Analysis of growth factors in renal cell carcinoma

Vol. 153, No. 2,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 818-824

June 16, 1988

ANALYSIS OF GROWTH FACTORS IN RENAL CELL CARCINOMA

Takahisa Nakamoto, Akihiro Usui, Kenji Oshima, Hideaki Ikemoto, Shinji Mitani*, and Tsuguru Usui

Department of Urology, Hiroshima University School of Medicine, i-2-3 Kasumi, Minami-ku,

Hiroshima 734, Japan

*Department of Urology, Kure National Hospital, 3-i, Aoyama, Kure 737, Japan

Received April 19, 1988

SUMMARY: Tissue extracts prepared from human renal tissue, renal cell carcinoma and serum-free conditioned media of ACHN cells and A498 cells, cell line originated from human renal cell carcinoma, stimulated DNA synthesis of BALB/c 3T3 cells. The activity (growth factor activity) was significantly higher in renal cell carcinoma than in normal tissues. Radioreceptor assay revealed that the contents of epidermal growth factor and type ~ transforming growth factor in the tissue extracts from renal cell carcinoma and conditioned media from renal cell carcinoma cell lines were below detectable level. Most of growth factor activity of the tissue extracts and conditioned media showed high affinity for heparin-Ultrogel, indicating that the major growth factor activity was due to heparin-binding growth factor(s). In addition, renal cell carcinoma contained growth factor activity for ACHN cells, which did not show specific affinity for heparin-Ultrogel. © 198~ Ao~de~io P r e s s , Z~o.

One of the most essential characters of malignant cells is uncontrolled growth. In this point, a hypothesis that the growth of malignant cells is enhanced by a growth factor or growth factors in autocrine or paracrine system is postulated(l,2). Growth factors have been identified in extracts of cancer tissues, cancer cells, and conditioned media of cell lines established from cancer tissues(3). Although there have been several reports on growth factors in renal cell carcinoma(4,5,6), the details of growth factor(s) in renal cell carcinoma is unclear. 0006-29lX/88

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In the present study, we demonstrate the presence of growth factor activities,

DNA synthesis stimulating activity for quiescent BALB/c 3T3

cells and renal cell carcinoma cell line, in renal cell carcinoma and determined some biochemical characteristics

of the growth factors.

MATERIALS AND METHODS i. Tissue and preparation of tissue extracts Six specimens of human renal cell carcinoma and adjacent normal kidney were obtained at radical nephrectomy. Renal tissue was homogenized in 5 volumes (v/w) of i0 m_M Tris-HCl (pH 7.5) and was centrifuged at 105,000 g for i hr. The resulting supernatant was used as a tissue extract. 2. Cells and preparation of serum-free conditioned media ACHN(7) and A498(8) cells were grown to confluence on 150 cm 2 plastic culture flask (Corning, New York) with Dulbecco's modified Eagle's medium (DME; Flow Laboratories, Irvine, Scotland) containing 10% calf serum (Flow Laboratories). The monolayers were washed 3 times with serum-free DME and replaced with 30 mi of the medium. Three days later, serum-free conditioned medium was collected and stored at -20 C until use. 3. Assay of growth factor activity Growth factor activity was assayed by measuring the ability of samples to stimulate the incorporation of ~H-thymidine into the DNA of quiescent BALB/c 3T3-3K cells as described by Nishi and associates(9). Activity was expressed as cpm/well/3 hr. Assay of growth factor activity using renal cell carcinoma cell line (ACHN) was carried out by the same procedure. 4. Radioreceptor assay of epidermal growth factor (EGF) and type transforming growth factor (TGF-~) Competition of 12sI labeled EGF (i00 Ci/~g, Amersham International plc, Backinghamshire) binding to normal rat kidney cell (NRK-52E) by unlabeled EGF or TGF-~ (recombinant TGF-~ was a gift from Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan)was determined according to the method described previously(lO). 5. Determination of heparin-binding growth factor content and affinity chromatography on a heparin-Ultrogel column Tissue extracts or serum-free conditioned media were supplemented with 4 M NaCI to make the final concentration of 0.5 M and then mixed with an equal volume of heparin-Ultrogel A4R (IBF biotechnics, Villeneuve-la-Garenne, France) which was equilibrated with I0 mM TrisHCI (pH 7.5) containing 0.5 M NaCI. The mixture was gently shaken for 3 hr at room temperature followed by centrifugation at 1,500 g for 5 min. The resulting supernatant was subjected to assay of growth factor activity. Control sample was treated with Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden). Some tissue extracts were analyzed by heparin-affinity chromatography. Tissue extract was applied to a heparin-Ultrogel column (1.2 x 7.5cm) equilibrated with i0 mM Tris-HCl (pH 7.5). The column was washed with the equilibration buffer and eluted with a gradient of 0-3.0 M NaCI. 819

