ data). presence

woe 2 10 20

”

0

30

40

EGF

0

10

20

30

40

(nghnl)

FIG. 5. Effect of EGF on anchorage-independent growth in soft agar of NRK and N-3 cells. Cells at 3000 of NRK (A) and N-3 (B) were plated in 35-mm dishes in 1 ml DMEM containing 0.3% agar in the presence of various doses of EGF with (0) or without (0) 1 rig/ml TGF-0. Colonies larger than 50 pm in diameter were counted after 2 weeks of incubation. Each value is the average of duplicate dishes.

whereas there appeared no stimulation of the 170-kDa band by EGF of N-3 cells (Fig. 6). The phosphorylated band is supposed to be EGF receptors: anti-phosphotyrosine antibody precipitates membranous EGF receptor with a molecular weight of 170 kDa when cells are incubated with EGF [4P]. These data suggest that the EGF receptor of N-3 cells is not autophosphorylated in the presence of EGF. Expression

of TGF-p and EGF

One could question why large number of colonies of N-3 appear in the presence of TGF-fl alone. A plausible mechanism for tbe appearance of N-3 colonies in soft agar might be due to enhanced expression of EGF by TGF-0. VanObberghen-Schilling et al. [42] have reported that addition of TGF-P or EGF to culture medium enhances TGF-P mRNA levels of NRK cells as well as the secretion of TGF-6 into the medium. Northern blot analysis with TGF-6 cDNA probe was done. As seen in Fig. 7A, treatment of NRK cells with EGF in(creased expression of TGF-P mRNA, but there appeared no significant change in mRNA levels in N-3 and N-9 cells treated with, or without EGF. Immunoprecipitates of biosynthetically labeled secretory proteins with monoclonal antibody against anti-rat EGF were examined by SDS-PAGE containing p-mercaptoethanol. As seen in Fig. 7B, there appeared no band corresponding to EGF in secretory proteins of NRK and N-9 cells treated with or without EGF and TGF-0. By contrast there appeared a significant amount of EGF (or TGF-a) secreted into culture medium when N-3 cells were treated with EGF or TGF-P. A malignantly transformed counterpart of NRK, N/MT-6, was also found to secrete EGF or TGF-CL These bands could not be detected when immunoprecipitation was carried out in the

TGF-ou, which is closely related to play a role in oncogenesis by inducing control of malignantly transformed cells ]3, 351. The transformation capacity of NRK cells to grow in soft agar by TGF-P is completely dependent upon exogenously added EGF or TGF-cu [4,17, andt or TGF-a induces rapid signaling events: elevation ofintracellular calcium contents, stirnn~at~o~ of phospholtpase C and of cellular uptake of glucose as well as amino acids, and subsequent onset of DNA synthesis and cell division [ 38,40,43]. By using EGF receptor-nonrespo~sive variants lacking EGF receptor from mouse Swiss 3T3 cells, stimulation of cell division as welt as of DNA synthesis has been reported by this study, we isolated, as clo NRK cells defective in the response to EGF tion in the presence of both colchicine and EGF. As seen in Table 1, DNA synthesis of N-3 and Nstate is only slightly stimulated if at NRK is significantly stimulated. D stimulated by TGF-P in the parental also stimulates cellular uptake of the NRK cells, but only slightly in N-3 and N-9 cells. TGF/3 however stimulates the uptake in both N-3 and N-9 cells, suggesting that the TGF-P receptor of N-3 and N9 cells is functional. NRK cells bave about 20,000 TGFp receptors on the cell surface 1371 and these receptors have no kinase activity ]45]. Both assays for DNA synthesis an ghtcose uptake indicate abortive signalings of EGF in N-3 and N-9 cells, possibly due to defective EGF receptor activity (Table 1). VanObberghen-Schihing et 611. [42] have reported

kDa

FIG. 6Stimulation of tyrosine phosphorylation of membranous proteins by EGF. NRK and N-3 cells at quiescent state were cultured without or -with 5 rig/ml EGF in MEM containing 0.5% FBS for 18 h and phosphorylated with 32Pi for 6 h, and then membrane proteins were prepared. Phosphorylated membrane proteins were immunoprecipitated with anti-phospbotyrosine antibody and subjected to 6% SDS-PAGE. Samples corresponding to 200,000 cpm were loaded on each lane.

88

HAMANAKA

123456

1 NRK rEGF

I-

1 N-3 +I-

1 N-S +I

-

+

‘. 1

NiK _

1 +I-

N-3 -

1 +I-

N-S -

IN/MT-61 +I-

-1

FIG. 7. Expression of TGF-P mRNA in NRK, N-3, and N-9 cells (A) and metabolic labeling and immunoprecipitation of EGF (B). In (A), cytoplasmic RNAs were extracted from NRK, N-3, and N-9 cells which were cultured in the absence or presence of 5 rig/ml EGF for 18 h, and these RNAs at 20 fig were hybridized to 3ZP-labeled TGF-P cDNA. Positions of rRNAs at 28 and 18 S are shown and equal amounts of RNAs were loaded on filters when stained with ethidium bromide. Arrow indicates TGF-P mRNA. In (B), NRK, N-3, N-9, and N/MT-6 cells incubated in the absence or presence of 5 rig/ml EGF or 1 rig/ml TGF-fl for 18 h were then labeled with [35S]cysteine for 20 h. The media were subjected to immunoprecipitation using rat EGF specific monoclonaI antibody, and SDS-PAGE (15% agar) and fluorography were performed. Lane 11 shows the control with preimmune serum when media of N/MT-6 cells are tested. Arrow indicates the band of EGF from the position of “‘1-EGF on a separate gel lane.

