Gastroenterologia Japonica Copyright 9 1992 by The Japanese Socie{y of Gastroenterology
Vol. 27, No. 4 Printed in Japan
Correlation between epidermal growth factor receptor concentration and the growth of human gastric cancer xenografts in nude mice Teruo KIYAMA 1, Masahiko ONDA l, Akira TOKUNAGA 1, Itsuro FUJITA 1, Takeshi OKUDA l, Takashi MIZUTANI 1, Toshiro YOSHIYUKI 1, Yasuhito SHIMIZU 1, Keigo NISHI 1, Norio MATSUKURA 1, Noritake TANAKA 1, Yuko TODOME 2, Hisashi OHKUNI 2, and Goro ASANO 3
1First Department of Surgery, 2Department of Microbiology and SDepartment of Pathology, Nippon Medical School, Tokyo, Japan Summary: Seven human gastric cancer xenografts with different concentrations of EGF receptor were established in nude mice. The expression of EGF receptor in the tumors was demonstrated by Western blotting with anti-EGF receptor antibody, binding assay with laSI-EGF and immunohistochemistry with anti-EGF receptor antibody. Western blotting revealed EGF receptor doublet bands at molecular masses of 150 KDa and 170 KDa in all of the samples. The concentration of 12SI-EGF binding activity in the tumors ranged from 36.0 to 11,000 fmol/mg protein, with a mean of 345 fmol/mg protein. EGF receptor was also demonstrated immunohistochemically on the apical border of the glands and the cell membrane of the tumor cells. There seemed to be a close correlation between the concentration of 12SI-EGF binding activity and the doubling time of these tumors in nude mice (y = --0.68). However, no definite correlation was observed between EGF ligand binding and histological features of intestinal type or diffuse type. The expression of EGF receptor appears to facilitate the growth of human gastric cancer xenografts in nude mice. Gastroenterol Jpn 1992;27:459-465. Key words: E G F receptor; gastric cancer; nude mice.
Introduction Epidermal growth factor (EGF) has been found in h u m a n urine and body fluids and is present in salivary glands and Brunner's glands of the duoden u m 1'2. E G F has been shown to stimulate cell growth in the gastrointestinal tract in v i v o 3 and inhibit gastric acid secretion 4. E G F in vitro appears to both stimulate and inhibit the growth of cancer cells with a high concentration of E G F receptor. The concentration of E G F that stimulates the growth of normal keratinocytes inhibits the proliferation of A431 epidermoid carcinoma cells in monolayer culture s. In contrast, E G F stimulates the growth of A431 cells in soft agar 6, and also w h e n xenografted in nude mice 7. E G F was
immunohistochemically detected m o r e often in h u m a n gastric cancer and EGF-positive cells showed a higher degree of local invasion and lymph node metastasis 8'9. E G F binds to a specific cell m e m b r a n e receptor (EGF receptor), which is a glycoprotein with a molecular weight of 170,0001~. The structure of the E G F receptor is homologous with the product of avian erythroblastosis virus oncogene v-erbn, which possesses tyrosine kinase activity. A n increasing a m o u n t of data has indicated that E G F receptor is expressed in a variety of cancers including those of the vulva, esophagus, breast and brain 121s. E G F receptor is found mainly in squamous cell carcinoma or epidermoid carcin o m a and is thought to regulate the growth of
Received July 16, 1991. Accepted February 28, 1992. Address for correspondence: Teruo Kiyama,M.D., First Department of Surgery, Nippon Medical School, 1-1-5Sendagi, Bunkyo-ku,Tokyo 113, Japan.
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these cancer cells ~6. Recent studies on surgically resected gastric cancer tissues, considered to have a low concentration of EGF receptor, have revealed the presence of EGF receptor immunohistochemically and shown that cases positive for EGF receptor have a poorer prognosis 17. These results raise the question of whether increased expression of EGF receptor in gastric cancers is associated with their growth. We therefore attempt to identify EGF receptor in human gastric cancer xenografts in nude mice and to examine whether the expression of EGF receptor is associated with the growth of these tumors. Materials and Methods
Animals Four-week-old male athymic nude mice were obtained from Sankyo Lab. Inc. (Tokyo, Japan). They were maintained under specific pathogenfree conditions, which involved the use of a laminar air-flow rack and sterilized food, water, bedding and cages. Experiments were begun after observing the mice for two weeks. Human gastric cancer xenografts Seven human gastric cancer xenografts, originally obtained from patients with gastric cancer, were established in our laboratory 18. Histological classification of the xenografts was done according to Laur6n 19. The implantation techniques used for serial transfer have been described previously2~ Briefly, fresh tissues removed from dorsal tumors of nude mice were washed with saline and cut into pieces 2 mm in diameter. Each piece was implanted by trocar into the flank of a nude mouse. The implantation sites were carefully inspected and measured twice a week. Relative tumor volume (mm 3) was calculated as 1/2ab 2 (a: long diameter, b: short diameter) 21. The data for exponentially growing tumors were analyzed by linear regression, and the doubling time was calculated as described by Giavazzi et al. 22. Identification of EGF receptor in human gastric cancer xenografts from nude mice Detection of EGF receptor by Western blotting
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Frozen tissues were scraped into buffer containing 20 mM HEPES, pH 7.4, 5 mM EDTA, 1 mM PMSF, and 1% Trasylo123, homogenized in a glass homogenizer with a Teflon pestle, and then centrifuged at 190,000 • g for 45 min at 5~ The pellet was suspended in buffer containing 20 mM HEPES, pH 7.4, 5 mM EDTA, 1% (v/v) Triton X100, 10% (v/v) glycerol, 1 mM PMSF and 1% Trasylol at 4~ and after centrifugation, the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). SDS-PAGE was carried out by the method of Laemmli 24. After electrophoresis, the gel was electroblotted onto a nitro-cellulose membrane by the method of Towbin et al. 2s. The membrane was incubated with anti-EGF receptor antibody (GR-01, Oncogene Science Inc., NY, USA) at a dilution of 1:50, at room temperature for 2 h. Alkaline phosphatase-conjugated antiprimary antibody (Kirkegaard & Perry Laboratories, Inc., MD, USA), at a dilution of 1 : 500, was used as secondary antibody for the identification of EGF receptor. The total protein content was determined by the method of Lowry et al. 26.