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RESULTS i) Growth factor activity in renal tissue and conditioned media of renal cell lines All the specimens examined in the present study enhanced DNA synthesis of quiescent BALB/c 3T3 cells in a dose-dependent manner. Typical dose response curves for growth factor activity of tissue extracts and serum-free conditioned media were shown in figure i. The maximum stimulation of DNA synthesis induced by the tissue extracts of renal cell carcinoma, normal kidney and conditioned media of ACHN and A498 were about 3 times, 2 times and 1.7 times higher than that induced by calf serum, respectively. Contents of growth factor activity per i00 pg protein of the tissue extracts prepared from renal cell carcinoma and normal kidney were summarized in figure 2. The tissue extracts from renal cell carcinoma contained growth factor activity significantly higher than those from normal kidney (Fig. 2).

• 1 ~'n

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] p <0.01

4

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.................•

.9

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Cancer

Normal

Protein (mg/ml)

Fig. i. Dose-response curves for growth factor activity in tissue extracts from renal cell carcinoma and normal kidney, and the serum-free conditioned media from renal cell carcinoma cell lines (ACHN and A498). •, tissue extract of renal ceil carcinoma of patient number 2; [], tissue extract of normal kidney of patient number 2; • , serum-free conditioned medium from ACHN cells; •, serum-free conditioned medium from A498 cells; ....., calf serum. Fig. 2. Growth factor activity of tissue extracts from renal cell carcinoma and normal kidney. The growth factor activity was represented as stimulation of DNA synthesis in iOO~g protein of sample. The activity in tissue extract of renal cell carcinoma was significantly higher than those of normal kidney (p< 0.01).

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90 o

50

E

L,

i;

Idoo

Concentration of EGF or TGF-~(ng) F i g . 3. C o m p e t i t i o n f o r ~2SI-EGF b i n d i n g by u n l a b e l e d EGF and TGF-~. The

concentration of EGF or TGF-~ and the percent binding to total binding were represented through a log-logit transformation, e, EGF; o, TGF-~.

2) Estimation of EGF and TGF-~ contents by radioreceptor assay

125I-EGF binding for NRK-52E cells was inhibited by unlabeled EGF and TGF-~. The relationship between the concentration of unlabeled EGF (l-lO0ng) or TGF-s (6.8-680ng) and the percent binding to total binding obtained through a log-logit transformation was linear (Fig. 3). One hundred microgram protein of tissue extracts from renal cell carcinoma or normal kidney, 2.0 ~g protein of serum-free conditioned medium from ACHN cells and 13.0 ~g protein of that from A498 cells did not inhibit 12sI-EGF binding for NRK-52E cells (Table i), indicating the contents of EGF and TGF-~ in the samples were below detection limit of the assay system.

3) Heparin-binding growth factor content in the tissue extracts and conditioned media and analysis of growth factor by heparin-Ultrogel affinity chromatography

The content of heparin-binding growth factor was more than 70% of the total growth factor activity in most specimens

examined (Table I).

When tissue extract of renal cell carcinoma (No. 4 in Table I) was subjected to heparin-Ultrogel affinity chromatography, almost all the protein was recovered from flow-through fractions, whereas about 76% of

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Table i. Biochemical characteristics of growth factor in tissue extracts from human renal cell carcinoma and normal kidney, and serum free conditioned media from renal cell carcinoma cell lines Patients

Competition for IzSI-EGF binding (B/Bo) (lO0pg protein)

Relative contents of heparin-binding growth factor (% of total activity)

Cancer 1 2 3 4 5 6

89.6 82.9 97.2 95.1 94.9 i00

67.4 87.3 84.9 67.6 84.8 88.5

Normal 1 2 3 4 5 6

85.2 98.2 98.6 91.2 95.5 i00

33.5 76.0 57.7 77.1 58.1 80.4

Conditioned media ACHN .4.498

i00 (2.0 pg protein) 95.6 (13.0 ~g protein)

73.4 70.8

growth factor activity was bound to the column (Fig. 4). The growth factor activity bound to the column was eluted as at least two distinct components; one was eluted with 1.1-1.2 M NaCI (about 30% of the total growth factor activity applied) and the other with 1.4-1.5 M NaCI (about 46%). In addition to the growth factor activity detected with BALB/c 3T3 cells, flow-through fractions contained growth factor activity for ACHN cells (Fig. 4).