that synthesis of TGF-6 mRNA and secretion of TGF-P in NRK cell are stimulated by exogenous EGF or TGF,& Consistent with this report, we confirmed that EGF enhanced TGF-@ mRNA in NRK cells (Fig. 6 and Table 1). In contrast, addition of EGF to culture medium of N3 and N-9 cells failed to cause enhanced expression of TGF-P-mRNA, supporting the notion of defective signaling of EGF in N-3 or N-9 cells (Fig. 7). EGF stimu-

TABLE

ET

AL.

lates tyrosine phosphorylation of the EGF receptor and tyrosine kinase of the receptor is essential for the stimulation of EGF-induced signaling events [46]. Treatment of NRK cells with EGF stimulated phosphorylation of a membranous protein band of molecular size 170 kDa, supposedly the EGF receptor, but not that of N-3 cells (Fig. 6). Scatchard analysis for EGF receptor binding indicated that the number of EGF receptors on the cell surface of N-3 cells is estimated to be about 50% or less than that of NRK cells. However, the receptor function might be defective in N-3 cells. Two EGF-nonresponsive variants, N-3 and N-9, show different properties in their secretion activities of EGF in response to growth factors (Table 1). N-3 cells secreted EGF in response to either TGF-P or EGF, but there appeared to be no such stimulatory effect by TGFp or EGF on the secretion of EGF from NRK and N9 cells (Fig. 7). The anchorage independence of N-3 is observed in the presence of TGF-P alone. The growth of N-3 in soft agar can be supported by secreting EGF. Addition of polyclonal antibody against rat EGF reduced the appearance of colonies of N-3 in soft agar to less than 30% of the control when TGF-@ was present (R. Hamanaka, unpublished data), supporting the above notion. However, if secretory EGF from TGF-P-treated N-3 cells supports their anchorage-independent growth in the presence of TGF-P alone, one can argue how EGF exerts its signal transduction. A cellular route to induce EGF appears to be specifically magnified in N-3 by TGF@or EGF while its EGF-induced pathway is modified. EGF receptors might still be able to mediate signal(s) of EGF which is secreted from the TGF-P-treated N-3 cells. Alternatively TGF-P-induced signal transduction for anchorage-independent growth may compensate the EGF-induced pathway in N-3 cells, even when its EGF receptor is completely inert. It remains to be seen which pathway, in the presence of TGF-P, is actually operational in N-3 cells. Further study is necessary in order to elucidate the molecular mechanism responsible for the autocrine growth controls in TGF-P-treated N-3 cells. On the other hand, the incapacity of N-9 cells to grow in soft agar in the presence of both EGF and TGF-/3 might

1

Comparison of Biological Properties of NRK, N-3, and N-9 Cells in the Presenceof EGF and TGF-P N-3

NRK Biological Anchorage-independent DNA synthesis Glucose uptake TGF-@ synthesis EGF secretion

activities growth

N-9

E”

T

E+T

E

T

E+T

E

T

E+T

-b + + + -

-

++ + ++ nt nt

++

+ -

++ -

-

-

+ nt +

+ nt nt

-

+ nt -

+ nt nt

a E, EGF; T, TGF-@; E + T, EGF plus TGF-6. b (-) No effect; (+) significant effect; (++) twofold

+ nt -

or more

effect

greater

than

(+)

effect;

nt, not tested.

EGF-NONRESPONSIVE

NRK

suggest the missing factor in N-9 that blocks its transformation. Inducibility of EGF by EGF or TGF-P in N3 is the only property which is not observed in N-9, suggesting the absence of signal transduction by EGF in N9 cells. Transfection of total EGF receptor cDNA into N-9 cells may help to confirm this possibility.

LINES

J., Downward, and Seeburg,

“* 22’

8

J., Mayes, E. L. V., Whittle, P. H. (1984) Nature ilondon)

Ozaene, B., Fulton, R. J., and Kaplan, P. L. (1980) 9. Cell. Physiol. 105, 163. Kaplan, I?. L., Anderson, M., and Ozanne, B. (1982) Proc. Nacl. Acad. Sci. USA 79,485.

24.

Kuratomi, Ohkuma,

25.

Derynck, R., Jarrett, J. A., Chen, E. Y., Eaton, D. H., Bell, Assoian, R. K., Roberts, A. B., Sporn, M. B., and Goeddel, (1985) Nature (London) 316,7Oi.

26.

Robey, P. G., Young, M. F., Flanders, K. C., Roche, daiah, P., Reddi, A. II., Termine, J. D., Sporn, erts, A. B. (1987) J. Cell. Biol. BOB, 457.

27.

Hagino, Y., Mawatari, M., Yosbimura, A., Kohno, K., Kobayashi, and KUTQ~O, M. (1988) Japan. 3. Cancer Res. ‘i’9,74

28.

Seguchi, T., Yoshimura, A., Ono, M., Ebina, Y., Rutter, W. J., and Kuwano,

and

29.

M. W., Hood, L. E., Devare, S. G., S. A., and Antoniades, H. N. (1983)

Maniatis, T., Fritscb, E. F., and Sambrook, J. (1982) in Molecular Cloning, pp. 194-195, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

30.

Kohno, K., Kikuchi, J., Sato, S., Takano; H., Saburi, Y., Asoh, K., and Kuwano, M. (1988) Japan. J. Cancer Res. ?‘9,1238.

31.

Ono,

32.

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