leSl-EGF ligand binding assay Frozen tissues were homogenized with buffer containing 5 mM sodium phosphate, pH 7.5, 10% glycerol, 0.5 mM dithiothreitol and 1 mM PMSF, using a Polytron homogenizer, and the homogenate was centrifuged at 1,000 x g for 10 min at 4~ The supernatant was then centrifuged at 100,000 x g for 35 min at 4~ The precipitate was resuspended in the same buffer and used for the assay. One hundred microliters of sample was incubated with 200 ~1 of EGF (Wakunaga, Hiroshima, Japan) and 100 V1 of 125I-EGF (Amersham Lab., Amersham, UK) for 1 h at 25~ with different concentrations of EGF ranging from 0.16 to 80 nM, and 20,000 dpm 125I-EGF. The sample was incubated with 1 ml of reagent containing 0.3% bovine ~,-globulin and 16% polyethyleneglycol, for 30 rain at 4~ and then centrifuged at 1800 x g for 30 rain at 4~ the radioactivity of the pellet was counted in a gamma counter. The concentration of EGF binding activity was calculated by Scat-
EGF receptor in gastric cancer xenografts
August 1992
5000
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~
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~
NMSI2
461
--- 200K
--- 92.5K
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.---69K
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----46K 10(
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3
4
5
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Figure 1. Growth rate of human gastric cancer xenografts in nude mice. The xenograft tumors proliferated exponentially after a latent period. (Mean_+SD).
chard analysis 27 and expressed as fmol per. mg membrane protein.
Immunohistochemical demonstration of EGF receptor Immunohistochemical study of EGF receptor was carried out using the avidin-biotin-peroxidase complex (ABC) m e t h o d of Hsu et al. 28. Cryostat sections (4 ~m) were immersed in methanol containing 0.03% hydrogen peroxidase for 30 min to block any endogenous peroxidase activity, and then treated for 60 min at room temperature with anti-EGF receptor antiserum (GR-01, Oncogene Science Inc., NY, USA). Avidin and biotin reagents were obtained from BioGenex Laboratories (Biotin-Streptavidin Immunostaining Kit, Dublin, CA). Controls for ABC immunohistochemistry were run simultaneously as follows: (1) replacement of primary antiserum with normal n o n - i m m u n e mouse IgG, (2) use of EGF receptor antiserum absorbed with EGF, (3) omission of secondary antiserum. The sections were counterstained with hematoxylin. Results
Growth of human gastric cancer xenografts in nude mice Seven h u m a n gastric cancer xenografts were
"'8OK
Figure 2. The expression of EGF receptor in a human gastric cancer xenograft in nude mice (NMS 2) by Western blotting using anti-EGF receptor antibody (GR-01, OSI). The doublet bands are positive; a dominant band at 170 KDa is seen, paired with a smaller band at 150 KDa.
established. The tumor of all xenografts in nude mice grew slowly during the latent period, and then began to grow exponentially, as shown in Figure 1. The mean relative volume at 6 weeks after inoculation ranged from 1.5 • 102 to 4.7 • 103 (mm3), and the t u m o r doubling time of the 7 xenografts was different from each other.
Identification of EGF receptor in human gastric cancer xenografts of nude mice Detection of EGF receptor by Western blotting EGF receptor was detected as doublet bands at 150 KDa and 170 KDa in all of the xenografts by Western blotting, as shown in Figure 2. 1esI-EGF ligand binding assay Table 1 shows the concentration of 125I-EGF binding in h u m a n gastric cancer xenografts from nude mice. The specific binding was calculated by Scatchard analysis, as shown in Figure 2. EGF binding activity was examined for each xenograft, and the binding concentration was found to have
462
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Vol. 27, No. 4
Table 1. Expression of EGF receptor in human gastric cancer xenografts in nude mice
Histologya) NMS
2 6 10 11 12 13 24
intestinal intestinal intestinal diffuse diffuse diffuse intestinal
Placenta
-
a91
Doubling time (days)
EGF binding capacity (fmol/mg protein)
Western blotting for EGF receptor
Immunohistochemistry for EGF receptor
12.2 b) 28.4 14.1 18.0 7.8 9.6 30.6
11,000(4) c) 36.0(2) 117(3) 48.9(3) 799(7) 537(2) 60.2(3)
)ositive ~ositive ~ositive 9ositive )ositive )ositive )ositive
positive positive positive positive positive positive positive
2,180(5)
positive
NTdl
-
to the classification of Laur6n. b)Mean, e)Mean (Number of experiments), d)NT; Not tested. 3 0 -! o
20h
ml0_
o
20
40 6'0 EGF bound (fmol) Figure 3. Scatchard analysis of 1251-1abeledEGF binding capacit,/in human gastric cancer xenograffs (NMS2). Kd= 1.95x 10 -~2 EGF binding capacity=7,820 fmol/mg protein.