DISCUSSION In order to clarify control mechanisms of the growth of renal cell carcinoma, growth factors in renal cell carcinoma, normal kidney and cell lines derived from renal cell carcinoma were analyzed using BALB/c 3T3 cells as a target. The tissue extracts from renal cell carcinoma contained significantly higher level of growth factor activity than those from normal kidney. It was reported that the content of EGF in human renal tissue was as high as that of salivary glands(ll). In addition, the observations that a large amount of TGF-~ was excreted in

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co

4

3 o x

o co

E o v

1

20

60

40 Fraction

80

number

Fig. 4. Affinity chromatography of the tissue extract from renal cell carcinoma of patient number 4 on a heparin-Ultrogel column. Tissue extract (12 ml; 13.3 mg protein/ml) was applied to a heparin-Ultrogel column (1.2 x 7.5 cm). After washing, a gradient (240 m l) of 0-3.0 M NaCI was applied. Fraction volumes was 4ml. • , DNA synthesis stimulating activity for BALB/c 3T3 calls; o , DNA synthesis stimulating activity for ACHN cells; ....., absorbance at 280 nm.

urine in patients with disseminated cancer(12) and that TGF-~ cDNA could be cloned from the cell line derived from renal cell carcinoma(5) were reported. Although these strongly suggested that the growth factor activity in renal tissues and the cell lines was due to EGF and/or TGF~, radioreceptor~assay revealed that the tissues and cell lines did not contain considerable amount of neither factor. The majority of growth factor activity in both renal carcinoma and normal kidney was appeared to be due to heparin-binding growth factor(s) as judged from the fact that more than 70% of the activity in most specimen was absorbed to heparin-Ultrogel in the presence of 0.5 M NaCI and was eluted with NaCI higher than 1 M

(in the case of renal cell carcinoma). There is a

possibility that heparin-binding growth factor in renal cell carcinoma and normal kidney was derived from stromal or blood component which were equally involved in both tissues. However, the results with serum-free conditioned media of renal cell carcinoma cell lines indicated that renal cell carcinoma cells secrete heparin-binding growth factor, though the secretion might not be specific for renal cell carcinoma cells. The significance of the presence of heparin-binding growth factor in renal cell carcinoma cells is currently unknown. There is a

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possibility that the growth factor enhances the growth of renal cell carcinoma cells through autocrine manner. Heparin-binding growth factor, however, failed to stimulate DNA synthesis of ACHN cells (Fig. 4). Thus the growth promoting effect of heparin-binding growth factor on renal cell carcinoma cells, if any, was not a direct action but via growth stimulation on stromal or vascular component. In addition to heparin-binding growth factor, renal cell carcinoma contained a growth factor which stimulated DNA synthesis of renal cell carcinoma cell line, ACHN.

Although the nature of the growth factor is

totally unknown except that it is distinct from heparin-binding growth factor, this factor is thought to be one of the candidates for autocrine growth factor of renal cell carcinoma.

REFERENCE i. Kuru,M., and Kosaki,G. (1961) Jap. J. Cancer Clin. 7, 70-81. 2. Sporn,M.B., and Todaro,G.J. (1980) New Engl. J. Med. 303, 878-880. 3. Nishikawa,K., and Masuda,Y. (1987) Jap. Clin. Endocrinol. 35, 12051209. 4. Bard,R.H., Mydlo,J.H., and Freed,S.Z. (1986) Urology 27, 447-450. 5. Derynck, R., Roberts,A.B., Winkler,M.E., Chen,E.¥., and Goeddel,D.¥. (1984) Cell 38, 287-297. 6. Walther,P.J., Little,N.A., Webb,K.S., and Durham,N.C. (1987) J. Urol. 137, 251A. 7. Borden,E.C., Hogan,T.F., and Voelkel,J.G. (1982) Cancer Res. 42, 4948-4953. 8. Giard,D.J., Aaroson,S.A., Todaro,G.J., Arnstein,P., Kersey,J.H., Dosik, H., and Parks,W.P. (1973) J. Natl. Cancer Inst. 51, 1417-1423. 9. Nishi,N., Matuo,Y., Muguruma,Y., Yoshitake,Y., Nishikawa,K., and Wada,F. (1985) Biochem. Biophys. Res. Commun. 132, 1103-1109. i0. Usui,T., Moriyama,N., Ishibe,T., and Nakatsu,H° (1984) Biochem. Biophys. Res. Commun. 120, 879-884. ii. Sumiyoshi,H., Taniyama,K., Yasui,W., Tahara,E., Sakamoto,T., Hayashi,H., and Nagara,S. (1987) Tr. Soc. Pathol. Jap. 76, 135-136. 12. Sherwin,S.A., Twardzik, D.R., Bohn,W.H., Cockley, K.D., and Todaro,G.J. (1983) Cancer Res. 43, 403-407.

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