a broad range with a mean of 345 fmol/mg protein. The EGF binding activity of NMS 2 was higher than that of normal placental tissue. The dissociation constant (Kd) of E G F for the samples ranged from 1.87 x 10-12 to 20.51 • 10-12 M. These values were close to those of placental tissue. There was no definite correlation between the concentration of EGF receptor and histological features of intestinal type or diffuse type.
Immunohistochemical staining of EGF receptor All of seven xenografts were positive for EGF receptor immunohistochemically, as revealed by staining predominantly on the apical border of the glands, the cell membrane and in the cytoplasm of the cancer cells, as shown Figure 4.
Concentration of EGF receptor and the doubling time of human gastric cancer xenografts The tumor doubling time of h u m a n gastric cancer xenografts ranged from 7.8 to 30.6 days (Table 1). There seemed to be an inverse correlation between the concentration of EGF receptor and the doubling time of the 7 xenografts, with a correlation coefficient o f - 0 . 6 8 , as shown in Figure 5. All tumors having more than 100 fmol/mg EGF receptor protein had a doubling time of less than 14 days. Conversely, all tumors having less than 100 fmol/mg EGF receptor protein showed a t u m o r doubling time of more than 14 days. Discussion EGF receptor was demonstrated by Western blotting, 125I-EGF ligand binding and immunohistochemistry in h u m a n gastric cancer xenografts of nude mice. Western blotting revealed E G F receptor as doublet bands at 150 KDa and 170 KDa in all of seven xenografts, thus corresponding to the published molecular weights for EGF receptor protein 29. nSI-EGF binding assay showed that a large n u m b e r of E G F binding sites were present in h u m a n gastric cancer xenografts. I m m u n o h i s tochemical studies demonstrated that E G F receptor was clearly positive predominantly on the apical border of the glands, the cell m e m b r a n e and in the cytoplasm of the cancer cells in the xenografts. The results of Western blotting matched those of immunohistochemistry in all specimens. Recently, EGF receptor was detected i m m u n o -
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463
EGF receptor in gastric cancer xenografts
Figure 4.
Immunohistochemistry of EGF receptor in a human gastric cancer xenograft from nude mice (NMS2) using anti-EGF receptor antibody (GR-01, OSi). EGF receptor is positive predominantly on the apical border of the glands, and the cell membrane of the tumor cells. (x 200).
10, 000 5, 000
~o -~ 1,000 c o. =5 c E ~
500
t
tO0
t
50
ib
fs
~o
fs
~0
Tumor doubling time (days)
Figure 5. EGF binding capacity and the growth of human gastric cancer xenografts in nude mice. log Y=3.55-0.067X, Correlation coefficient= -0.68.
histochemically in 9 of 15 surgically resected gastric cancer specimens 3~ and increased levels of EGF receptor in gastric cancer have been shown by I:5I-EGF binding assay 31'32. We examined EGF receptor in surgical specimens of stomach cancer by ]2SI-EGF binding assay, and found that 23 (46%) of 50 specimens were EGF receptor-positive33. Thus, reports on the presence of EGF receptor in human gastric cancers have been accumulating. EGF receptor was initially shown to be present more frequently in squamous cell carcinomas
than in adenocarcinomas, and the gene for EGF receptor was found to be more often amplified in the former than the latter 16. Squamous cell carcinoma of the lung, however, did not contain a greater concentration of EGF receptor than adenocarcinoma of the lung 34. The degree of EGF receptor expression in cancer cells may vary from tissue to tissue. The present study showed that some human gastric adenocarcinomas among the xenografts had a large amount of EGF receptor, the highest receptor concentration being far greater than that of normal placental tissue. An inverse correlation was observed between EGF binding activity and the doubling time of the 7 xenografts, indicating an effect on tumor growth. The growth of xenografts having a large amount of EGF receptor, more than 100 fmol/mg protein, was faster than that ofxenografts having a small amount of EGF receptor, less than 100 fmol/mg protein. Tumor tissues exhibited higher EGF receptor mRNA expression levels than those in the adjacent normal tissues 3s. The degree of gene amplification and concentration of EGF receptors have been shown to be directly correlated with the growth of A431 cells transplanted into athymic mice 36. Thus, the expression
464
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of EGF receptors appears to facilitate the growth of human gastric cancer xenografts in nude mice. EGF, via its receptor, has both stimulative and inhibitory effects on the in vitro growth of A431 depending on culture conditions; while EGF inhibits anchorage-dependent growth in monolayer culture, it stimulates anchorage-independent growth in soft agar 6. The ability of cells to grow as colonies in soft agar has been shown to correlate highly with tumorigenicity in nude mice 37. EGF was found to stimulate the in vivo growth of EGF receptor-hyperproducing A431 cells 7. It will be necessary to examine the effect of EGF on the growth of human gastric cancer xenografts in nude mice having different concentrations of EGF receptor, since EGF-EGF receptor stimulation may contribute to the growth of these tumor cells. This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and the Science Research Promotion Fund from the Japan Private School Promotion Foundation, Japan. The authors are grateful to Mr. Mitsuhiro Kudoh for excellent technical assistance and Ms. Kanna Mizuno for animal care.
References 1. Elder JB, Williams G, Lacey E, et al. Cellular localization of human urogastrone/epidermal growth factor. Nature 1978;271: 466-467. 2. Heitz PU, Kasper M, Noorden SV, et al. Immunohistochemical localization of urogastrone to human duodenal and submandibular glands. Gut 1978;19:408-413. 3. Dembinski A, Gregory H, Konturek SJ, et al. Trophic action of epidermal growth factor on the pancreas and gastroduodenal mucosa in rats. J Physiol 1982;325:35-42. 4. Konturek SJ, Cieszkowski M, Jaworek J, et al. Effects of epidermal growth factor on gastrointestinal secretions. Am J Physiol 1984;246:580-586. 5. Gill GN, Lazer CS. Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells. Nature (London) 1981;293:305-307. 6. Lee K, Tanaka M, Shigeno C, et al. Epidermal growth factor stimulates the anchorage-independent growth of human squamous cell carcinomas overexpressing its receptors. Biochem Biophys Res Commun 1990;168:905-911. 7. Ozawa S, Ueda M, Ando N, et al. Stimulation by EGF of the growth of EGF receptor-hyperproducing tumor cells in athymic mice. Int J Cancer 1987;40:706-710. 8. Onda M, Tokunaga A, Nishi K, et al. The correlation of epidermal growth factor with invasion and metastasis in human gastric cancer. Jpn J Surg 1990;20:269-274. 9. Tahara E, Sumiyoshi H, Hata J, et al. Human epidermal growth
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factor in gastric carcinoma as a biologic marker of high malignancy. Jpn J Cancer Res 1986;77:145-152. 10. Hunter T. The epidermal growth factor receptor gene and its product. Nature 1984;311:414-416. 11. Downward J, Yarden Y, Mayes E, et al. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 1984;307:521-527. 12. Merfino GT, Xu YH, Ishii S, et al. Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells. Science (Wash. DC) 1984;224:417-419. 13. Ozawa S, Ueda M, Ando N, et al. High incidence of EGF receptorhyperproduction in esophageal squamous cell carcinomas. Int J Cancer 1987;39:333-337. 14. Fitzpatrick SL, LaChance MP, Schultz GS. Characterization of epidermal growth factor receptor and action on human breast cancer cells in culture. Cancer Res 1984;44:3442-3447. 15. Libermann TA, Razon N, Bartal AD, et al. Expression of epidermal growth factor receptors in human brain tumors. Cancer Res 1984;44:753-760. 16. Yamamoto T, Kamata N, Kawano H, et al. High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell fines. Cancer Res 1986;46:414416. 17. Yasui W, Hata J, Yokozaki H, et al. Interaction between epidermal growth factor and its receptor in progression of human gastric carcinoma. Int J Cancer 1988;41:211-217. 18. Yoshiyuki T, Shimizu Y, Onda M, et al. Immunohistochemical demonstration of epidermal growth factor in human gastric cancer xenografts in nude mice. Cancer 1990;65:953-957. 19. Laur6n P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal type carcinoma. Acta Pathol Microbiol Scand 1965;64:31-49. 20. Kiyama T, Onda M, Tokunaga A, et al. Changes in serum and tissue carcinoembryonic antigen of a human gastric cancer xenograft in nude mice. Jpn J Cancer Res 1990;81:58-62. 21. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24:148-154. 22. Giavazzi R, Campbell DE, Jessup JM, et al. Metastatic behavior of tumor cells isolated from primary and metastatic human colorectal carcinomas implanted into different sites in nude mice. Cancer Res 1986;46:1928-1933. 23. Slieker LJ, Lane MD. Post-translational processing of the epidermal growth factor receptor. J Biol Chem 1985;260:687-690. 24. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685. 25. Towbin H, Staehefin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:43504354. 26. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 27. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1949;51:600-672. 28. Hsu SM, Raine L, Fanger H. Use of avidin-peroxidase complex (ABC) in immunoperoxidase techniques; a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577-580. 29. Cohen S, Ushiro H, Stoschek C, et al. A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 1982;257:1523-1531.
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EGF receptor in gastric cancer xenografls
30. Sakai K, Mori S, Kawamoto T, et al. Expression of epidermal growth factor receptors on normal human gastric epithelia and gastric carcinoma. J Natl Cancer Inst 1986;77:477-483. 31. Yasui W, Sumiyoshi H, Hata J, et al. Expression of epidermal growth factor receptor in human gastric and colonic carcinomas. Cancer Res 1988;48:137-141. 32. Pfeiffer A, Rothbauer E, Wiebecke B, et al. Increased epidermal growth factor receptors in gastric carcinomas. Gastroenterology 1990;98:961-967. 33. Nishi K, Onda M, Tokunaga A, et al. Expression of epidermal growth factor receptor in human gastric cancer. Proc 48th Ann Meet Jpn Cancer Assoc 1989;1333. (in Japanese)
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34. Veale D, Kerr N, Gibson G J, et al. Characterization of epidermal growth factor receptor in primary human non-small cell lung cancer. Cancer Res 1989;49:1313-1317. 35. Yoshida K, Kyo E, Tsujino T, et al. Expression of epidermal growth factor, transforming growth factor-a and their receptor genes in human gastric carcinomas; implication for autocrine growth. Jpn J Cancer Res 1990;81:43-51. 36. Santon JB, Cronin MT, Macleod CL, et al. Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice. Cancer Res 1986;46:4701-4705. 37. Kahn P, Shin SI. Cellular tumorigenicity in nude mice. J Cell Biol 1979;82:1-16.
Vol. 27, No. 4 Printed in Japan
Correlation between epidermal growth factor receptor concentration and the growth of human gastric cancer xenografts in nude mice Teruo KIYAMA 1, Masahiko ONDA l, Akira TOKUNAGA 1, Itsuro FUJITA 1, Takeshi OKUDA l, Takashi MIZUTANI 1, Toshiro YOSHIYUKI 1, Yasuhito SHIMIZU 1, Keigo NISHI 1, Norio MATSUKURA 1, Noritake TANAKA 1, Yuko TODOME 2, Hisashi OHKUNI 2, and Goro ASANO 3
1First Department of Surgery, 2Department of Microbiology and SDepartment of Pathology, Nippon Medical School, Tokyo, Japan Summary: Seven human gastric cancer xenografts with different concentrations of EGF receptor were established in nude mice. The expression of EGF receptor in the tumors was demonstrated by Western blotting with anti-EGF receptor antibody, binding assay with laSI-EGF and immunohistochemistry with anti-EGF receptor antibody. Western blotting revealed EGF receptor doublet bands at molecular masses of 150 KDa and 170 KDa in all of the samples. The concentration of 12SI-EGF binding activity in the tumors ranged from 36.0 to 11,000 fmol/mg protein, with a mean of 345 fmol/mg protein. EGF receptor was also demonstrated immunohistochemically on the apical border of the glands and the cell membrane of the tumor cells. There seemed to be a close correlation between the concentration of 12SI-EGF binding activity and the doubling time of these tumors in nude mice (y = --0.68). However, no definite correlation was observed between EGF ligand binding and histological features of intestinal type or diffuse type. The expression of EGF receptor appears to facilitate the growth of human gastric cancer xenografts in nude mice. Gastroenterol Jpn 1992;27:459-465. Key words: E G F receptor; gastric cancer; nude mice.
Introduction Epidermal growth factor (EGF) has been found in h u m a n urine and body fluids and is present in salivary glands and Brunner's glands of the duoden u m 1'2. E G F has been shown to stimulate cell growth in the gastrointestinal tract in v i v o 3 and inhibit gastric acid secretion 4. E G F in vitro appears to both stimulate and inhibit the growth of cancer cells with a high concentration of E G F receptor. The concentration of E G F that stimulates the growth of normal keratinocytes inhibits the proliferation of A431 epidermoid carcinoma cells in monolayer culture s. In contrast, E G F stimulates the growth of A431 cells in soft agar 6, and also w h e n xenografted in nude mice 7. E G F was
immunohistochemically detected m o r e often in h u m a n gastric cancer and EGF-positive cells showed a higher degree of local invasion and lymph node metastasis 8'9. E G F binds to a specific cell m e m b r a n e receptor (EGF receptor), which is a glycoprotein with a molecular weight of 170,0001~. The structure of the E G F receptor is homologous with the product of avian erythroblastosis virus oncogene v-erbn, which possesses tyrosine kinase activity. A n increasing a m o u n t of data has indicated that E G F receptor is expressed in a variety of cancers including those of the vulva, esophagus, breast and brain 121s. E G F receptor is found mainly in squamous cell carcinoma or epidermoid carcin o m a and is thought to regulate the growth of
Received July 16, 1991. Accepted February 28, 1992. Address for correspondence: Teruo Kiyama,M.D., First Department of Surgery, Nippon Medical School, 1-1-5Sendagi, Bunkyo-ku,Tokyo 113, Japan.
460
T. Kiyama et al.
these cancer cells ~6. Recent studies on surgically resected gastric cancer tissues, considered to have a low concentration of EGF receptor, have revealed the presence of EGF receptor immunohistochemically and shown that cases positive for EGF receptor have a poorer prognosis 17. These results raise the question of whether increased expression of EGF receptor in gastric cancers is associated with their growth. We therefore attempt to identify EGF receptor in human gastric cancer xenografts in nude mice and to examine whether the expression of EGF receptor is associated with the growth of these tumors. Materials and Methods
Animals Four-week-old male athymic nude mice were obtained from Sankyo Lab. Inc. (Tokyo, Japan). They were maintained under specific pathogenfree conditions, which involved the use of a laminar air-flow rack and sterilized food, water, bedding and cages. Experiments were begun after observing the mice for two weeks. Human gastric cancer xenografts Seven human gastric cancer xenografts, originally obtained from patients with gastric cancer, were established in our laboratory 18. Histological classification of the xenografts was done according to Laur6n 19. The implantation techniques used for serial transfer have been described previously2~ Briefly, fresh tissues removed from dorsal tumors of nude mice were washed with saline and cut into pieces 2 mm in diameter. Each piece was implanted by trocar into the flank of a nude mouse. The implantation sites were carefully inspected and measured twice a week. Relative tumor volume (mm 3) was calculated as 1/2ab 2 (a: long diameter, b: short diameter) 21. The data for exponentially growing tumors were analyzed by linear regression, and the doubling time was calculated as described by Giavazzi et al. 22. Identification of EGF receptor in human gastric cancer xenografts from nude mice Detection of EGF receptor by Western blotting
Vol. 27, No. 4
Frozen tissues were scraped into buffer containing 20 mM HEPES, pH 7.4, 5 mM EDTA, 1 mM PMSF, and 1% Trasylo123, homogenized in a glass homogenizer with a Teflon pestle, and then centrifuged at 190,000 • g for 45 min at 5~ The pellet was suspended in buffer containing 20 mM HEPES, pH 7.4, 5 mM EDTA, 1% (v/v) Triton X100, 10% (v/v) glycerol, 1 mM PMSF and 1% Trasylol at 4~ and after centrifugation, the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). SDS-PAGE was carried out by the method of Laemmli 24. After electrophoresis, the gel was electroblotted onto a nitro-cellulose membrane by the method of Towbin et al. 2s. The membrane was incubated with anti-EGF receptor antibody (GR-01, Oncogene Science Inc., NY, USA) at a dilution of 1:50, at room temperature for 2 h. Alkaline phosphatase-conjugated antiprimary antibody (Kirkegaard & Perry Laboratories, Inc., MD, USA), at a dilution of 1 : 500, was used as secondary antibody for the identification of EGF receptor. The total protein content was determined by the method of Lowry et al. 26.
leSl-EGF ligand binding assay Frozen tissues were homogenized with buffer containing 5 mM sodium phosphate, pH 7.5, 10% glycerol, 0.5 mM dithiothreitol and 1 mM PMSF, using a Polytron homogenizer, and the homogenate was centrifuged at 1,000 x g for 10 min at 4~ The supernatant was then centrifuged at 100,000 x g for 35 min at 4~ The precipitate was resuspended in the same buffer and used for the assay. One hundred microliters of sample was incubated with 200 ~1 of EGF (Wakunaga, Hiroshima, Japan) and 100 V1 of 125I-EGF (Amersham Lab., Amersham, UK) for 1 h at 25~ with different concentrations of EGF ranging from 0.16 to 80 nM, and 20,000 dpm 125I-EGF. The sample was incubated with 1 ml of reagent containing 0.3% bovine ~,-globulin and 16% polyethyleneglycol, for 30 rain at 4~ and then centrifuged at 1800 x g for 30 rain at 4~ the radioactivity of the pellet was counted in a gamma counter. The concentration of EGF binding activity was calculated by Scat-
EGF receptor in gastric cancer xenografts
August 1992
5000
z
~
J
~
NMSI2
461
--- 200K
--- 92.5K
100t
.---69K
500
..4-----I
"Ms
----46K 10(
5 0 84
1
2
3
4
5
6
weeks after mocuPatlon
Figure 1. Growth rate of human gastric cancer xenografts in nude mice. The xenograft tumors proliferated exponentially after a latent period. (Mean_+SD).
chard analysis 27 and expressed as fmol per. mg membrane protein.
Immunohistochemical demonstration of EGF receptor Immunohistochemical study of EGF receptor was carried out using the avidin-biotin-peroxidase complex (ABC) m e t h o d of Hsu et al. 28. Cryostat sections (4 ~m) were immersed in methanol containing 0.03% hydrogen peroxidase for 30 min to block any endogenous peroxidase activity, and then treated for 60 min at room temperature with anti-EGF receptor antiserum (GR-01, Oncogene Science Inc., NY, USA). Avidin and biotin reagents were obtained from BioGenex Laboratories (Biotin-Streptavidin Immunostaining Kit, Dublin, CA). Controls for ABC immunohistochemistry were run simultaneously as follows: (1) replacement of primary antiserum with normal n o n - i m m u n e mouse IgG, (2) use of EGF receptor antiserum absorbed with EGF, (3) omission of secondary antiserum. The sections were counterstained with hematoxylin. Results
Growth of human gastric cancer xenografts in nude mice Seven h u m a n gastric cancer xenografts were
"'8OK
Figure 2. The expression of EGF receptor in a human gastric cancer xenograft in nude mice (NMS 2) by Western blotting using anti-EGF receptor antibody (GR-01, OSI). The doublet bands are positive; a dominant band at 170 KDa is seen, paired with a smaller band at 150 KDa.
established. The tumor of all xenografts in nude mice grew slowly during the latent period, and then began to grow exponentially, as shown in Figure 1. The mean relative volume at 6 weeks after inoculation ranged from 1.5 • 102 to 4.7 • 103 (mm3), and the t u m o r doubling time of the 7 xenografts was different from each other.
Identification of EGF receptor in human gastric cancer xenografts of nude mice Detection of EGF receptor by Western blotting EGF receptor was detected as doublet bands at 150 KDa and 170 KDa in all of the xenografts by Western blotting, as shown in Figure 2. 1esI-EGF ligand binding assay Table 1 shows the concentration of 125I-EGF binding in h u m a n gastric cancer xenografts from nude mice. The specific binding was calculated by Scatchard analysis, as shown in Figure 2. EGF binding activity was examined for each xenograft, and the binding concentration was found to have
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Table 1. Expression of EGF receptor in human gastric cancer xenografts in nude mice
Histologya) NMS
2 6 10 11 12 13 24
intestinal intestinal intestinal diffuse diffuse diffuse intestinal
Placenta
-
a91
Doubling time (days)
EGF binding capacity (fmol/mg protein)
Western blotting for EGF receptor
Immunohistochemistry for EGF receptor
12.2 b) 28.4 14.1 18.0 7.8 9.6 30.6
11,000(4) c) 36.0(2) 117(3) 48.9(3) 799(7) 537(2) 60.2(3)
)ositive ~ositive ~ositive 9ositive )ositive )ositive )ositive
positive positive positive positive positive positive positive
2,180(5)
positive
NTdl
-
to the classification of Laur6n. b)Mean, e)Mean (Number of experiments), d)NT; Not tested. 3 0 -! o
20h
ml0_
o
20
40 6'0 EGF bound (fmol) Figure 3. Scatchard analysis of 1251-1abeledEGF binding capacit,/in human gastric cancer xenograffs (NMS2). Kd= 1.95x 10 -~2 EGF binding capacity=7,820 fmol/mg protein.
a broad range with a mean of 345 fmol/mg protein. The EGF binding activity of NMS 2 was higher than that of normal placental tissue. The dissociation constant (Kd) of E G F for the samples ranged from 1.87 x 10-12 to 20.51 • 10-12 M. These values were close to those of placental tissue. There was no definite correlation between the concentration of EGF receptor and histological features of intestinal type or diffuse type.
Immunohistochemical staining of EGF receptor All of seven xenografts were positive for EGF receptor immunohistochemically, as revealed by staining predominantly on the apical border of the glands, the cell membrane and in the cytoplasm of the cancer cells, as shown Figure 4.
Concentration of EGF receptor and the doubling time of human gastric cancer xenografts The tumor doubling time of h u m a n gastric cancer xenografts ranged from 7.8 to 30.6 days (Table 1). There seemed to be an inverse correlation between the concentration of EGF receptor and the doubling time of the 7 xenografts, with a correlation coefficient o f - 0 . 6 8 , as shown in Figure 5. All tumors having more than 100 fmol/mg EGF receptor protein had a doubling time of less than 14 days. Conversely, all tumors having less than 100 fmol/mg EGF receptor protein showed a t u m o r doubling time of more than 14 days. Discussion EGF receptor was demonstrated by Western blotting, 125I-EGF ligand binding and immunohistochemistry in h u m a n gastric cancer xenografts of nude mice. Western blotting revealed E G F receptor as doublet bands at 150 KDa and 170 KDa in all of seven xenografts, thus corresponding to the published molecular weights for EGF receptor protein 29. nSI-EGF binding assay showed that a large n u m b e r of E G F binding sites were present in h u m a n gastric cancer xenografts. I m m u n o h i s tochemical studies demonstrated that E G F receptor was clearly positive predominantly on the apical border of the glands, the cell m e m b r a n e and in the cytoplasm of the cancer cells in the xenografts. The results of Western blotting matched those of immunohistochemistry in all specimens. Recently, EGF receptor was detected i m m u n o -
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EGF receptor in gastric cancer xenografts
Figure 4.
Immunohistochemistry of EGF receptor in a human gastric cancer xenograft from nude mice (NMS2) using anti-EGF receptor antibody (GR-01, OSi). EGF receptor is positive predominantly on the apical border of the glands, and the cell membrane of the tumor cells. (x 200).
10, 000 5, 000
~o -~ 1,000 c o. =5 c E ~
500
t
tO0
t
50
ib
fs
~o
fs
~0
Tumor doubling time (days)
Figure 5. EGF binding capacity and the growth of human gastric cancer xenografts in nude mice. log Y=3.55-0.067X, Correlation coefficient= -0.68.
histochemically in 9 of 15 surgically resected gastric cancer specimens 3~ and increased levels of EGF receptor in gastric cancer have been shown by I:5I-EGF binding assay 31'32. We examined EGF receptor in surgical specimens of stomach cancer by ]2SI-EGF binding assay, and found that 23 (46%) of 50 specimens were EGF receptor-positive33. Thus, reports on the presence of EGF receptor in human gastric cancers have been accumulating. EGF receptor was initially shown to be present more frequently in squamous cell carcinomas
than in adenocarcinomas, and the gene for EGF receptor was found to be more often amplified in the former than the latter 16. Squamous cell carcinoma of the lung, however, did not contain a greater concentration of EGF receptor than adenocarcinoma of the lung 34. The degree of EGF receptor expression in cancer cells may vary from tissue to tissue. The present study showed that some human gastric adenocarcinomas among the xenografts had a large amount of EGF receptor, the highest receptor concentration being far greater than that of normal placental tissue. An inverse correlation was observed between EGF binding activity and the doubling time of the 7 xenografts, indicating an effect on tumor growth. The growth of xenografts having a large amount of EGF receptor, more than 100 fmol/mg protein, was faster than that ofxenografts having a small amount of EGF receptor, less than 100 fmol/mg protein. Tumor tissues exhibited higher EGF receptor mRNA expression levels than those in the adjacent normal tissues 3s. The degree of gene amplification and concentration of EGF receptors have been shown to be directly correlated with the growth of A431 cells transplanted into athymic mice 36. Thus, the expression
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of EGF receptors appears to facilitate the growth of human gastric cancer xenografts in nude mice. EGF, via its receptor, has both stimulative and inhibitory effects on the in vitro growth of A431 depending on culture conditions; while EGF inhibits anchorage-dependent growth in monolayer culture, it stimulates anchorage-independent growth in soft agar 6. The ability of cells to grow as colonies in soft agar has been shown to correlate highly with tumorigenicity in nude mice 37. EGF was found to stimulate the in vivo growth of EGF receptor-hyperproducing A431 cells 7. It will be necessary to examine the effect of EGF on the growth of human gastric cancer xenografts in nude mice having different concentrations of EGF receptor, since EGF-EGF receptor stimulation may contribute to the growth of these tumor cells. This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and the Science Research Promotion Fund from the Japan Private School Promotion Foundation, Japan. The authors are grateful to Mr. Mitsuhiro Kudoh for excellent technical assistance and Ms. Kanna Mizuno for animal care.
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factor in gastric carcinoma as a biologic marker of high malignancy. Jpn J Cancer Res 1986;77:145-152. 10. Hunter T. The epidermal growth factor receptor gene and its product. Nature 1984;311:414-416. 11. Downward J, Yarden Y, Mayes E, et al. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 1984;307:521-527. 12. Merfino GT, Xu YH, Ishii S, et al. Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells. Science (Wash. DC) 1984;224:417-419. 13. Ozawa S, Ueda M, Ando N, et al. High incidence of EGF receptorhyperproduction in esophageal squamous cell carcinomas. Int J Cancer 1987;39:333-337. 14. Fitzpatrick SL, LaChance MP, Schultz GS. Characterization of epidermal growth factor receptor and action on human breast cancer cells in culture. Cancer Res 1984;44:3442-3447. 15. Libermann TA, Razon N, Bartal AD, et al. Expression of epidermal growth factor receptors in human brain tumors. Cancer Res 1984;44:753-760. 16. Yamamoto T, Kamata N, Kawano H, et al. High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell fines. Cancer Res 1986;46:414416. 17. Yasui W, Hata J, Yokozaki H, et al. Interaction between epidermal growth factor and its receptor in progression of human gastric carcinoma. Int J Cancer 1988;41:211-217. 18. Yoshiyuki T, Shimizu Y, Onda M, et al. Immunohistochemical demonstration of epidermal growth factor in human gastric cancer xenografts in nude mice. Cancer 1990;65:953-957. 19. Laur6n P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal type carcinoma. Acta Pathol Microbiol Scand 1965;64:31-49. 20. Kiyama T, Onda M, Tokunaga A, et al. Changes in serum and tissue carcinoembryonic antigen of a human gastric cancer xenograft in nude mice. Jpn J Cancer Res 1990;81:58-62. 21. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24:148-154. 22. Giavazzi R, Campbell DE, Jessup JM, et al. Metastatic behavior of tumor cells isolated from primary and metastatic human colorectal carcinomas implanted into different sites in nude mice. Cancer Res 1986;46:1928-1933. 23. Slieker LJ, Lane MD. Post-translational processing of the epidermal growth factor receptor. J Biol Chem 1985;260:687-690. 24. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685. 25. Towbin H, Staehefin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:43504354. 26. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 27. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1949;51:600-672. 28. Hsu SM, Raine L, Fanger H. Use of avidin-peroxidase complex (ABC) in immunoperoxidase techniques; a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577-580. 29. Cohen S, Ushiro H, Stoschek C, et al. A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 1982;257:1523-1531.
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30. Sakai K, Mori S, Kawamoto T, et al. Expression of epidermal growth factor receptors on normal human gastric epithelia and gastric carcinoma. J Natl Cancer Inst 1986;77:477-483. 31. Yasui W, Sumiyoshi H, Hata J, et al. Expression of epidermal growth factor receptor in human gastric and colonic carcinomas. Cancer Res 1988;48:137-141. 32. Pfeiffer A, Rothbauer E, Wiebecke B, et al. Increased epidermal growth factor receptors in gastric carcinomas. Gastroenterology 1990;98:961-967. 33. Nishi K, Onda M, Tokunaga A, et al. Expression of epidermal growth factor receptor in human gastric cancer. Proc 48th Ann Meet Jpn Cancer Assoc 1989;1333. (in Japanese)
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34. Veale D, Kerr N, Gibson G J, et al. Characterization of epidermal growth factor receptor in primary human non-small cell lung cancer. Cancer Res 1989;49:1313-1317. 35. Yoshida K, Kyo E, Tsujino T, et al. Expression of epidermal growth factor, transforming growth factor-a and their receptor genes in human gastric carcinomas; implication for autocrine growth. Jpn J Cancer Res 1990;81:43-51. 36. Santon JB, Cronin MT, Macleod CL, et al. Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice. Cancer Res 1986;46:4701-4705. 37. Kahn P, Shin SI. Cellular tumorigenicity in nude mice. J Cell Biol 1979;82:1-16.
